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"Nippleareola complex NAC reconstruction is a technique used in breast reconstructive surgerywhich is performed during the final stage of breast reconstruction after total mastectomy of primary breast cancerComposite nipple grafts utilizing the contralateral NAC are common however to our knowledge there are noreports of new primary invasive ductal carcinoma development within the graft Here we describe one such casefor the first timeCase presentation A 54yearold woman was referred to us by the Department of Plastic and ReconstructiveSurgery in our medical center for further evaluation of right nipple erosion She had undergone total mastectomyof the right breast following a breast cancer diagnosis years ago at which time tumor biological profilingrevealed the following estrogen receptor ER positive progesterone receptor PgR negative and humanepidermal growth factor receptor HER2 undetermined She received adjuvant chemotherapy and endocrinetherapy She defaulted endocrine therapy for a few years and years after surgery she underwent autologousbreast reconstruction with a deep inferior epigastric perforator DIEP flap In the following year NAC reconstructionwas performed using a composite graft technique Seven years after the NAC reconstruction erosion appeared onthe nipple grafted from its contralateral counterpart scrape cytology revealed malignancy The skin on the rightside of her chest around the NAC and subcutaneous fat tissue consisted of transferred tissue from the abdomen asthe DIEP flap and grafted nipple were located on the graft skin The right nipple carcinoma arose from the tissuetaken from the left nipple Magnetic resonance imaging MRI or computed tomography showed no malignantfindings in the left breast As the malignant lesion seemed limited to the area around the grafted right nipple onMRI surgical resection with sufficient lateral and deep margins was performed around the right nipple Pathologicalfindings revealed invasive ductal carcinoma with comedo ductal components infiltrating the graft skin andunderlying adipose tissue Immunohistochemistry revealed positive for ER PgR and HER2Continued on next page Correspondence mar016outlookjp1Department of Breast and Thyroid Surgery Yokohama City UniversityMedical Center Urafunecho Minamiku Yokohama Japan4Department of Gastroenterological Surgery Yokohama City UniversityGraduate School of Medicine Yokohama JapanFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 0cKimura Surgical Case Reports Page of Continued from previous pageConclusions To our knowledge this is the first case involving the development of invasive ductal carcinoma in anipple graft constructed on the skin of a DIEP flap with the origin from the contralateral breasts nippleKeywords Breast cancer Autologous breast reconstruction Nipple graft Contralateral breast cancer Nippleareolacomplex reconstruction Deep inferior epigastric perforator flap Reconstructed nipple Donor nipple Compositenipple grafting Invasive cancer of graftBackgroundAutologous breast reconstruction ABR after mastectomy for breast cancer has become common over thepast decades [] An ABR with a perforator flap is a goodoption for a lot of patients who may be concerned abouttheir body image and nippleareola complex NAC reconstruction is performed in women who have undergone a total or skinsparing mastectomy which differsfrom a nipplesparing mastectomy It is an integral partof the breast reconstruction process as patients associatethis stage with the end of their plastic surgery treatmentand a sense of completion [] It was once a concernthat surgical trauma could activate dormant micrometastases however more recent reports have shown no increased risk of breast cancer recurrence following breastreconstruction [] New primary carcinomas arisingfrom the grafted breast tissue have rarely been reportedfollowing surgery for breast cancer Here we present arare case of a new primary carcinoma in a reconstructednipple which originated from the contralateral nipplewithout any malignancy detected through imaging in either the left nipple or left mammary glandCase presentationA 54yearold woman was referred to us by the Department of Plastic and Reconstructive Surgery in our medical center for further evaluation of prolonged rightnipple erosion She had previously received a diagnosisof right breast cancer and undergone total mastectomyand axillary dissection years ago in another hospitalHistopathology identified an invasive ductal carcinomawith a tumor diameter of cm and a nuclear grade of one of lymph nodes showed metastasis Tissue profiling revealed the following estrogen receptor ERpositive progesterone receptor PgR negative and human epidermal growth factor receptor HER2 undetermined As adjuvant therapy she received six cyclesof cyclophosphamide methotrexate and 5fluorouracilCMF followed by tamoxifen for years Then shedefaulted her endocrine therapy Seven years after thesurgery the doctors from our medical center performedABR with a deep inferior epigastric perforator DIEPflap at another facility The following year her right nipple was reconstructed by Vshaped resection of the leftnipplegraftingnipplesharingautologousandantechnique The right areola was reconstructed with apenetrating skin graft from the proximal thigh and leftareola Concurrent mastopexy was performed for the leftbreast Fig 1aAfter years right nipple erosion appeared and shevisited the Department of Plastic and ReconstructiveSurgery in our medical center At first it appeared thatshe had an eczematous nipple lesion caused by an infection and she was treated with antibiotics however theerosion progressed and enlarged over the course of afew months She was eventually referred to our department The skin on the right side of her chest around theNAC and the subcutaneous adipose tissue consisted oftransferred tissue from her abdomen as the DIEP flapand grafted nipple were constructed on the skin graft Inthe right nipple normal tissue was almost completely affected by erosion and there was no abnormality itchingor pain in the right areola Fig 1b Scrape cytology revealed malignancy of the epithelial cells and that theright nipple carcinoma originated from the tissue takenfrom the left nipple On magnetic resonance imagingMRI the malignant lesion seemed limited to the areaaround the grafted right nipple Fig 2a with no malignancy observed in the left breast on MRI and computedtomography CT Fig 2b In addition no distant metastases were observed on CT Pagets disease was clinically suspected and we performed surgical treatmentThough the standard surgical operation for mammaryPagets disease is mastectomy we performed partialbreast excision including the right nipple with sufficientlateral and deep margins Fig 3ac because there wasno mammary tissue in the right reconstructed breast except for the nipple and areola The incision was closedwith investing suturesThe specimen submitted for surgical pathology wascomposed of epithelial and adipose tissue grafted fromthe abdomen areolar tissue grafted from the base of thethigh and left areola and a nipple graft from the contralateral side Macroscopically the lesion spread aroundthe nipple and adipose tissue Fig 4a Pathologicalexamination identified invasive ductal carcinoma with afew comedo ductal components within the nipple extensive infiltration of grafted epithelial and adipose tissueFig 4b and a tumor diameter of mm The nucleargrade score was nuclear atypia score was and 0cKimura Surgical Case Reports Page of Fig a This is an image obtained about months after autologous breast reconstruction ABR with a deep inferior epigastric perforator DIEPflap followed by nippleareola complex NAC reconstruction of the right breast b Image of the right NAC shows erosion and marginal crustingwith a small amount of normal tissue at the upper right side of the grafted nipple The erosion was hemorrhagic on scraping The grafted areolahad no abnormalitymitotic count score was there was no lymphatic orvascular invasion and the lateral and deep margins werenegative Immunohistochemical staining showed strongpositive for ER weak positive for PgR positive for HER2with a score of and cells showing positive Ki67stainingAs the biological profile classified the tumor as a luminal HER2 type weekly paclitaxel trastuzumab andendocrineadjuvantadministered astherapy weretherapies No distant metastases or local recurrence wereseen year after the surgeryDiscussionNAC reconstruction is a technique used in the finalstage of breast reconstruction following total mastectomy for primary breast cancer Composite nipple grafting has been described in the literature since the early1970s and the technique is now widely performed Tilldate only one report of breast cancer originating in aFig a Magnetic resonance imaging MRI shows enhancement of the lesion of the right nipple and slight subcutaneous adipose tissue bThere is no abnormal enhancement in the left breast 0cKimura Surgical Case Reports Page of Fig Preoperative image of the patient a and markings for skin incision b Intraoperative image after excision of the lesion with sufficientlateral and deep margins cnipple reconstructed from tissue obtained from thecontralateralunaffected side has been identified via aPubMed search however that case involved Pagets disease not an invasive carcinoma [] A de novo carcinoma developed years after the NAC reconstructionand the donor nipple had no identifiable lesion similarto the findings in this case To our knowledge that report documented the first case of Pagets disease arisingfrom a grafted nipple in the US literatureBreast cancer is considered to develop from the epithelia of terminal duct lobular units TDLUs includingcases of in situ and invasive carcinomas TDLUs usuallyexistin distal mammary glands through a series ofbranches On the other hand major lactiferous ductsterminate in and exit from the mammary glands at thenipple although there are cases where TDLUs exist inadjacent lactiferous ducts in the nipple Kryvenko et aland occultinvestigated thefrequency of TDLUsneoplastic epithelial proliferation in grosslyclinically unremarkable nipples [] They observed TDLUs in ofnipples of tissue specimens from therapeutic or prophylactic mastectomies while occult neoplastic epithelialproliferation was seen in of grosslyclinically unremarkable therapeutic mastectomy nipples They also reported that the nipples were unremarkable in all cases ofprophylactic mastectomies Moreover occult neoplasticproliferationsin grossly unremarkable nipples werelargely correlated with underlying carcinomas but onlytwo patients had a primary malignant neoplasm in theirnipples Based on these reports it is difficult to confidently state whether grossly unremarkable donor nipplesare afflicted by malignant neoplasms or notThis case is also rare in terms of the origin of the invasive ductal carcinoma of the nipple Very little has beenpublished about breast cancers developing within thenipple especially invasive ones The first large series ofFig Histopathological findings a The boundary of the tumor is delineated with a red line on the macroscopic image The tumor is localizedwithin epithelial and subcutaneous adipose tissue The macroscopic image shows extensive infiltration and growth of tumor cells b At the lowerperipheral area tumor cells are infiltrating into the adipose tissue across the desmoplastic border black arrow The infiltrating tumor cells showtrabecular sheetlike acinar and nesting growth patterns with a few ductal carcinomas in situ with comedo necrosis red dotted circle 0cKimura Surgical Case Reports Page of carcinomas originating in the nipple was published in [] Of approximately cases of malignantdisease of the breast in patients undergoing surgery atthe Mayo Clinic in years only of cases originated in the nipple Secondly Sanders have reported common features ofinvasive primary breastcarcinomas originating in the nipple [] The frequencyof those carcinomas was in the report and only patients among more than breast carcinoma patients investigated in their study presented with symptoms related to the nipple Of these patients hadepithelial changes associated with in situ ductal carcinomas involving the skin of the nipple Pagets diseasewith small foci of invasion into the dermis The rest ofthe patients presented with a nipple mass with or without skin changes Consistent with the case reported hereHER2 positivity was observed in tissue obtained from of patients with Pagets disease and of patientswith a nipple mass The likelihood of lymph node metastases was not higher than that for carcinomas of similarsizes and there was no disease recurrence followingproper adjuvant therapy Thus invasive primary nipplecarcinomas are rare but conventionaltherapies forbreast cancer treatment are also useful in such cases Inour case we initially suspected Pagets disease becauseof nipple erosion and the fact that no solid tumor wasidentified by palpation The patient exhibited pagetoidspread of an invasive ductal carcinoma with comedoductal components without the presence of a palpablesolid tumor However contrast scrape cytology suggested a ductal carcinoma because no melanin granuleswere observed in the malignant cells [] In contrastconsidering that HER2 positivity is more frequent inPagets disease than in invasive ductal carcinoma it cannot be denied thatthis carcinoma originated fromPagets disease in terms of the immunoprofile HER2 isoverexpressed in ER and PgR are positive in approximately and respectively in Pagets disease[] in contrast with positive for HER2 in invasive ductal carcinoma However there were only a fewPaget cells within the squamous epithelium of the nipple and most malignant cells had directly infiltrated thenipple surface resulting in ulceration In addition theinfiltrating malignant cells were notlarge cells withabundant eosinophilic cytoplasm like Paget cells Therefore they had likely originated from the invasive ductalcarcinoma rather than Pagets diseaseThere has been a lot of discussion about the oncological safety of ABR after mastectomy for invasivebreast cancer however a current literature review failedto show significant risks of either concurrent or delayeddistant or local recurrence following ABR relative to therisk in the absence of ABR In this case there were noremnant breast tissues in the right breast except for thenipple and areola and the malignant neoplasm arosefrom the flap of abdominal skin from a DIEP flap []Thus the malignancy was not defined as a local recurrence but as a new primary carcinoma considering therewere no right breast tissue components such as skinblood and lymphatic vessels around the malignant neoplasm Although total mastectomy is usually recommended in cases of local recurrence of breast cancer inthis case we performed a partial resection because thelesion was quite localized and there was no mammarygland except for localized tissue beneath the nipple ofthe right breast based on imaging and the carcinomaseemed not to be related to lymphovascular invasion ofthe initial breast cancerIt was unclear whether the de novo carcinoma arosebefore or after the NAC surgery Surgical treatmentmight trigger the occurrence of malignant changes although there are few case reports describing cancers developing in transferred tissues to support this scenarioCancer development is a multifaceted dynamic seriesof events and some new carcinogenic hypotheses havebeen described Recently the role of the microenvironment in driving tumor progression has been increasinglyrecognized [] Tumor formation begins when geneticabnormalities occur in cells that undergo rapid unchecked proliferation However a tumor is not solelycomprised of cancer cells it is a heterogeneous collection of both cancer cells and surrounding noncancerousor stromal cells that work in concert with one anotherto promote unrestricted growth infiltration and propagation of malignancy throughout the body In the microenvironment of carcinomas tumor cells recruit varioustypes of stromal cells such as fibroblasts inflammatorycells and endothelial cells and their cellular interactionsare important drivers of progressive tumor growth []Expansion of the tumor stroma is often observed in invasive carcinomas these changes result in desmoplasiaswhere tumors and stroma actively interact Pathologically our case presented scirrhous spread of a carcinomain the absence of a palpable tumor thus we assumedthat there was an expansion of a desmoplasia aroundthose tissuesA hypoxic environment is one of the key physiologicaland microenvironmental characteristics that differentiatetumors from normal tissues The transcription factorhypoxiainducible factor 1α and angiogenesis are important factors that regulate hypoxiainduced signalingcascades [] Hypoxia can drive and maintain geneticinstability resulting in a mutated phenotype Moreoverhypoxia along with acidosis increases clonal selectionresulting in aggressive cancer phenotypes As a resultstressorsincluding surgery may induce oncogenicchanges in tissue In contrast nipple banking was formally performed to rebuild a nipple which is a 0cKimura Surgical Case Reports Page of technique in which a nipple is taken from a site on theipsilateral breast banked in the groin and then laterreturned to the chest [ ] Nevertheless the nipplewas taken in the case without apparent involvement ofthe nipple with carcinoma some cases of the development of heterotopic carcinoma in the transplanted nipple were reported This indicates that cancer cells in thetransplanted nipple could survive in a hypoxic or ischemic environment In our case surgical stressors mightalso have affected the donor tissue of the left nipplethough the timing of generation of malignant cells wasnot definitive Generally careful preoperative screeningfor contralateral breast cancer should be performed before NAC reconstruction whether malignantlesionsexist though there were no signs of malignancy in theleft breast in this case Moreover postoperative examinations are important because of the possibility of development of occult malignant tumors in the left breastthus we have continued to monitor the patients leftbreast carefullyConclusionsTo our knowledge this is the first report of an invasiveductal carcinoma developing in a grafted nipple following ABR This might be a rare case but clinicians mustconsider the possibility that carcinomas can develop in agraft as long as there are remnants of mammary glandtissue within the graftAbbreviationsABR Autologous breast reconstruction CMF Cyclophosphamidemethotrexate and 5fluorouracil 5FU CT Computed tomographyDIEP Deep inferior epigastric perforator ER Estrogen receptor HER2 Humanepidermal growth factor receptor MRI Magnetic resonance imagingNAC Nippleareola complex PgR Progesterone receptor TDLU Terminalduct lobular unitAcknowledgementsWe would like to thank Editage wwweditagecom for English languageeditingConsent to participateThis study was carried out in accordance with the principles of theDeclaration of HelsinkiAuthors contributionsMK wrote the manuscript and performed the breast cancer surgery whicharose in the grafted nipple in this case in which KN assisted and instructed TSperformed all plastic surgery operations which were mentioned in this reportfor this patient and supervised this manuscript from the point of plastic surgeryKN TI and IE supervised this manuscript from the point of oncology and breastsurgery MT and YI supervised this manuscript from the point of pathology HSSI MM SA AY KS and YI served as the attending physicians of the presentedpatient All authors read and approved the final manuscriptFundingThe authors have no financial contributions to discloseEthics approval and consent to participateNot applicableConsent for publicationThe patient described in this report provided informed consent for thepublication of the case detailsCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Breast and Thyroid Surgery Yokohama City UniversityMedical Center Urafunecho Minamiku Yokohama Japan2Plastic and Reconstructive Surgery Yokohama City University MedicalCenter Yokohama Japan 3Diagnostic Pathology Yokohama City UniversityMedical Center Yokohama Japan 4Department of GastroenterologicalSurgery Yokohama City University Graduate School of Medicine YokohamaJapan 5Department of Oncology Yokohama City University Graduate Schoolof Medicine Yokohama Japan 6Department of Breast Oncology and SurgeryTokyo Medical University Shinjuku Tokyo JapanReceived January Accepted July ReferencesNahabedian MY Patel K Autologous flap breast reconstruction surgicalalgorithm and patient selection J Surg Oncol Sisti A Grimaldi L Tassinari J Cuomo R Fortezza L Bocchiotti MA et alNippleareola complex reconstruction techniques a literature review Eur JSurg Oncol Geers J Wildiers H Van Calster K Laenen A Floris G Vandevoort M et alOncological safety of autologous breast reconstruction after mastectomy forinvasive breast cancer BMC Cancer Basu CB Wahba M Bullocks JM Elledge R Paget disease of a nipple graftfollowing completion of a breast reconstruction with a nipplesharingtechnique Ann Plast Surg Kryvenko ON Yoon JY Chitale DA Lee MW Prevalence of terminal ductlobular units and frequency of neoplastic involvement of the nipple inmastectomy Arch Pathol Lab Med Congdon GH Dockerty MB Malignant lesions of the nipple exclusive ofPagets disease Surg Gynecol Obstet Sanders MA Brock JE Harrison BT Wieczorek TJ Hong X Guidi AJ et alNippleinvasive primary carcinomas clinical imaging and pathologicfeatures of breast carcinomas originating in the nipple Arch Pathol LabMed Sakorafas GH Blanchard K Sarr MG Farley DR Pagets disease of the breastCancer Treat Rev Hoon Tan P Ellis I Allison K Brogi E Fox SB Lakhani S The WHOclassification of tumours of the breast Histopathology Allen RJ Treece P Deep inferior epigastric perforator flap for breastreconstruction Ann Plast Surg Spaw M Anant S Thomas SM Stromal contributions to the carcinogenicprocess Mol Carcinog Yamaguchi Y Hayashi S Estrogenrelated cancer microenvironment ofbreast carcinoma Endocr J Bristow RG Hill RP Hypoxia DNA repair and genetic instability Nat RevCancer Cucin RL Gaston JP Case report implantation of breast cancer in atransplanted nipple a plea for preoperative screening CA Cancer J ClinSnyderman RK Nipple banking CA Cancer J Clin Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
Molecular Medicine The role of selenium metabolism andselenoproteins in cartilage homeostasisand arthropathiesDonghyun Kang Jeeyeon Lee Cuiyan Wu3 Xiong Guo3 Byeong Jae Lee24 JangSoo Chun5 andJinHong Kim AbstractAs an essential nutrient and trace element selenium is required for living anisms and its beneï¬cial roles in humanhealth have been well recognized The role of selenium is mainly played through selenoproteins synthesized by theselenium metabolic system Selenoproteins have a wide range of cellular functions including regulation of seleniumtransport thyroid hormones immunity and redox homeostasis Selenium deï¬ciency contributes to various diseasessuch as cardiovascular disease cancer liver disease and arthropathyKashinBeck disease KBD and osteoarthritisOA A skeletal developmental disorder KBD has been reported in lowselenium areas of China North Korea and theSiberian region of Russia and can be alleviated by selenium supplementation OA the most common form of arthritisis a degenerative disease caused by an imbalance in matrix metabolism and is characterized by cartilage destructionOxidative stress serves as a major cause of the initiation of OA pathogenesis Selenium deï¬ciency and dysregulation ofselenoproteins are associated with impairments to redox homeostasis in cartilage We review the recently exploredroles of selenium metabolism and selenoproteins in cartilage with an emphasis on two arthropathies KBD and OAMoreover we discuss the potential of therapeutic strategies targeting the biological functions of selenium andselenoproteins for OA treatmentIntroductionSelenium Se is an essential trace element in humans12Selenium is generally taken up from the diet through foodor other forms of external supplementation Dietaryselenium is obtained in the form of selenomethionineSeMet selenocysteine Sec selenite and selenate Signiï¬cant health beneï¬ts have been attributed to seleniummetabolic systems that play major physiological roles inthyroid hormone metabolism immunity and antioxidantdefense23 Selenium is required for the production ofthyroid hormonemetabolizing enzymes and seleniumCorrespondence JinHong Kim jinhkimsnuackr1Center for RNA Research Institute for Basic Science Seoul South Korea2Department of Biological Sciences College of Natural Sciences Seoul NationalUniversity Seoul South KoreaFull list of author information is available at the end of the These authors contributed equally Donghyun Kang Jeeyeon Leesupplementation is thought to improve the function ofthyrocytes and immune cells4 Selenium supplementationdemonstrated immunostimulant effects such as enhancedproliferation of activated T cells activation of naturalkiller cells and tumor cytotoxicity mediated by cytotoxiclymphocytes56 In contrast selenium deï¬ciency is associated with the occurrence virulence and disease progression of viral infections7Selenium inadequacy can lead to various types ofdiseases most notably cardiovascular disease8 cancer13 hepatopathy1617 and arthropathy Cardiovascular diseases are associated with systemic seleniumlevel with a higher risk at or μgL seleniumconcentration in the blood10 A type of endemic cardiomyopathy Keshan disease is linked to selenium deï¬ciency811 Keshan disease occurs in lowselenium areasin Chinasodium seleniteand is prevented by The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Korean Society for Biochemistry and Molecular Biology 0cKang Experimental Molecular MedicinePage of studiesEpidemiologicalsupplementation12 Lowselenium status is correlatedwith a significantly increased risk of cancer incidenceand mortality13haveprovided evidence on the cancerpreventing effects ofselenium18 Selenium deï¬ciency is also characterizedby elevated levels of oxidative stress markers in the liver21which significantly contribute to liver injury17 The oxidative stress caused by selenium deï¬ciency further plays adetrimental role in joint development Selenium deï¬ciency is the main cause of endemic KashinBeck diseaseKBD which is mainly reported in lowselenium areas ofChina North Korea and the Siberian region of RussiaMoreover there is a growing body of evidence suggestingthat the pathogenesis of osteoarthritis OA the mostcommon form of arthritis may be associated with selenium deï¬ciency by resulting in oxidative stress22However it is noteworthy that excessive selenium intakecan also cause selenosis2930 which accompanies adversesymptoms including fatigue diarrhea nausea increasedheart rate necrosis in liver and kidney and neurologicaldamage Chroniccompromisesimmune and reproductive systems in humanseventuallyselenosisOA is characterized by progressive loss of cartilageextracellular matrix ECM and pathological changes inother joint tissues such as subchondral bone sclerosisosteophyte formation and synovial ammation31 Cartilage destruction is considered a hallmark of OA and is aresult of increased production of catabolic effectors32and reduced matrix biosynthesis by chondrocytes36 OA isassociated with multiple etiologies involving systemicfactors such as age37 as well as local factors such asmechanical stress38 driven by weightbearing and jointinstability Both OAcausing factors have been found tocause oxidative stress in chondrocytes Oxidative stressresults from the abnormal production of reactive oxygenspecies ROS and the loss of cellular antioxidant capacityMany preclinical and clinical studies have indicated theaccumulation of oxidative burden in chondrocytesundergoing osteoarthritic changes3940 Emerging evidence suggests that oxidative stress is mechanisticallylinked to the initiation of osteoarthritic changes inchondrocytes through the acquisition of senescent phenotypes36 Therefore restoring redox homeostasis canserve as a rational therapeutic strategy to alleviate OAprogression Here we review the role of selenium metabolism in cartilage and bone and the signiï¬cance ofmaintaining its homeostasis in the context of joint diseases such as KBD and OAOverview of the selenium metabolic systemThe selenium metabolic system and the biosynthesis ofselenoproteinsSelenium metabolism is a systemic process that includesandtransformationtransportationabsorptiontheOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyexcretion of selenium Fig Selenium is obtained inanic formsSeMet and Secand inanic formsselenite and selenatefrom diet Selenium is taken up bythe liver that synthesizes and exports SELENOP whicheventually circulates through the bloodstream SELENOPwith multiple Sec residues41 transports selenium to othertissues and ans42 and the transported selenium isconverted to selenophosphate by intracellular seleniummetabolic pathways Selenium is excreted through exhalation and urine in the form of smallmolecule metabolites formed by sequential methylation4344Selenium plays biological roles predominantly in theform of selenoproteins synthesized by the seleniummetabolic system Ingested inanic selenium is ï¬rstreduced to hydrogen selenide H2Se via glutathioneGSH and thioredoxin TXN systems Selenide is furtherconverted to Sec amino acids for incorporation intospeciï¬c sites of selenoproteins such as the catalytic sites ofa selenoenzyme Mechanistically selenophosphate synthetase SEPHS2 catalyzes the production of selenophosphate through the reduction of hydrogen selenideThe subsequent reaction with phosphoseryltRNA PSertRNA[Ser]Sec yields SectRNA[Ser]Sec Sec amino acids areincorporated into polypeptidethrough themachinery utilizing the UGA codon Selenocysteineinsertion sequence binding protein SBP2 binds toselenocysteine insertion sequence SECIS element whichis located in the ²untranslated region ²UTR of selenoprotein mRNA and mediates the transfer of SectRNA[Ser]Sec to the Asite of ribosome which recognizesthe UGA codon as the Sec integration codon Collectivelythe selenoprotein translation machinery consists of SECISelement SBP2 Secspeciï¬c eukaryotic elongation factorEEFSEC and aminoacylated SectRNA[Ser]Sec therebyenabling UGA to be recognized as a Sec codon and utilized for translation into the growing polypeptidechainsSelenoproteinssome ofSelenoprotein is deï¬ned as a protein containing Secamino acid residue The biological functions of seleniumare mostly exerted through selenoprotein domains thatcontain Sec residues Twentyï¬ve selenoprotein geneshave been identiï¬ed in the human genome45 In mice atotal of selenoproteins have been characterized46 andtargeted deletion ofthese selenoproteinsdemonstrated their essential roles in developmental processes and in disease pathogenesis Selenoproteins can beclassiï¬ed into subfamilies based on their cellular functionssuch as those implicated in antioxidation GPX1 GPX2GPX3 GPX4 redox regulation TXNRD1 TXNRD2TXNRD3 MSRB1 SELENOH SELENOM SELENOWthyroid hormone metabolism DIO1 DIO2 DIO3 selenium transport and storage SELENOP selenophosphatesynthesis SEPHS2 calcium metabolism SELENOK 0cKang Experimental Molecular MedicinePage of Fig Selenium metabolic system in mammals Selenium is absorbed from the diet undergoes several conversion steps and is incorporated intopolypeptide chains completing selenoprotein synthesis Dietary sources of selenium uptake exist in inanic form such as selenate and selenite andanic form such as Sec and SeMet Inanic forms are reduced by TXNRDTRX or GRXGSH systems and anic forms are cleaved by SCLYforming selenide Selenophosphate is synthesized from selenide by SEPHS2 and the subsequent reaction with PSertRNA[Ser]Sec mediated by SEPSECSyields SectRNA[Ser]Sec SectRNA[Ser]Sec is transferred to the Asite of ribosome mediated by SBP2 which binds to SECIS located in the ²UTR of aselenoprotein mRNA Finally the UGA codon is recognized as the Sec integration codon Abbreviations SeMet selenomethionine Secselenocysteine GRX glutathione reductase TRX thioredoxin TXNRD thioredoxin reductase GSH glutathione MGL methionine gammalyase SCLYselenocysteine lyase SEPHS2 selenophosphate synthetase SARS seryltRNA synthetase PSTK phosphoserylSeptRNA kinase SEPSECS SeptRNASectRNA synthase EEFSEC Secspeciï¬c eukaryotic elongation factor SBP2 SECIS binding protein SELENOT myogenesis SELENON protein foldingSELENOF SELENOI SELENOS and protein AMPylation SELENOO4748 The functions of other selenoproteins such as GPX6 and SELENOV still remain unclearGlutathione peroxidases GPXs such as GPX1 cytosolicGPX GPX2 gastrointestinal GPX and GPX4 phospholipid hydroperoxide GPX catalyze the decompositionof a great variety of peroxides thus protecting cellsagainst oxidative damage4950 Thioredoxin reductasesTXNRDs employ NADPH as an electron donor to revertoxidized TXN to a reduced dithiol the oxidation status ofwhich is critically implicated in regulating various cellbehaviors including proliferation and apoptosis51 Thephysiological signiï¬cance of TXNRDs is further supported by the embryonic lethality of Txnrd1 or Txnrd2knockout mice5253 Deiodinases DIOs regulate thyroidhormone metabolism by catalyzing the conversion ofthyroid hormones from precursor thyroxine T4 to biologically active triiodothyronine T3 or inactive reverseT3 rT354 The expression levels of several selenoproteinsOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyare uenced by the extent of selenium uptake Forexample seleniumdeï¬cient animals and human cell linesexhibit reduced transcription of selenoproteins such asGPX1 DIOs SELENOI and SELENOW55 A subset ofselenoproteins such as GPX1 and SELENOW is moresensitive to selenium supplementation or deï¬ciency Thehierarchy of selenoprotein expression is more apparentwhen the intracellular level of selenium is limited1Seleniumresponsive genesgenesareseleniumcontainingSeleniumresponsivethe genes whoseexpression patterns are uenced by supplementationwith selenium orcompoundsTreatment of a cancer cell line with methylseleninic acidin genes58 Theseinduced expression changesresponsive genes were closely associated with annotationsrelated to cell cycle regulation androgenresponsive genesand phase II detoxiï¬cation pathway Selenium supplementation of macrophages diminished the expression oflipopolysaccharide LPSinduced proammatory genes 0cKang Experimental Molecular MedicinePage of such as cyclooxygenase2 COX2 and tumor necrosisfactorα TNFα59 suggesting that selenium has antiammatory effects on the immune system The CTDdatabase httpctdbase reports the effect of environmental chemicals including selenium on gene expression proï¬les in various human tissuesThe role of selenium and selenoproteins incartilage development and KBDSelenium levels and its role in joint tissuesJoints are composed of various types of connective tissues including cartilage bone synovium meniscus andligament Among these tissues cartilage is the maincomponent that absorbs mechanical stress cushioningbones from impacting each other during various weightbearing activities In the human knee joint the seleniumconcentration in cartilage is approximately μgkg dryweight whereas the selenium concentrations in ligamentand meniscus are and μgkg dry weight respectively6061 The requirement of adequate physiologicalselenium levels for maintaining cartilage homeostasis hasbeen recognized Selenium deï¬ciency retards the growthand development of cartilage and bone62 Growthretardation was observed in rats after two generations ofselenium deï¬ciency62 Mice fed a diet deï¬cient in selenium resulted in ï¬brocartilage formation at the articularsurface ultimately showing degeneration of articularcartilage63 Selenium deï¬ciency induced the expression ofthe chondrocyte hypertrophy marker gene type X collagenCOLX in articular cartilage64 The expression of parathyroid hormonerelated protein PTHrP which controlschondrocyte maturation during endochondral ossiï¬cation was enhanced in both articular cartilage andhypertrophic growth plate following selenium deï¬ciencyThese changes were in line with the phenotypic changesobserved in the cartilage of KBD patients64 However itshould be noted that growth retardation caused by selenium deï¬ciency may also be associated with the deregulation of bone metabolism65 In a study by Cao et alselenium deï¬ciency severely compromised bone microarchitecture as a result of increased bone resorption66Abnormalities in selenium metabolism and skeletaldevelopment diseasesSelenium deï¬ciency is regarded as one of the initiatingfactors of KBD which is an endemic osteoarthropathycaused by the premature closure of epiphyseal plate andthe impaired skeletal development Skeletal deformities inhands ï¬ngers knees and elbows and in severe casesdwarï¬sm and movement disorders are the symptoms ofKBD22 The KBD area roughly coincides with lowselenium areas including a geological belt extendingfrom northeast to southwest China North Korea andeastern Siberia22 A metaanalysis showed that seleniumOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologylevels in the water soil cereal and corn in KBD endemicregions were lower than they were in nonendemicregions supporting the fact that the level of selenium intissue is predominantly affected by dietary intake23 In linewith this ï¬nding selenium levels in the whole bloodserum hair and urine of KBD patients were markedlylower than those of healthy controls24Selenoprotein gene polymorphisms are associated withincreased susceptibility to KBD There were significantdifferences in the allelic frequency of GPX1 Pro198Leurs1050450 between the KBD and control group67 Inaddition the mRNA level of GPX1 and enzyme activity oftotal GPX in blood were lower in the KBD group thanthey were in the control group67 Haplotypes of TCCTTC and TTT of rs1050450 rs3811699 and rs1800668in GPX1 gene also had a significant link to KBD68 Asinglenucleotide polymorphism SNP in the promoterregion of SELENOS rs28665122 105G A was relatedto the increased risk of KBD and upregulation of PI3KAktsignaling in patients with KBD69 In this study tertbutylhydroperoxide tBHPtreatmentinduced chondrocyteapoptosis was mitigated by selenium supplementation viasodium selenitetreatment which suppressed thePI3KAkt pathway The minor Aallele of SELENOFrs5859 was associated with a significantly higher incidenceof KBD70The animals fed a seleniumdeï¬cient diet recapitulatedsome of the pathological manifestations of KBD stronglysupporting the notion that selenium deï¬ciency is criticallyassociated with the development of this endemic arthropathy Selenium deï¬ciency impaired bone and cartilagegrowth with the exhibition of premature chondrocytehypertrophy as evidenced by an increased expression ofCOLX compatible with the phenotypes in KBD cartilage64The lowselenium condition in combination with threemycotoxins deoxynivalenol DON nivalenol NIV and T yielded procatabolic changes and hypertrophic phenotype of chondrocytes as evidenced by the loss of aggrecanand type II collagen COLII and the increase in COLX andmatrix metalloproteinases MMPs expressionrespectively71 In contrast selenium supplementation partiallyalleviated these mycotoxininduced damages in chondrocytes71 In rats dietary selenium deï¬ciency over twogenerations caused the onset of physiological seleniuminsufï¬ciency72 In this condition pathological changes inthe epiphyseal plate were observed with the decreasedexpression of COLII and GPX1 in the chondrocytes suggesting a possible association of reduced chondrocyte anabolism and antioxidant capacity with the epiphyseal platelesions observed in KBD72 The relevance ofimpairedselenium metabolism to the onset of KBD was furthervalidated using a mouse genetic deletion model Targeteddeletion of SectRNA[Ser]Sec Trsp gene in osteochondroprogenitor cells from embryonic stage caused the 0cKang Experimental Molecular MedicinePage of depletion of selenoproteins in skeletal systems causinggrowth retardation abnormalities in the epiphyseal growthplate delayed endochondral ossiï¬cationand chondronecrosis which recapitulated the major pathologicalfeatures of KBD73As a prophylactic treatment selenium supplementationswere given to children living in a KBD area The supplemented group showed elevated physiological seleniumlevels in their hair samples and exhibited a substantiallylower prevalence of KBD74 A metaanalysis including ï¬verandomized controlled trials RCTs and ten prospectivenonRCTs statistically demonstrated the beneï¬ts of selenium supplementation in preventing KBD in children75Selenium metabolism and OAPhysiological signiï¬cance of oxidative stress inchondrocytesOA is the most common form of arthritis and is primarilycharacterized by the loss of cartilagespeciï¬c ECM and otherpathological changes in joints including subchondral bonesclerosis osteophyte formation and synovial ammation31Articular cartilage is composed of abundant proteoglycans inwhich sulfated glycosaminoglycan chains such as chondroitinsulfates are bound to a core protein such as aggrecan Loss ofcartilage matrix during OA progression is a combined resultof increased catabolic process in cartilage and reduced anabolic activity of chondrocytes The molecularlevel understanding of OA pathogenesis has led to the identiï¬cation ofmajor catabolic enzymes ADAMTS576 MMP377 andMMP1378 which mediate the degradation of cartilagematrix Proammatory cytokines drive the expression ofthese catabolic factors in chondrocytes through the activationof transcription factors such as HIF2α32 and NFκB79Abnormalities in various metabolic pathways such as glucose80 or amino acid metabolic system81 in chondrocyteshave been implicated in activating catabolic cascades inosteoarthritic cartilage82 Moreover increased cellular uptakeof Zn2 through the upregulation of zinc transporter ZIP8activates metalregulatory transcription factor1 MTF1which in turn induces the expression of matrixdegradingenzymes in chondrocytes3383 Regulation of catabolism bythefurthershowed the association of metabolic abnormalities with thecatabolic process of OA34cholesterolCH25HCYP7B1RORαaxisMeanwhile the upstream regulatory mechanism eliciting an imbalance in OA matrix homeostasis needs furtherinvestigation OAcausing factorssuch as age andmechanical stress lead to excessive oxidative stress inchondrocytes3738 Consistently clinical and preclinicalOA studies indicated a cumulative oxidative burden inosteoarthritic chondrocytes3940 Emerging evidence suggests that oxidative stress plays a significant role in OAdevelopment and the disease progression can be mitigatedby counteracting oxidative stress3684In generalOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyoxidative stress results from the abnormal production ofROS and the loss of cellular antioxidant capacity Synovialï¬uid from patients with latestage OA who were undergoing knee joint replacement had a lower level of oxidoreductases than that from healthy controls87 In partthe increase in oxidative stress is attributable to mitochondrial dysfunction in OA chondrocytes8889 OAchondrocytes displayed reduced mitochondrial DNAcontent mitochondrial dysfunction and diminishedexpression of NRF2 which regulates the transcription ofoxidoreductase genes89 Similarly chondrocytes fromaged individuals exhibited increased ROS burden andmitochondrial and genomic DNA damage90 Therefore the proper maintenance of redox homeostasis canpotentially serve as a rational therapeutic strategy toprotect against OA progressionPotential roles of selenium metabolism in OAThe protective effect of selenium in OA has beenexplored in a large number of epidemiological and geneticstudies Table The concentration of selenium in serumwas significantly lower in OA patients than that of normalcontrols25 Similarly the results from a populationbasedcohort study demonstrated the linkage between lowselenium levels in toenails with OAassociated pain anddisease severity2627 Several studies have indicated thatcartilage matrix homeostasis is impaired in seleniumdeï¬ciency Lowselenium status diminished COLIIexpression level regulated by SOX9 which is known as amaster regulator required for maintaining cartilage matrixIn fact SOX9 was destabilized by thehomeostasisdownregulation ofseleniumresponsive PRMT5 thatsustains SOX9 stability via methylation93 In anotherstudy rats fed a seleniumdeï¬cient diet exhibited lowsulfotransferase activity which resulted in diminishedforcontents ofmechanicalcartilagematrix28 In contrast selenium supplementation ameliorated the spontaneous degeneration of articular cartilagein STR1 N mice by increasing the expression of GPXs94In cultured chondrocytes pretreatment with SeMetmarkedly inhibited nitric oxide NO and prostaglandinE2 PGE2 production in response to proammatorycytokine IL1β95 Expression of SBP2 a factor recognizingSECIS element had a positive correlation with GPX1 andGPX4 expression and antioxidant capacity in chondrocytes96 Oxidation resistance mediated by SBP2 wasdiminished in response to IL1β treatment in vitro and indamaged regions of cartilage in OA patients96 Downregulation of selenoprotein mRNAs including GPX397GPX1 and GPX49698 and Selenop99 was observed inhuman and mouse OA chondrocytessulfated glycosaminoglycan essentialstressabsorbingpropertyofGenetic factors such as SNPs in selenoproteins wereidentiï¬ed to be risk factors for OA development A GAG 0cKang Experimental Molecular MedicinePage of Table List of selenoproteins associated with the pathogenesis of arthropathies KBD and OAGeneGPX1GPX3GPX4DIO2DIO3SELENOFSELENOPSELENOSFunctionExpression in OASNPAntioxidantReduction of hydrogen peroxide and anic peroxidesDownregulatedPlasma antioxidantDetoxiï¬cation of lipid hydroperoxidesMetabolism of lipidsActivation of hormonesDeiodination of T4 to T3Inactivation of hormonesConversion of T4 to rT3Protein foldingStorage and transport of SeAntioxidant propertiesProtein foldingERassociated protein degradationDownregulatedDownregulatedUpregulatedDownregulatedrs1050450 KBDrs3811699 KBDrs1800668 KBDrs225014 OArs12885300 OArs945006 OArs5859 KBDrs28665122 KBDRefhaplotype in SELENOS gene was significantly associatedwith increased levels ofammatory factors in OApatient plasma100 SNPs in DIO2 which converts precursor thyroid hormone T4 to its active form T3 were alsorelated to genetic susceptibility to OA developmentLevels of DIO2 mRNA and protein were markedly upregulated in OA cartilage101 A common DIO2 haplotypecomposed of the minor Callele of SNP rs225014 and thecommon Callele of SNP rs12885300 was significantlyassociated with advanced hip OA as indicated by a higherodds ratio101 Locus rs225014 which confers risk toOA was associated with the differential methylation ofCpG located in the upstream region of DIO2 gene andwas correlated with upregulated DIO2 expression inOA104 Meanwhile DIO3 depletes the resources that canbe utilized for the production of active thyroid moleculesby catalyzing the conversion of T4 and T3 into inactivemetabolites The minor Gallele of the DIO3 variantrs945006 was associated with a protective effect againstOA development105However a few aspects regarding the relationshipbetween selenium and OA remain controversial Firstseveral studies indicate that there are no differences inselenium levels between OA and normal tissues Theselenium concentrations in synovial ï¬uid and plasma of OA patients were not significantly different from thoseof healthy controls106 Similarly no significant difference in selenium concentration was noted between sixdogs with posttraumatic OA and six control dogs107Second the beneï¬cial effect of selenium supplementationin alleviating OA symptoms has been debated The resultsfrom a controlled doubleblind trial of patientsOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologyrevealed that the supplementation of a formulation containing selenium with vitamins A C and E SeACE didnot have any remarkable curative effect compared to aplacebo108 In a study with an independent cohort theprevalence of radiographic knee OA was not significantlyassociated with dietary selenium intake109Nonethelessit is apparent that selenium deï¬ciencydysregulation of selenoproteins and genetic variations inselenoprotein genes serve as potential risk factors for OAThe vital role of selenium metabolism in maintainingcartilage homeostasis is expected considering its criticalinvolvementin regulating cellular processes such aschondrogenic differentiation of progenitor cells maintenance of redox homeostasis and DNA damage repair inchondrocytes which are covered in the next sectionIntracellular roles of selenium metabolism andselenoproteins in cartilageChondrogenic differentiation programs of progenitor cellsSelenium exerts various beneï¬cial effects to promoteproliferation and differentiation of chondrogenic progenitorcells110111 Selenium supplementation stimulated the proliferation of ATDC5 chondrogenic cells even under serumdeprivation by inducing cyclin D1 expression110 Deï¬ciencyof SELENOO interfered with the chondrogenic differentiation of ATDC5 cells by suppressing the expression ofchondrogenic genes SOX9 COLII and aggrecan anddecreasing the activity of alkaline phosphatase112 Knockdown of Gpx1 reduced the chondrogenic differentiation ofATDC5 cells by modulating intracellular GSHoxidizedGSH GSSG ratio113 Selenop was differentially upregulatedduring the chondrogenic differentiation of micromass 0cKang Experimental Molecular MedicinePage of culture of mesenchymal cells isolated from mouse limbbuds114 In line with the effects of selenium metabolism andselenoproteins in chondrogenic progenitor cells observedin vitro deï¬cient uptake of selenium severely affectedchondrogenic differentiation of mesenchymal lineage cellsin mice64andOsteochondroprogenitorspeciï¬c deletion of Trsp genesignificantly impaired chondrogenic programs causingabnormalities in bone and cartilage development in mice73endochondralossiï¬cationthusAntioxidant defense and redox homeostasisfunction ofattributed to theThe protective effects of selenium on cartilage are primarilyantioxidantdefense115 The metabolism and survival of chondrogenic progenitors and chondrocytes are greatly compromised by ROS including free radicals peroxides andsuperoxide anions118 Recent studies strongly supportthe notion that mitochondrial dysfunction and oxidativestress are the main drivers of OA pathogenesis37Although ROS play essential roles in the maintenance ofbasal cellular activities such as chondrocyte proliferationand matrix remodeling in cartilage excessive oxidativestress causes detrimental events such as cellular senescence36121 dedifferentiation122 and apoptosis123 ROScause oxidative damage to various cellular componentsand disrupt the balance between ECM catabolism andanabolism119 ROS suppress mitochondrial oxidativephosphorylation and ATP production which are essentially required to sustain cartilage matrix synthesis124 Inaddition ROS induce matrix degeneration through theupregulation of matrixdegrading enzyme expressionwhile this effecttreatment123125 The detrimental effects of ROS on cartilagehomeostasis can be effectively alleviated by augmentingcellular antioxidant activity under stress conditions andseveral attempts have been made to treat OA by targetingthe regulators involved in oxidative stress production incartilage84is abolished by antioxidantThe protective role of selenium metabolism is thoughtto be exerted through the neutralization of ROS viaantioxidant activities of selenoproteins including GPXsand TXNRDs Bone marrow stromal cells cultured inmedium supplemented with low selenite concentrationexhibited ROS accumulation along with the reducedexpression of GPXs TXNRDs and other seleniumindependentinmicronuclei generation which is an indication of chromosome damage126 Both GPX1 expression and activitywere substantially lower in mice fed a seleniumdeï¬cientdiet than those in mice fed a normal dietleading todecreased trabecular number reduced femoral trabecularvolumetotal bone volume ratio and trabecular separation66 The rats exposed to a seleniumdeï¬cient diet withT2 toxin showed increased lipid peroxidation level andoxidoreductaseenzymesresultingOfï¬cial journal of the Korean Society for Biochemistry and Molecular Biologydecreased antioxidant GPX activity in their serum andcartilage127 A seleniumdeï¬cient dietinduced theexpression of miR1385p which in turn suppressed theexpression of SELENOM that has antioxidant functionand caused mitochondrial dysfunction and apoptosis ofchondrocytes128 Lead Pbinduced oxidative stress andtoxicity reduced the expression of selenoprotein mRNAsand the effect was mitigated by selenium supplementation129 In summary the antioxidant properties of selenoproteins showed therapeutic potential by counteractingthe accumulation of damage induced by oxidative stress incartilageDNA damage repairIt is well known that DNA damage pathways play substantial roles in the progression of arthropathies119 Theexpression of genes related to DNA damage was changedin the cartilage of KBD patients130131 Chronic DNAdamage induces the initiation of apoptosis or cellularsenescence in chondrocytes36132133 Selenium has apotential to reduce DNA damage and increase DNArepair capacity134 In part the beneï¬cial effect of seleniumon genomic stability is associated with the antioxidationeffect of selenoproteins such as GPXs and TXNRDswhich remove ROS before they cause DNA damage134Cancer cells supplemented with selenium nM sodiumselenite or μM SeMet showed elevated levels of GPX1and TXNRD1 enzyme activity effectively protectingagainst DNA strand breaks induced by ultraviolet A orH2O2induced oxidative stress135 SeMet reduced theextent of DNA damage and enhanced DNA repair capacity by inducing repair complex formation in DNAdamaged cells through U | Thyroid_Cancer |
"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagans nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming Peoples Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expb·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expbD f · expbD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Beggs test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deeks plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Beggs testor Deeks test indicated the potential of publication bias[] Inconsistency index I2 and Cochrans Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagans nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The Ï2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ± NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA ± NA ± Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Beggs Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of BeggsTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Beggs Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Beggs Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deeks funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagans nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagans nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming PeoplesHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L | Thyroid_Cancer |
Lung cancer has high mortality often accompanied with systemic metabolicdisorders The present study aimed at defining values of transnodules crossclinical phenomic and lipidomic network layers in patients with adenocarcinoma ADC squamous cell carcinomas or small cell lung cancer SCLCWe measured plasma lipidomic profiles of lung cancer patients and found thataltered lipid panels and concentrations varied among lung cancer subtypes genders ages stages metastatic status nutritional status and clinical phenomeseverity It was shown that phosphatidylethanolamine elements and were SCLC specific whereas lysophosphatidylcholine and snposition1 and phosphatidylcholine and were ADCspecific There were statistically more lipids declined in male ages latestage metastasis or body mass index Clinical transomics analyses demonstrated that one phenome in lung cancer subtypes might be generated from multiple metabolic pathways and metabolites whereas a metabolic pathway andmetabolite could contribute to different phenomes among subtypes althoughthose needed to be furthermore confirmed by bigger studies including larger population of patients in multicenters Thus our data suggested that transomic profiles between clinical phenomes and lipidomes might have the value to uncoverthe heterogeneity of lipid metabolism among lung cancer subtypes and to screenout phenomebased lipid panels as subtypespecific biomarkersK E Y WO R D Slipidomics lung cancer phenomes subtypes transomicsINTRODUCTIONLung cancer is a systemic and aggressive disease withhigh morbidity and mortality and it is often accompanied with systemic metabolic disorders for exampleup or downregulated expression of mechanismassociatedgenes or activation of metabolismdependent enzymes Forexample metabolismassociated genes of small cell lungThis is an access under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided theoriginal work is properly cited The Authors Clinical and Translational Medicine published by John Wiley Sons Australia Ltd on behalf of Shanghai Institute of Clinical BioinformaticsClin Transl Med 202010e151101002ctm2151wileyonlinelibrarycomjournalctm2 of 0c of ZHU et alcancer SCLC cells altered after mitogenactivated proteinkinase MAPK kinase module MEK5ERK5 was blockedaccompanied by dysfunctions of several lipid metabolismpathways like the mevalonate pathway for cholesterolsynthesis1 Lipids mainly including subclasses of phosphatidic acid PA phosphatidylcholines PC phosphatidylethanolamine PE phosphatidylglycerol PGphosphatidylinositol PI and phosphatidylserine PShave multiple important biological functions such asbiomembrane composition vesicular trafficking adhesion migration apoptosis energy storage neurotransmission signal transduction and posttranslational modification They have alterations under circumstance of lungcancer Circulating levels of PCs and PEs in patients withnonsmall cell lung cancer NSCLC differed from thosewith noncancer lung diseases or health and were suggested as diagnostic biomarkers of early NSCLC2 Theheterogeneity of circulating lipidomic profiles was foundto exist among patients with squamous cell carcinomasSCC adenocarcinoma ADC or SCLC and there was aclear correlation between genomic and lipidomic profilesof lipidassociated proteins and enzymes3 As the part ofclinical transomics the lung cancerspecific and subtypespecific lipidomics in the circulation were defined and evidenced by integrating lipidomic data with genomic expression of lipid proteins among lung cancer subtypesClinical transomics is defined as a new subject tointegrate clinical phenomes with molecular multiomicsfor understanding molecular mechanisms of diseases inmultiple dimensions4 Clinical transomics becomes moreimportant as a new and novel approach for the discovery of diseasespecific biomarkers and therapeutic targetsalthough there are still many obstacles to be overcomefor example specificity and decisive role of transnodulesamong multiomic networks for intra and intercellular communication56 Recent studies applied the transomics among phosphorproteomics transcriptomics genesequencing and genomics for new molecular category ofliver cancer to provide a new therapeutic strategy7 As thepart of clinical transomics clinical lipidomics was considered as one of major metabolic profiles for identificationand validation of lung cancerspecific biomarkers by integrating clinical phenomes with lipidomic profiles89 Clinical lipidomics could demonstrate the complexity of thelipidome in metabolic diseases and lung cancer and presented the variation among diseases and subtypes of lungcancer1012Our previous study demonstrated the difference oflipidomic profiles among patients with different lungcancer subtypes and the potential association betweenlipidomic phenotypes and gene expression oflipidmetabolismassociated proteins and enzymes as a conceptevaluation3 The present study furthermore investigatedthe values of transnodules crossclinical phenomic andlipidomic network layers in the recognition of lung cancersubtypes ADC SCC and SCLC in order to understandclinical phenomeassociated lipid changes or lipidomicphenotypeassociated clinical phenomes We also evaluated the differences of lipidomic profiles between maleand female various ages early and late stages with orwithout metastasis body mass index BMI or and digital evaluation scores less or more than METHODS AND MATERIALSChemical agentsThe internal standard cocktails were subscribed fromAvanti Lipids Polar Alabaster AL USA the acetone acetonitrile ammonium bicarbonate dithiothreitol formicacid iodoacetamide and Tris base Analytical Gradefrom SigmaAldrich St Louis MO USA and ammonium acetate NH4OAc hexane isopropyl alcohol IPAmethanol and highperformance liquid chromatographygrade chloroform CHCl3 from Merck Millipore Billerica MA USAPatient populationThe study designed as a casecontrol approach wasapproved by the Ethical Evaluation Committee of Zhongshan Hospital ethical code B2018187 The subjectsgave informed consent for clinical data collection andlipids analysis before all the other procedures The studyincluded lung cancer patients diagnosed according topathology of whom were ADC SCC SCLC and other healthy people The stage and severity of lung cancerwere defined according to the Eighth Edition of TNMClassification for Lung Cancer13 Patients were recruitedduring October to March Healthy controlsparticipated were blood donors in Zhongshan HospitalSubjects with other respiratory diseases or family historyof lung cancer were excluded Fasting blood was drawnfrom healthy controls and lung cancer patients on theday of entering hospital to harvest plasma All the clinicaldata including symptoms signs laboratory tests imagespathologic information and survival status years laterwere collected and followed upDigital evaluation score systemThe Digital Evaluation Score System DESS is a scoreindex system by which clinical descriptive information of 0cZHU of each phenome can be translated into clinical informatics14When the severity of each component was scored as or of which represented the most severe condition whereas indicated normal physiological range Thegross DESS scores ranged from to points the higherthe score the severer the condition A total of clinical phenomes were collected and scored in each of threelung cancer group including histories symptoms signs laboratory measurements image features and pathologic indexes as listed in Table S1spectrometry analysisLipid extraction for massAbout µL plasma was collected into a glass tubeinto which µL internal standard was added and then mL of methanolchloroformformic acid asreported previously315 This mixture was incubated at ¦C overnight after vigorous shaking Two milliliters ofHajras reagent M H3PO4 M KCl were droppedblended and centrifuged at rpm for min Afterstratification chloroform in the lower layer was pipettedto another glass tube and concentrated to µL withthe nitrogen flow where the liquid with isopropyl alcoholhexane100 mM ammonium acetate at the ratio of was added till mL The sample was then centrifugated at rpm at ¦C for min The normalphaseliquid chromatography coupled TripleQuadrupole massspectrometer QTRAP SCIEX Framingham MAUSA was used for lipid extraction by the positive and negative electrospray ionization mode In the multiple reactionQTrap was utilized to scan the precursorproduct ion andexamine the mode operation Each test was repeated threetimes The peak area of each pair was quantified with multiple reaction monitoring data by the software MultiQuantAB SCIEXPurification of plasma lipidsLipid samples were derived through Ultimate SiO2 mm à mm µm Agilent Technologies Santa ClaraCA USA with mLmin flow rate highpurity heliumIn the meanwhile µL was added with the split ratio of at the ignition chamber temperature of ¦C and theinjection port temperature of ¦C It was started at temperature ¦C which gradually increased ¦Cmin to¦C and kept for min The mass spectrometry was subjected to liquid chromatographymass spectrometer analysis FOCUS DSQTM II Thermo Fisher Scientific mainlyunder the following conditions Electron Ionization EI asionization source ion source temperature at ¦C ionization voltage at eV multiplier voltage at kV minsolvent delaying and amu of scan rangeIdentification of lipidomic profilesLipid extracts were loaded onto an Ultremex silica column mm à mm µm which was fitted with a mmà mm silica guard cartridge Phenomenex TorranceCA USA and then eluted The sample was enriched ata gradient of nLmin In the mins run B phasewas from to min then rose to from to min linearly ramped for min as to return from to min until the end The QTrap was conductedin the multiplereaction monitoring mode and the different precursorproduct ion pairs were scanned in thepositivenegative electrospray ionization mode Up to lipids of plasma samples were carried out to get possiblelipids chemical structureslipidomic profilesComprehensive analyses ofMultiQuant software AB SCIEX was used to process dataafter lipids were identified by mass spectrometry Further MetaboAnalyst software wwwmetaboanalystcawas utilized for conducting multivariate statistical analysis cluster analysis dimensionality reduction and makingheat mapphenome and lipidome network layersTransnodule analyses crossThe typespecific lipids were identified as more than twotimes elevated or declined significantly compared withother lung cancer subtypes fold change and Pvalue whereas the coexpression lipids were identified as those similarly changed in all lung cancer subtypesas compared with healthy controls The expression quantitative trait locus eQTL model was utilized to evaluatetransnodules between lipidomic profiles and clinical phenomesStatistical analysisData were presented as mean ± SE The means of eachgroup were used for calculation and comparison Statistical significance of differences between two groupsor among multiple groups was determined by Studentsttest or oneway ANOVA test respectively Statistical 0c of ZHU et alsignificance was affirmed when Pvalue We alsoseparately calculated mean values of each phenomesDESS score in different lung cancer subtypes which wereranked to obtain top clinical phenomes of those threegroups of patients Volcano maps showed the significantlyelevated or declined lipids in ADC SCC or SCLC patientsA VIP plot was further exploited to sort the lipids according to their importance to differentiate the four groups Toexplore the correlation between lipid elements and clinical phenomes we applied the lipidquantitative trait locimodel modified from eQTL model Besides MatrixlQTL Rpackage was used to acquire the significant phenomelipidpairs and corresponding Pvalues Moreover GraphPadPrism was utilized to make the receiver operating characteristic curve to evaluate the earlydiagnostic value andaccuracy of clinical phenomespecific lipid elements inADC SCC or SCLC The present study furthermore analyzed the significant differences of lipids among differentages eg and between female and maleearly and late stage metastasis and nonmetastasis highand low DESS scores ¤ and and high and lowBMI ¤ and RESULTSwith lung cancer subtypesClinical phenomes of patientsEighteen female and male lung cancer patients wereenrolled in the present study aged from to ± years old including ADC SCC and SCLC The totalscores of DESS were ± ± and ± in patientswith ADC SCC and SCLC respectively The DESS values of SCLC group were significantly higher than those ofhealthy control group P Top clinical phenomesof ADC SCC or SCLC patients as well as patients survivedor nonsurvived during study period were listed in Table Stages at primary diagnosis and recruitment period for thestudy lymphatic metastasis N12 in ipsilateral paratracheal hilum or mediastinum and enhanced images egfocus enhanced in CT or hypermetabolism in PETCTwere shown in all three subtypes of lung cancers In addition thyroid transcription factor1 TTF1 Napsin A keratin and location of tumor were noticed in ADC obscureboundary emphysema tumor size the cycle number offirst line chemotherapy obstructive pneumonia atelectasis and pulmonary nodule in SCC as well as number ofmetastatic lymph nodes in SCLC separately Top clinicalphenomes were similar between survived and nonsurvivedpatients but the total amounts of DESS of nonsurvivedpatients were significantly higher than those of survivedpatients Table Of total clinical phenomes hadthe statistical significance of each two groups inbetweenTable P or lesswith lung cancer subtypesLipidomic profiles of patientsTotal lipid elements of plasma were identified qualitatively and quantitatively mainly including PAs PCs PEs PGs PIs PSs lysophosphatidylcholineslysoPC lysophosphatidylethanolamines lysoPE lysophosphatidylglycerols lysoPG lysophosphatidylinositols lysoPI lysophosphatidylserines lysoPS ninediacylglycerides and triacylglycerols TAG Levels ofsome lipid elements in ADC SCC or SCLC patients weresignificantly higher Table S2 or lower Table S3 as compared with healthy control twofold P The majority of those elevated lipid elements were PC PA and lysoPC in ADC PE PC PS and PG in SCC or PS PE PG lysoPS and lysoPI in SCLC Of those declinedlipid elements were PS in ADC whereas and were PA in SCC and SCLC respectively Table demonstrates lung cancer subtypespecific lipid elements identified by those lipid elements elevated or declined exclusively in each lung cancer subtype for example some oflysoPC and PC in ADC whereas lysoPI lysoPS PE andPA in SCLC By partial least squares discrimination analysis PLSDA analysis top lipid elements were definedon the basis of variable import in project VIP score of eachgroup TAG565 lysoPG182 and PG and increased in ADC lysoPG181 PA140245 PI384180PA and and PE385PE180 increased inSCLC and PI362PI180 and lysoPG182 decreased in SCCdetail in Figure 1A There was a clear distribution oftop lipid elements among lung cancer groups as compared with the healthy control Figure 1B Of those significantly increased lipid elements in patients with lungcancers top six lipids of each group were identified Figure levels of LysoPC sn2 sn1 and sn1 in ADC Figure 2A PS363 in SCC Figure 2B andPA and and PI and inSCLC Figure 2C were significantly higher than in otherthree groups PLSDA component analysis demonstratedthat five principal components selected were and Figure 3A In the atom map whichwas based on the expression of major C atom numbersin various lipid types levels of lipids with carbons and increased whereas those with carbons decreased as compared with healthy control Figure 3BAs compared with the healthy control Figure 4A wenoticed that PI mainly declined in ADC Figure 4B PA 0cZHU of TA B L E carcinoma SCLC as well as lung cancer patients survived or nonsurvivedTop clinical phenomes of patients with adenocarcinoma ADC squamous cell carcinomas SCC or small cell lungPatients with ADCStage at primarydiagnosis ± Stage at recruitmenttime ± TTF1 ± N2 ipsilateralmediastinum ± Napsin A ± Enhanced image ± Location ± N1 ipsilateralparatracheal ± N1 ipsilateral hilum ± CK7Patients with SCCN1 ipsilateral hilum ± Enhanced image ± Stage at recruitmenttime ± Stage at primarydiagnosis ± obscure boundary ± Emphysema ± T tumor ± L1 cycle ± Obstructivepneumoniaatelectasis ± pulmonary nodulePatients with SCLCStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralParatracheal ± T tumor ± N1 ipsilateral hilum ± N2 ipsilateralmediastinum ± Enhanced image ± pulmonary nodule ± N LN ± MaintenancetreatmentPatients survivedStage at primarydiagnosis ± N2 ipsilateralmediastinum ± Enhanced image ± Stage at recruitmenttime ± N1 ipsilateral hilum ± TTF1 ± Napsin A ± Location ± N1 ipsilateralparatracheal ± LobularPatientsnonsurvivedStage at recruitmenttime ± Stage at primarydiagnosis ± N1 ipsilateralparatracheal ± N2 ipsilateralmediastinum ± N1 ipsilateral hilum ± Enhanced image ± N2 below carina ± TTF1 ± N LN ± T tumor ± ± Abbreviations N degrees of lung cancer metastasis to lymph nodes of TNM category N1 degree that has metastatic lymph nodes near pulmonary center andside of main bronchia N2 degree that has metastatic lymph nodes in the same side of the mediastinum as lung cancer TTF1 thyroid transcription factor1 asan immunohistochemical biomarker for adenocarcinoma CK7 keratin as an immunohistochemical biomarker for epithelial cells L1 cycle number of the firstline chemotherapy cycles ± ± ± in ADC and SCC Figure 4C and lysoPG in SCLC Figure 4D whereas PG and TAG increased in ADC and SCCPE in SCLC and PC in SCC The volcanic map demonstrated the clear patterns of lipid elements significantlyincreased or declined between heathy controls with ADCFigure 4E SCC Figure 4F or SCLC Figure 4G and varied among different subtypes of lung cancerspatient gendersDifferent lipidomics betweenAbout or lipid elements significantly increased and or declined more than twofold in male or femalelung cancer patients as compared with male or femalehealthy controls respectively Tables S4 and S5 Of thosePC and PE mainly elevated in male and female patientswhereas PA declined in both although the number ofPA in male patients was more than in female patientsTable demonstrates genderspecific lipid elements identified by those lipid elements elevated or declined exclusively in either male or female lung cancer patients forexample some of lysoPS PC and PS elevated in malepatients whereas lysoPI and PE in female patients Therewere about or increased or declined lipid elementsdiffered between male and female lung cancer patientsTable 0c of ZHU et alTA B L E adenocarcinoma ADC squamous cell carcinoma SCC or small cell lung cancer SCLC patientsComparisons of clinical phenomes in increased folds and statistical significance Pvalues between each two groups ofTTF1Napsin ABullaeP40HemoptysisEmphysemaSputumCK7HbCoughEGFRVacuole cavityCEAN2 ipsilateral mediastinumNew metastasisP63Cyfra211Obstructive pneumonia atelectasisSmokingPleural pullThirdlineWBCL1 cyclePDL1 tumorPTBronchiectasisPD1 tumorL2 chemo regimenSynMaintenance treatmentNSEN1 ipsilateral ParatrachealCD56CHGT tumorPD1 interstitialSum of all tumors mmN LNKi67Bronchial stenosisN3 opposite sideBurrNeuL2 cyclePulmonary noduleCK56ADC vs SCCFoldsNANANANANANANANAPvaluesNANASCC vs SCLCFoldsNANANANANANANANANAPvaluesNANANAADC vs SCLCFoldsNANANANANANANANAPvaluesNANANANA 0cZHU of Lung cancer subtypeassociated lipid elements significantly elevated or declined alone in patients with adenocarcinomaFoldsPvalues LipidsFolds PvaluesSquamous cell carcinomad181SoC1P240d181S1PPS363TA B L E squamous cell carcinoma or small cell lung cancer more than twofold as compared with healthy control PvaluesSmall cell lung cancerAdenocarcinomaLipidsLipidsElevated twofoldlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPE lysoPG lysoPG lysoPS lysoPS lysoPS PA PA PA PC PC PC 332e PC 161e181PC 352e PC 160e202PC PC lysoPG lysoPI sn1lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PA PA PE PE orFoldsPvaluesPC PC or PC PC PC PC orPE PE Declined twofoldPG PS PS SM240PG PS401PE PE PI PI PI PI PI PI PIP PS PS TAG PA PA PA PA PA PA PA PA PA 0c of ZHU et alF I G U R E Scores of altered lipid elements in variable import in project VIP chart A where top lipid elements were defined amongpatients with adenocarcinoma ADC squamous cell carcinomas SCC small cell lung cancer SCL and healthy controls CON The xaxisrepresents the VIP score and the yaxis represents the lipid elements corresponding to the VIP score The right color grid stands for the relativeconcentration of lipid elements in four groups The degree of altered concentrations increased from green to red The heatmap B describesthe top lipid elements at the high concentration and the degree of lipid elements increased from blue low to brown highF I G U R E less than and respectively as compared with the healthy controlTop six significantly increased lipid elements in patients with ADC A SCC B and SCLC C and stand for the Pvalue 0cZHU of F I G U R E Histography of five component distributions and percentages A measured by partial least squares discrimination analysisPLSDA and the carbon atom map B in healthy controls red and patients with ADC green SCLC orange and SCC blue Each ofselected five principal components represents as the model to interpret that values of abscissa and ordinate represents the distance from thesample nodule to the origin of the center after projecting to a plane in multidimensional space A The atom map describes the expression ofmajor carbon atom number between and in various lipid types BF I G U R E The proportion of main lipid elements of healthy controls A ADC B SCC C and SCLC D and volcanic mapbetween heathy controls with ADC E SCC F or SCLC G respectively The lipid elements were identified on the basis of statistical significance The abscissa represents log values of fold changes where the left side of the first dotted line perpendicular to the abscissa represents fold changes and the right side of the second dotted line represents 2fold changes The vertical coordinate represents log10 Pvalue Theupper side of the dotted line perpendicular to the ordinate stands for Pvalue less than as compared with healthy controls 0c of ZHU et alFoldsPvaluesPvaluesFemale patients with lung cancerLipidsFoldsTA B L E Genderassociated lipid elements significantly elevated or declined alone in male or female patients with lung cancer morethan twofold as compared with healthy control PvaluesMale patients with lung cancerLipidsElevated twofoldlysoPI sn1lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS lysoPS PC PC or PC PC PC 375ePC 160e225180e205PC PC PC PC PC PC PC PC PC PC PC PC PC C1P120 MeanC1P160 MeanC1P240 MeanCer120d171Sod180Sa1Pd181S1Pd181SolysoPC sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS PC PC PE 355p PE 160p204PE 356p PE 160p205PE PE PE 376p PE 180p205 orPE PE PG PG PI 311p PI 160p160PS PS PS PS PS PS Declined twofoldlysoPS PA PA PA PA PA PA PA PA PA 181p204 160e226PE 377p PE 160p226PE PE PE PE orPI PI or or PS PS PA PA Continues 0cZHU et alContinuedTA B L E Male patients with lung cancerLipidsPG PG PS PS PS FoldsPvalues of Female patients with lung cancerLipidsFoldsPvaluesDifferent lipidomics among patientages stags metastases and survival statusAbout and lipid elements significantly elevatedor and declined in lung cancer patients at years old respectively as compared with healthycontrols P We noticed that elements of PG andPS mainly increased in lung cancer patients at all agegroups for example and at 60year group and at to 70year group and and at year group lysoPC and PC increased at 60year group and and at 70year group and PEincreased at to 70year group and at 70yeargroup as detailed in Table S6 Elements of PA mainlydeclined in lung cancer patients at all ages Table S7 Ofthose significantly altered lipid elements and appeared only at 60year to 70year and 70yeargroups respectively and considered as agespecific lipidelements Table LysoPC and lysoPI mainly increasedin 60year and to 70year old patients whereas lysoPEdeclined in 60year group We also compared lipidomicprofiles between patients at early and late stages of lungcancer and found and lipid elements significantlyincreased at early and late stages respectively of whichPE PG ad PS increased in both stages lysoPI in earlystage and PC in late stage Table S8 About and elements declined at early and late stages where the majority was PA Table S9 Table demonstrates stagespecificlipid elements identified by those lipid elements elevatedor declined exclusively at early and late stages of lung cancer for example some of lysoPI and PE elevated at earlystage and lysoPC and PE at late stageWe noticed about or lipid elements significantlyincreased in patients without or with metastasis of whichlysoPI mainly elevated in patients without metastasiswhereas PC and PE in patients with metastasis Table S10The declined number of lipid elements especially PA inpatients with metastasis was significantly higher than inpatients without metastasis Table S11 There were about or elevated or declined lipid elements in patients withmetastasis of which PA was majority of declined elementsin patients with metastasis Table Lipidomic panel also differed between survived andnonsurvived patients There were only eight lipids exclusively elevated in nonsurvived patients that is lysoPS140PC PC PE PE or PE PE PS330 PS372 andPS387 However far more lipids31 elevated alone in survived patients mainly elements of lysoPC lysoPG lysoPIlysoPS and PS On the contrary there were no lipidsdeclined alone in survived patients while lipids in nonsurvived patients of which were PA elements Table clinical phenomes and lipidomesTransomic profiles betweenWe also compared the difference of lipidomic profilesbetween general metabolism statuses of patients indicatedby BMI and between degrees of clinical phenomes measured by DESS scores Levels of lysoPC or lysoPI mainlyelevated in patients with BMI ¤ or respectivelyTable S12 whereas the number of declined PA in patientswith BMI ¤ was higher than that in patients withBMI Table S13 About BMIassociated lipid elements significantly elevated or declined exclusively inpatients with BMI ¤ and about in patients withBMI Table Levels of lysoPC and PE or PG and PSmainly increased in patients with DESS ¤ or TableS14 The number of declined PA n in patients withDESS ¤ was lower than that in patients with DESS n Figure demonstrates the variation of transomicprofiles among lung cancer subtypes indicated by transomic nodules cross significant networks of clinical phenome and lipidome layersDISCUSSIONThe present study preliminarily found the differencesof lipidomic profiles among patients of different lungcancer subtypes genders ages stages metastatic statusbody qualities and clinical phenome severities Besides itinitially demonstrated clinical phenomeassociated lipid 0c of ZHU et alFolds Pvalues LipidsPatient age Folds Pvalues LipidsPatient age TA B L E Ageassociated lipid elements significantly elevated or declined alone in each age group of patients with lung cancer morethan twofold as compared with healthy control PvaluesPatient age LipidsElevated twofoldC1P120 MeanlysoPC sn2lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPG d181S1PlysoPC sn1lysoPE190lysoPE191PC PC PC PE 356p PE160p205PE PE PE PE orlysoPG140lysoPI sn2lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1Folds PvalueslysoPS lysoPS150lysoPS161lysoPS170lysoPS201lysoPS202lysoPS220PA PE PE orPE PE PE PE PE PE orPI 311p PI160p160PI PI PI PI PI361TAG PA PA PG393lysoPS lysoPS lysoPS PC PE PE PG PG PG PS Declined twofoldlysoPE sn1lysoPE sn1lysoPE sn1lysoPE sn2PA PA PS PS PE PE orPE PE PG351PS354PS372PA elements and lipid elementassociated clinical phenomesusing clinical transomics Studies on lipidomic profilesof lung cancer patients have experienced three phasesto detect the difference of lipidomic profiles betweenhealthy and lung cancer patients16 the association ofmultiomics among lung cancer subtypes3 and themolecular mechanism of clinical lipidomicsbased targetlipid elements17 Of those lipidomicsbased data limitedinformation could be adopted to understand the diseaseoccurrence and development phone progression and 0cZHU of Stageassociated lipid elements significantly elevated or declined alone in patients with lung cancer at the early stage or lateFolds Pvalues LipidsPatients at late stageFolds Pvalues LipidsTA B L E stage more than two folds respectively as compared with healthy control PvaluesPatient at early stageLipidsElevated twofoldlysoPE lysoPG lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPC sn1lysoPG lysoPS lysoPS lysoPS lysoPS PC PC orlysoPI sn1lysoPI sn1lysoPI sn1lysoPI sn1lysoPS lysoPS lysoPS lysoPS PC PC PE PE PE PE PE 356p PE 160p205PE PE PE PE PE PE PI PI 311p PI 160p160PI PI or or PS PS PC PC PC 375e PC 160e225 or180e205PC PC PC PC PC PC PC PC PC PC PC PC PC PC orPC PC PC PE 355p PE 160p204PE 376p PE 180p205 or181p204 or 160e226PE 377p PE 160p226PE PE PE PE orPE PE PE PE PG PG PG PG PG PG PG PG Patients at late stageFoldsDeclined twofoldlysoPS PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PA PG PG PS PS PvaluesContinues 0c of ContinuedTA B L E Patient at early stageLipidsPatients at late stageFolds Pvalues LipidsPG PS PS PS ZHU et alPvaluesPatients at late stageFoldsFolds Pvalues Lipidsresponse to therapy due to the lack of link between omicsdata and clinical phenomes Like other omics investigations most genomic data were not tied with clinicalinformation so that with little values to be understoodand applied for clinical precision medicine18 In orderto face the major challenge that most clinical information was descriptive and unmatched with the digitalquantity of omics data clinical phenomes were scoredby DESS and integrated with genomic and proteomicdata of patients with acute respiratory distress syndromeand chronic obstructive pulmonary disease19 Clinicalphenomes were furthermore integrated with lipidomicprofiles in patients with pulmonary embolism acutepneumonia and acute exacerbation of chronic obstructive pulmonary diseases based on clinical transomicsprinciple15Lipidomic profiles difference between health and lungcancer has been defined and it depends upon methodologies of measurement and analysis sample preparationsand sources and patient populations and status8 Forexample serum levels of lysoPC C260 and C261 and PCC424 and C344 were different between stage I NSCLCand healthy patients22 Some elements and pathwaysof serum PC and PE profiles increased in patients withlung benign disease and earlystage NSCLC as comparedwith healthy whereas few eg PC significantlyelevated in earlystage NSCLC patients alone2 It seemsthat patterns of lipid elements may be associated with thespecificity of lung cancer and stage rather than the intactlipid pathways We performed | Thyroid_Cancer |
"Follicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that mostly occurs in systemiclymph nodes FDCS in the uterine cervix has not yet been reportedCase presentation A 49yearold woman was referred to our department with a cervical tumor which washistologically suspected to be undifferentiated carcinoma She underwent hysterectomy salpingooophorectomyand pelvic lymphadenectomy after neoadjuvant chemotherapy with paclitaxel and carboplatin The resectedspecimen contained high numbers of spindle cells and was immunohistochemically confirmed to be FDCS Thetumor was completely resected and recurrence was not detected at a 16month followupConclusion FDCS is an extremely rare malignant tumor in the uterine cervix and an accurate diagnosis andcomplete resection are essential for a good prognosisKeywords Follicular dendritic cell sarcoma Cervical cancerBackgroundFollicular dendritic cell sarcoma FDCS is a rare mesenchymal tumor that was initially reported in []Three types of tumors are derived from dendritic cellsFDCS derived from follicular dendritic cells that presentantigens to B lymphocytes in lymph follicles [] interdigitating dendritic cell sarcoma derived from the Tcellzones of lymphoid ans such as the paracortex anddeep cortex of the lymph nodes and fibroblastic reticular cell sarcoma derived from a reticular network oflymphoid ans [] An accurate diagnosis is challenging without an appropriate series ofimmunohistochemistry therefore some tumors may be diagnosed asundifferentiated carcinoma The accuracy of a diagnosisinfluences the prognosis of patients because of the lowresponse rate to established chemotherapy and radiotherapy [] Correspondence myoshihara1209mednagoyauacjp2Department of Obstetrics and Gynecology Nagoya University GraduateSchool of Medicine Tsurumacho Showaku Nagoya Aichi JapanFull list of author information is available at the end of the Although previous studies described FDCS in the cervical lymph nodes liver stomach and tonsils FDCS inthe uterine cervix has not yet been reported We hereinpresent a 49yearold woman diagnosed with FDCS inthe uterine cervix which was successfully treated bycomplete surgery without postoperative adjuvant therapybased on a precise pathological diagnosis This is thefirst case of FDCS arising from the uterine cervix andwe described the time course of this patient from herinitial admission to diagnosis and treatment We alsoemphasized the importance of considering FDCS as adifferential diagnosis for cervical tumors because an accurate diagnosis and complete resection are essential fora good prognosisCase presentationA 49yearold woman gravida para with abnormalgenital bleeding was referred to our department with acervical mass She had a previous history of uterine myomectomy and cervical polypectomy which were both benign diseases Pelvic magneticimagingrevealed a mm solid tumor developing from theresonance The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cNakamura BMC Women's Health Page of findingsrevealed aIntraoperativeand carboplatin AUC5 mgbody Massive genitalbleeding occurred months after the admission therefore the patient underwent extended total hysterectomybilateral salpingooophorectomy and pelvic lymphadenectomysmallamount of bloody ascites and the resected specimen hada necrotic mass at the posterior side of the cervix Fig Tumor cells exhibited the same histopathological characteristics as those in the previous biopsy Fig 3a Additionalimmunohistochemistry revealed cells that werepositive for both CD68 and FDC Fig 3b which wasconsistent with the diagnosis of FDCS The tumor wascompletely resected and lymphovascular invasion wasnot detected There was also no evidence of lymph nodemetastasis She did not receive adjuvant therapy and herCA19 level decreased to within almost normal limitsTable Tumor recurrence was not detected at the month followup after admission The consent of the patient for publication was recorded according to the Ethics Committee of Nagoya University and the principlesof the Declaration of HelsinkiDiscussionThis is the first case of FDCS in the uterine cervix Afterreported surgery and chemotherapy we reached a finaldiagnosis Our experience demonstrates the difficultiesassociated with accurately diagnosing FDCS due to alack of familiarity with the pathologies of rare tumorsincluding sarcoma Although FDCS in the gynecologicalsystem is markedly rarer than other carcinomas it needsto be considered as a differential diagnosis because of itspotentiallyappropriatetreatmentprognosiswithoutfatalFDCS has been reported in approximately casesworldwide with an age range of to years and amalefemale ratio of The primary lesion of FDCS isFig Surgical specimen of the tumor The tumor with a necroticlesion arose from the posterior side of the cervix arrowheadsFig Magnetic resonance imaging of the patient at the initialpresentation A solid tumor developing from the posterior of theuterine cervix was confirmed arrowheadsposterior of the uterine cervix Fig and and mm spaceoccupying lesions were also detectedaround the rectus which were suspected to be lymphnode metastases Chestabdominal computed tomography CT showed no enlarged lymph nodes or distantmetastasis except for the tumors already detected Positron emission tomographyCT revealed the accumulation offluorodeoxyglucose maximum standardizeduptake value at the cervical tumor only Serumtumor marker levels were as follows carcinoembryonicantigen ngml cancer antigen125 UmLcancer antigen199 Uml squamous cell carcinomaantigen ngml soluble interleukin2 receptor Uml The other results of the blood examination including renal function liver enzymes and electrolyteswere within normal limitsBiopsy of the cervical tumor was performed and ahistopathological examination indicated that the tumorhad atypically spindle ovoid and polygonal cells with aneosinophilic hyalinerich cytoplasm with whorled andcordlike forms Nuclei were oval to round and had mildatypia Inflammatory cells were scattered to various degrees around tumor cells The results of immunohistochemical staining were as follows cytokeratin AE1AE3positive epithelial membrane antigen EMA negativeCAM52 negative cytokeratinMNF116 positive S100negative vimentin positive actin negative desminnegative cluster of differentiation CD negative CD8negative CD10 negative CD5 negative CD20 negativeCD79a negative PgR PgR inhibin negative thyroid transcription factor1 negative EpsteinBarr virusencoded RNA in situ hybridization negativeSince the tumor was suspected to be undifferentiatedcarcinoma of the uterine cervix the patient receivedchemotherapy with four cycles of paclitaxel mgm2 0cNakamura BMC Women's Health Page of Fig The histopathology of the cervical tumor The tumor had atypically spindle ovoid and polygonal cells with an eosinophilic hyalinerichcytoplasm with whorled and cordlike forms Nuclei were oval to round and had mild atypia a Tumor cells were positive for FDC bmostly in the lymph nodes in the neck axilla and mediastinum The remainder of FDCS originate from extralymphatic lesions such as the liver mesenterium stomach smallintestine pharynx tonsils retroperitoneumand ovary [] however FDCS in the uterine cervixhas not yet been reported in the English literatureDue to the wide range of primary lesions there is noknown specific initial symptom of FDCS some patientsmay exhibit symptoms associated with an increase intumor volume such as lymph node swelling [] Sincethere are no specific findings in blood examinations orimaging studies difficulties are associated with makingan accurate diagnosis prior to the initiation of treatmentThe diagnosis of FDCS is pathologically confirmed usingbiopsy or resected specimens however due to the rarityof this tumor it is important to conduct appropriate immunohistochemistry in consideration of FDCS from thefindings of hematoxylin and eosin staining The tumorcells of FDCS are spindle to epithelioid in shape with thecoexistence of multinucleated cells Single nuclear inflammatory cells infiltrate and form bundles flowers andswirls structures Some giant cells and ReedSternbergcells are also detected in these tumors To reach a definitive diagnosis of FDCS FDC markers such as CD21CD35 KiM4p and CNA42 are used to distinguish itfrom other tumors that may have a mesenchymal structure such as undifferentiated carcinoma meningiomaand paraganglioma [ ] Alternatively FDCS may befound within lesions of Castlemans disease particularlyits hyalinevascular type [] It has been reported thatFDCS occurred after the excision of a lesion of hyalinevascular type Castlemans disease and occupied most ofthe lesions It is also important to distinguish FDCSfrom inflammatory pseudotumorlike FDCS associatedwith EpsteinBarr virus This tumor is often found in theabdominal ans particularly in the liver and spleenand has the characteristics of positive FDCS markersand the detection of EBV by in situ hybridization It progresses more slowly than FDCS and longterm survivalhas been reported even after recurrence []The treatment for cervical cancer principally involvessurgeryradiation and chemotherapy New surgicaltechniques such as laparoscopic radical hysterectomyand sentinel lymph node biopsy have also become available [ ] On the other hand a basic therapeuticstrategy for FDCS is prioritized to guarantee completesurgical tumor resection because of the low responserate to established chemotherapy and radiotherapy [] Saygin examined patients with FDCS andreported 2year survival rates of and forearlylocally advanced and distant metastasis diseaserespectively Patients who underwent complete tumorresection had a better prognosis than those with unresectable localized tumors Furthermore no prognosticdifference was observed between the surgery group andpostoperative radiotherapy groups A large tumor sizelarger than cm and lymphoplasmacytic cell invasionhave been identified as poor prognostic factors [] InTable Level of tumor markers in each followup periodTumor marker Normal rangeCA125 UmL AdmissionCA199 UmL CEA UmL SCC ngmL At surgery m after admissionAt final followup m after admissionsIL2R UmL Abbreviation CA cancer antigen CEA carcinoembryonic antigen SCC squamous cell carcinoma antigen sIL2R soluble interleukin2 receptor 0cNakamura BMC Women's Health Page of the present case as the tumor remained localized andwas completely resected the patient could follow favorable clinical course without any tumor recurrenceConclusionFDC sarcoma is a rare tumor that may develop in theuterine cervix FDCS needs to be considered when confirming a mesenchymal cervical tumor and appropriateimmunohistochemistry needs to be performed for bothan accurate diagnosis and selection of a therapeuticstrategyAbbreviationsFDC Follicular dendritic cell sarcoma FDCS Follicular dendritic cellCT Computed tomography CD Cluster of differentiationAcknowledgmentsThe authors thank Dr Sakata and Dr Hirata for supporting the managementof the case with patience and knowledgeAuthors contributionsTN and MY developed the study concept interpreted the data and wrotethe manuscript ST and HK participated in designing the study and plannedthe investigations FK drafted the study design and supervised all of thework All authors have approved the final manuscriptShia J Chen W Tang LH Carlson DL Qin J Guillem JG Extranodalfollicular dendritic cell sarcoma clinical pathologic and histogeneticcharacteristics of an underrecognized disease entity Virchows Arch Ruco LP Gearing AJ Pigott R Pomponi D Burgio VL Cafolla A et alExpression of ICAM1 VCAM1 and ELAM1 in angiofollicular lymph nodehyperplasia Castleman's disease evidence for dysplasia of folliculardendritic reticulum cells Histopathology Maeda K Matsuda M Suzuki H Saitoh HA Immunohistochemicalrecognition of human follicular dendritic cells FDCs in routinely processedparaffin sections J Histochem Cytochem Arber D Kamel O van de Rijn M Davis RE Medeiros LJ Jaffe ES et alFrequent presence of the EpsteinBarr virus in inflammatory PseudotumorHum Pathol Casarin J Bogani G Papadia A Ditto A Pinelli C Garzon S et alPreoperative Conization and risk of recurrence in patients undergoinglaparoscopic radical hysterectomy for early stage cervical cancer amulticenter study J Minim Invasive Gynecol Rossetti D Vitale SG Tropea A Biondi A Lagan AS New procedures for theidentification of sentinel lymph node shaping the horizon of futureManagement in Early Stage Uterine Cervical Cancer Updat Surg Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNo funding was obtained for this studyAvailability of data and materialsNot applicableEthics approval and consent to participateNot requiredConsent for publicationWritten consent to publish this information was obtained from studyparticipantsCompeting interestsAll authors declare that there are no competing interestsAuthor details1Department of Obstetrics and Gynecology Anjo Kosei Hospital Anjo AichiJapan 2Department of Obstetrics and Gynecology Nagoya UniversityGraduate School of Medicine Tsurumacho Showaku Nagoya AichiJapanReceived April Accepted August References Monda L Warnke R Rosai J A primary lymph node malignancy withfeatures suggestive of dendritic reticulum cell differentiation A report of cases Am J Pathol Tew JG Kosco MH Burton GF Szakal AK Follicular dendritic cells asaccessory cells Immunol Rev Saygin C Uzunaslan D Ozguroglu M Senocak M Tuzuner N Dendritic cellsarcoma a pooled analysis including cases with presentation of ourcase series Crit Rev Oncol Hematol Pileri SA Grogan TM Harris NL Banks P Campo E Chan JK Tumoursof histiocytes and accessory dendritic cells an immunohistochemicalapproach to classification from the international lymphoma study groupbased on cases Histopathology Chan JK Fletcher CD Nayler SJ Cooper K Follicular dendritic cell sarcomaClinicopathologic analysis of cases suggesting a malignant potentialhigher than currently recognized Cancer 0c" | Thyroid_Cancer |
breast cancer patients especially those with triplenegative breast cancer is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancer Although collagen type VIII alpha chain COL8A1 has been shown to be downregulated in BRIP1knockdown breast cancer cells its clinical role in breast cancer remains unknownMethods Gene microarrays and mRNA sequencing data were downloaded and integrated into larger matrices based on various platforms Therefore this is a multicentered study which contains breast cancer patients and controls COL8A1 mRNA expression in breast cancer was compared between molecular subtypes Inhouse immunohistochemistry staining was used to evaluate the protein expression of COL8A1 in breast cancer A diagnostic test was performed to assess its clinical value Furthermore based on differentially expressed genes DEGs and coexpressed genes CEGs positively related to COL8A1 functional enrichment analyses were performed to explore the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerResults COL8A1 expression was higher in breast cancer patients than in control samples standardized mean difference confidence interval [CI] Elevated expression was detected in various molecular subtypes of breast cancer An area under a summary receiver operating characteristic curve of CI with sensitivity of CI and specificity of CI showed moderate capacity of COL8A1 in distinguishing breast cancer patients from control samples Worse overall survival was found in the higher than in the lower COL8A1 expression groups Intersected DEGs and CEGs positively related to COL8A1 were significantly clustered in the proteoglycans in cancer and ECMreceptor interaction pathwaysConclusions Elevated COL8A1 may promote the migration of breast cancer by mediating the ECMreceptor interaction and synergistically interplaying with DEGs and its positively related CEGs independently of molecular subtypes Several genes clustered in the proteoglycans in cancer pathway are potential targets for developing effective agents for triplenegative breast cancerKeywords COL8A1 Breast cancer Immunohistochemistry staining MechanismCorrespondence fengzhenbo_gxmu163com chenganggxmueducn Wei Peng and JianDi Li contributed equally as first authors Department of Pathology The First Affiliated Hospital of Guangxi Medical University NO6 Shuangyong Road Nanning Guangxi Peoples Republic of ChinaFull list of author information is available at the end of the BackgroundBreast cancer poses a grave threat to female health According to the latest American cancer statistics breast cancer is estimated to be the most common cancer and the second most common cause of cancerassociated The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cPeng a0et a0al Cancer Cell Int Page of deaths in women [] Owing to higher distal metastatic and recurrent rates triplenegative breast cancer TNBC patients exhibit worse overall and diseasefree survival than any other type of breast cancer [] Etiology investigations suggest that hereditary factors account for nearly onetenth of breast cancer cases Other risk factors such as early or delayed menstruation nulliparity hormone replacement therapy and alcohol consumption also contribute to the prevalence of breast cancer [] Clinical practice guidelines recommend that females aged or who are at higher risk screen for breast cancer [] Imaging examinations such as bilateral breast Xray imaging positron emission tomographycomputed tomography and ultrasound histological findings and especially molecular pathology are the predominant methods of breast cancer diagnosis and assessment [ ] Based on the tumor burden optimal treatments namely breastconserving surgery radiotherapy chemotherapy and endocrine therapy are individually designed for breast cancer patients [] For TNBC patients anthracyclines and taxanes are preferred in the initial treatment and neoadjuvant therapy has been recognized as a standard strategy [] Unfortunately neither endocrine therapy nor trastuzumab treatment is effective for TNBC Targeted drugs for example vascular endothelial growth factor [VEGF] antibodies epidermal growth factor receptor [EGFR] inhibitors and mammalian target of rapamycin [mTOR] inhibitors are gradually being employed in TNBC treatment even though their therapeutic effects are unsatisfactory [] Therefore the situation faced by breast cancerespecially TNBCpatients is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancerMolecular events occurring in breast cancer help us better understand the onset and progression of breast cancer It is generally agreed that chromosome 1q amplification chromosome 16q deletion and PIK3CA mutations are the most common pathways leading to luminal breast cancer [ ] Moreover breast cancer gene BRCA1 and P53 mutations EGFR upregulation and cytokeratin downregulation have been associated with TNBC [ ] It has also been reported that circSEPT9 promotes tumor formation and TNBC progression [] Recently Np63 has been found to participate in breast cancer metastasis and dissemination [] On the other hand several genes protect patients from breast cancer progression For example ZNF750 miR5745p and circKDM4C can inhibit breast cancer progression by mediating the epigenetic regulation of prometastatic genes indirectly suppressing SKILTAZCTGF and miR548pPBLD axis regulation [] Based on these discovered molecular mechanisms some progress has been made in breast cancer treatment Delivering dual microRNA using CD44targeted mesoporous silica nanops proved to be effective in TNBC treatment [] Although many studies provided in a0vitro and in a0vivo a detailed molecular picture of TNBC cancers [] the underlying cause of breast cancer has not been fully understood Further research is required to elucidate the breast cancer mechanisms and discover effective therapeutic targets for TNBCCollagen type VIII alpha chain COL8A1 also named C3orf7 is located at chromosome and encodes alpha chain in collagen type VIII which is an essential component of extracellular matrix ECM [] Previous studies mainly addressed the relevance between COL8A1 and agerelated macular degeneration ADM as well as cell proliferation [] Recently limited studies demonstrate the deregulation of COL8A1 in various cancers Elevated COL8A1 expression was found in gastric cancer patients and higher COL8A1 correlated with advanced tumor stages and worse overall survival condition and COL8A1 was selected as a candidate diagnostic biomarker in gastric cancer [] Additionally upregulation of COL8A1 was also reported in adamantinomatous craniopharyngioma [] Furthermore COL8A1 proved to participate in the progression of colon adenocarcinoma possibly by mediating focal adhesionrelated pathways [] COL8A1 upregulation induced by TGFβ1 was found in renal cell carcinoma carcinogenesis and also correlated with poor prognosis [] Moreover elevated COL8A1 in hepatocellular carcinoma promoted tumor cells proliferation invasion and in a0vivo tumorigenicity [] Thus far only few studies mentioned COL8A1 in breast cancer COL8A1 was one of the key genes restored by epigallocatechin3gallate in a murine breast cancer model [] COL8A1 proved downregulated in both BRIP1knockdown breast cancer cells and MCF10A a0CDH1 noncancer breast cells [ ] However the role of COL8A1 in breast cancer remains unknownConsidering this knowledge gap our study aimed to explore the role of COL8A1 in breast cancer We were focused on investigating the expression of COL8A1 messenger RNA mRNA in various molecular subtypes of breast cancer by analyzing gene microarray and RNA sequencing data sets The COL8A1 protein expression level was validated by immunohistochemistry staining We also aimed to determine prognostic value of COL8A1 in breast cancer to pave the way for future clinical applications Moreover we explored the molecular mechanisms of COL8A1 underlying breast cancer to improve our knowledge of breast cancer carcinogenesis and progression 0cPeng a0et a0al Cancer Cell Int Page of MethodsExpression of a0COL8A1 mRNA in a0breast cancerWe integrated gene microarrays and mRNA sequencing data downloaded from Gene Expression Omnibus The Cancer Genome Atlas TCGA the GenotypeTissue Expression the Sequence Read Archive ArrayExpress and Oncomine The search formula based on MESH terms was as follows Breast OR mammary AND neoplasm OR cancer OR adenoma OR carcinoma OR tumor OR BRCA OR neoplasia OR malignant OR malignancy Studies were screened according to the following criteria i the studied species should be Homo sapiens ii the studied specimens should be tissue dissected from patients or healthy individuals rather than cell lines In the case of duplicated studies or samples the most recent version was retained The exclusion criteria were as follows i expression profiles not including COL8A1 ii breast cancer patients receiving hormone therapy or chemotherapy iii stromal rather than epithelial tumors and iv metastatic rather than primary tumors The included data sets were carefully checked and a log2 transformation was performed if any matrices had not been normalized Additionally the data sets were integrated into larger matrices according to various platforms and batch effects between studies were removed using the limmavoom package in R v361 Subsequently COL8A1 expression values were extracted and grouped according to specimen types Standardized mean difference SMD were calculated to compare the expression of COL8A1 mRNA between breast cancer patients and control samples using STATA v120 Heterogeneity between the included studies was assessed with the I2 statistic Statistical significance was set to an I2 value greater than with a Pvalue less than A random effects model was used in the case of significant heterogeneity Sensitivity analysis was performed to probe the potential source of heterogeneity and a publication bias test was used to evaluate the stability of the SMD results Subgroup analysis was performed to compare the COL8A1 expression levels between molecular subtypes luminal A luminal B human epidermal growth factor receptor 2positive [HER2 ] and TNBCDiagnostic value of a0COL8A1 in a0breast cancerA diagnostic test was performed to assess the clinical significance of COL8A1 in breast cancer Based on the expression value of COL8A1 a receiver operating characteristic ROC curve was plotted to compute the area under the curve AUC using IBM SPSS Statistics v190 AUC values of less than between and and greater than represented weak moderate and strong discriminatory capability of COL8A1 respectively between breast cancer patients and control samples The true positives false positives true negatives and false negatives rates were calculated and the cutoff values were identified A summary receiver operating characteristic sROC curve was drawn using STATA v120 to assess the general discriminatory capability of COL8A1 between breast cancer patients and control samples The significance of the area under the sROC curve was consistent with that of the ROC curve The diagnostic odds ratio DOR sensitivity specificity positive diagnostic likelihood ratio DLR P and negative diagnostic likelihood ratio DLR N were also calculated to precisely determine the accuracy and validity of COL8A1 in distinguishing breast cancer patients from control samplesPrognostic value of a0COL8A1 in a0breast cancerTo explore the relation between COL8A1 mRNA expression and prognosis of breast cancer patients information on clinicopathological parameters was collected The independent samples ttest or oneway analysis of variance was used to identify statistically significant differences in COL8A1 expression between two or more groups A Pvalue of was considered statistically significant KaplanMeier curves were used to compare high and low COL8A1 expression groups in terms of survival The logrank test was used to determine whether the prognostic difference was statistically significantInvestigation of a0COL8A1 protein expression in a0breast cancer by a0immunohistochemistry stainingA total of nonspecific invasive breast carcinoma and normal breast tissue specimens were obtained from the First Affiliated Hospital of Guangxi Medical University PRCHINA All patients had previously signed informed consent forms and our research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University The breast cancer and normal breast tissue specimens were fixed with formalin The two steps immunohistochemistry method was used to determine the protein expression of COL8A1 The primary antibody was polyclonal Antibody to COL8A1 concentrated dilution purchased from Wuhan Pujian COLTD Supervision TM MouseRabbitHRP Broad Spectrum Detection System Product No D300415 was purchased from Shanghai Long Island The experimental procedure conformed to the manufacturers instructions The patients clinicopathological information was analyzed to determine the relationship between COL8A1 protein expression and prognosisEvaluation of a0genetic alteration and a0mutation landscapesThe cBioPortal for Cancer Genomics httpcbiop ortal proved to be a powerful tool and facilitated our online search and cancer genomics data analysis Using 0cPeng a0et a0al Cancer Cell Int Page of cBioPortal we gained insight into the genetic alterations of COL8A1 in breast cancer patients and obtained information on the association between COL8A1 alterations and breast cancer patient survival We selected the Breast Invasive Carcinoma TCGA Firehose Legacy cohort which contains patients and used a method of mRNA expression zscores relative to diploid samples RNASeqV2 RSEM We also considered the mutation types of COL8A1 in breast cancer patients using the Catalogue of Somatic Mutations in Cancer COSMIC which has been recognized as the most detailed resource for somatic mutations in cancerIdentification of a0differentially expressed genes and a0COL8A1 coexpressed genes in a0breast cancerTo gain insight into the role of COL8A1 in breast cancer we identified DEGs and COL8A1 CEGs using the limmavoom package The DEG criteria were as follows ilog2FoldChange and ii adjusted Pvalue The CEG criteria were as follows irelation coefficient and ii Pvalue Upregulated DEGs and CEGs positively related to COL8A1 were intersected Similarly downregulated DEGs and CEGs negatively related to COL8A1 were intersectedMolecular mechanisms of a0COL8A1 underlying breast cancerOverlapping genes were used to perform function enrichment to shed light on the potential mechanisms of COL8A1 underlying breast cancer The R clusterProfiler package was used to conduct Gene Ontology GO Kyoto Encyclopedia of Genes and Genomes KEGG Disease Ontology DO and Reactome pathway analyses Proteintoprotein interaction PPI network was constructed using STRING https strin gdb to investigate protein interactions Hub genes and functional modules were identified using Cytoscape v361 Based on breast cancer patients the mutation landscapes of genes clustered in essential pathways were visualized using the TCGAmutations package in R v361ResultsUpregulation of a0COL8A1 mRNA in a0breast cancerAdditional file a0 Figure S1 shows the flow diagram of the study inclusion process A total of studies were included and integrated into larger platform matrices covering breast cancer patients and controls Table a0 COL8A1 was generally upregulated in breast cancer compared to normal breast tissue Thirteen of the twenty platforms showed much higher COL8A1 expression in breast cancer patients than in control samples Additional file a0 Figure S2 Because of significant heterogeneity I2 P a random effects model was used An SMD value of confidence interval [CI] showed that COL8A1 expression was significantly higher in breast cancer than in nonbreast cancer tissue Fig a0 Sensitivity analysis indicated that the included studies could not explain the source of heterogeneity Additional file a0 Figure S3a No publication bias existed Additional file a0 Figure S3b Subsequently we compared COL8A1 expression levels between different subtypes of breast cancer COL8A1 expression was universally higher in luminal A luminal B HER2 and TNBC patients than in control samples Additional file a0 Figure S4 Additional file a0 Figure S5 and Additional file a0 Figure S6a Furthermore three platforms showed significantly higher COL8A1 expression in nonTNBC than TNBC while only one showed lower expression in nonTNBC than TNBC Additional file a0 Figure S6b However an SMD of CI showed no difference in COL8A1 expression between TNBC and nonTNBC patients Additional file a0 Figure S7 Subgroup analysis of four molecular subtypes of breast cancer showed no significant differences in COL8A1 expression levels between them Fig a0Diagnostic and a0prognostic value of a0COL8A1 mRNA in a0breast cancerThe clinical value of COL8A1 in breast cancer was found to be promising Among the thirteen platforms showing high COL8A1 expression twelve platforms indicated the ability of COL8A1 in differentiating breast cancer patients and control samples where four platforms showed strong discriminatory capability of COL8A1 between breast cancer patients and control samples Additional file a0 Figure S8 An area under the sROC curve of CI with sensitivity of CI and specificity of CI displayed moderate capacity in distinguishing breast cancer patients from control samples Fig a03a A DOR of CI also highlighted the discriminatory ability of COL8A1 in breast cancer Fig a03b The DLR P and DLR N were CI and CI respectively Additional file a0 Figure S9 As shown in Additional file a0 Figure S10 and Additional file a0 Table a0S1 elevated COL8A1 expression correlated with race white black molecular subtypes of breast cancer luminal B luminal A TNBC ER PR and HER2 status Moreover KaplanMeier curves indicated worse overall survival in high compared to low COL8A1 expression groups Fig a0Expression levels and a0clinical significance of a0COL8A1 protein in a0breast cancerClinical data of breast cancer samples used to perform in immunohistochemistry was summarized 0cPeng a0et a0al Cancer Cell Int Page of ldnaoPESGi abarA iduaSESG ecnarFESG ASUESG inapSESG ecnarFESG ecnarFESG adanaCESGkramneDESGi eropagnSESGl yatIESG ASUESG ynamreGESG inapSESG ASUESGASUESG cil bupeRhcezCESG ldnaerIESGi eropagnSESGASUESG ailartsuAESG iocxeMESGi abarA iduaSESGASUESG ASULPGESGadanaCESG ASUESG adanaCESGASULPGESGASULPGESGadanaCESGianhCESG anhCi ESG ASUESG ASUESGianhCESGl aisyaaMESGi anhCESGadanaCESGi anhCESGASUESGi anhCESGASUESGi eropagnSESGl aisyaaMESGASUESG ESG ESG ESG ESG ESGASUESG ESGASU ESGASU ESGanhCi ESGynamreG ESGASU ESGLPGLPGLPGLPGLPGLPGLPGLPGLPGLPGLPGLPGLPGLPGxETGAGCTESGESGESGESGESGsrebmun latoTseirtnuoC seireSOEG NTNFPFPTleuavPfdtlortnoC ACRBnoisseccADSMNDSMN stesataddell a0 orneeht a0fonoitamrofni cisaB elbaT 0cPeng a0et a0al Cancer Cell Int Page of Fig General expression status of COL8A1 in breast cancer BRCA compared to nonBRCA tissues A standardized mean difference SMD value and 95CI with no overlap of zero indicated COL8A1 was significantly upregulated in BRCA compared to nonBRCA tissuesAdditional file a0 Table a0 S2 Protein expression confirmed the upregulation of COL8A1 in breast cancer The breast cancer patients whose specimens were analyzed in this study were aged between and mean and their followup durations ranged from to a0 days Immunohistochemistry staining showed varying coloration intensity of COL8A1 in breast cancer and normal breast tissue COL8A1 was negatively weakly moderately or strongly stained in normal breast epithelium Fig a05ad and breast cancer tissue Fig a0 5eh According to the staining intensity and color range percentages of breast cancer tissue specimens exhibited low and exhibited high COL8A1 expression whereas of normal breast tissue specimens exhibited low and exhibited high COL8A1 expression A Chi square test confirmed the significantly higher expression of COL8A1 in breast cancer than normal breast tissue Ï2 P Moreover elevated COL8A1 expression correlated with estrogennegative ER breast cancer P Genetic alterations and a0mutation kinds of a0COL8A1 in a0breast cancerAlterations and mutations of COL8A1 in breast cancer were relatively frequent Based on cBioPortal COL8A1 was altered in of breast cancer patients Additional file a0 Figure S11 Amplification and high and low mRNA were the main alterations No statistically significant difference in overall and diseasefree survival was found between high and low COL8A1 expression breast cancer groups P Furthermore according to COSMIC substitution missense mutations were the most frequent types Additional file a0 Table a0S3DEGs and a0COL8A1 CEGs in a0breast cancerA total of platform matrices were collected to determine the DEGs The approach has been aforementioned Initially upregulated DEGs downregulated DEGs CEGs positively related to COL8A1 and CEGs negatively related to COL8A1 were identified Additional file a0 Figure S12 shows partial DEGs and CEGs After being intersected 0cPeng a0et a0al Cancer Cell Int Page of Fig Subgroup analysis based on the subtypes of breast cancer The result indicated that the elevated COL8A1 expression shared no significant difference among Luminal A Luminal B HER and Three Negative Breast Cancer TNBC subgroupsthe DEGs and CEGs were divided into two gene sets overlapping upregulated DEGs and CEGs positively related to COL8A1 all genes appeared in no fewer than three data sets and overlapping downregulated DEGs and CEGs negatively related to COL8A1Potential mechanisms of a0COL8A1 underlying breast cancerThe GO KEGG DO and Reactome pathway analyses based on the intersected genes are shown in Additional file a0 Table a0S4 Regarding the overlapping upregulated DEGs and CEGs positively related to COL8A1 the following KEGG pathways were significantly aggregated 0cPeng a0et a0al Cancer Cell Int Page of Fig Diagnostic value of COL8A1 in breast cancer BRCA a Summary receiver operating characteristic sROC curve b Forest plot of diagnostic odd ratio DOR An AUC value and a DOR signified COL8A1 possessed moderate capability in distinguishing BRCA from nonBRCA patients AUC area under the curve 0cPeng a0et a0al Cancer Cell Int Page of Fig The prognostic value of COL8A1 mRNA in breast cancer tissues a GSE25307 b GSE35629GPL1390 c TCGA In the GSE25307 cohort high COL8A1 group possessed poor overall survival condition compared to low COL8A1 group in breast cancer patients 0cPeng a0et a0al Cancer Cell Int Page of See figure on next pageFig Protein expression levels of COL8A1 in breast cancer BRCA and normal breast tissues ad normal breast tissues eh BRCA tissues Magnification à COL8A1 was negatively stained in normal breast epithelium a and BRCA e COL8A1 was weakly stained in normal breast epithelium b and BRCA f COL8A1 was moderately stained in normal breast epithelium c and BRCA g COL8A1 was strongly stained in normal breast epithelium d and BRCA h According to staining intensity and percentage of color range BRCA tissues exhibited low COL8A1 expression and BRCA tissues exhibited high COL8A1 expression While normal breast tissues exhibited low COL8A1 expression and normal breast tissues exhibited high COL8A1 expressionFig a06a proteoglycans in cancer Additional file a0 Figure S13 ECMreceptor interaction Additional file a0 Figure S14 and several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly genes WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B BAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though is not in the top KEGG pathways Moreover DO analysis indicated that these genes are closely associated with myeloma and bone marrow cancer Fig a06b as well as renal cell carcinoma ovarian cancer renal carcinoma and other types of cancer Furthermore Reactome pathway analysis revealed extracellular matrix anization ECM proteoglycans integrin cell surface interactions and degradation of the extracellular matrix as the top four metabolic pathways Fig a0 6c Regarding GO enrichment extracellular matrix anization extracellular matrix and extracellular matrix structural constituent were the most clustered Biological Process BP Cellular Component CC and Molecular Function MF terms respectively Fig a07a The proteoglycans in cancer and ECMreceptor interaction pathways were selected to construct PPI networks Fig a07b c FN1 and ITGB1 were identified as the hub genes in the two networks respectively The mutation landscapes of the genes in these two important pathways are shown in Fig a0 In particular FN1 was altered in of breast cancer samples where missense mutations accounted for The regulatory networks of COL8A1 and enriched genes in the proteoglycans in cancer and ECMreceptor interaction pathways as well as the top two functional modules are displayed in Additional file a0 Figure S15 On the other hand the enrichment results regarding the overlapping CEGs negatively related to COL8A1 and downregulated DEGs showed no statistical significance these data are therefore not shown Additional file a0 Table a0S5DiscussionThe highlight of this study is that it comprehensively explored the upregulation of COL8A1 mRNA in breast cancer from multiple aspects based on breast cancer patients and controls Our study is multicentered because we collected breast cancer patients from Asia American Europe and Oceania covering different countries This is the first study to investigate the protein expression of COL8A1 in breast cancer using immunohistochemistry staining Moreover this study is the first to assess the clinical prognostic value of COL8A1 in breast cancer Furthermore our study sheds light on the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerThis study demonstrates the upregulation of COL8A1 in breast cancer We found higher COL8A1 expression in breast cancer than normal breast tissue based on large platform matrices integrated from data sets Subgroup analysis based on four molecular subtypes of breast cancer showed that elevated COL8A1 expression is independent of subtypes Further analysis also revealed universally higher COL8A1 expression levels in luminal A luminal B HER2 and TNBC patients than in control samples A comparison of COL8A1 expression between nonTNBC and TNBC patients showed no statistically significant difference Immunohistochemistry staining confirmed the upregulation of COL8A1 protein in breast cancer We thus concluded that COL8A1 expression is higher in breast cancer patients than in control samples and that upregulation is independent of molecular subtypes of breast cancer Though the expression of COL8A1 in tissue cannot reflect the early diagnostic value in breast cancer we assume that if COL8A1 is also differentially expressed in the bodily fluid of patients it will be possible to serve as a potential diagnostic marker for breast cancer Nevertheless no previous studies have demonstrated the expression level of COL8A1 in the bodily fluid of breast cancer patients until nowWe determined the clinical value of COL8A1 in breast cancer for the first time Our diagnostic test showed a moderate discriminatory capability of COL8A1 between breast cancer and normal breast tissue Higher COL8A1 expression levels in breast cancer patients correlated with worse survival outcomes Additionally COL8A1 expression was much higher in patients of the white than of the black race Our TCGA cohort analysis showed lower COL8A1 upregulation levels in TNBC than in the luminal A and B subtypes Furthermore high COL8A1 protein levels were related to ER breast cancer Thus COL8A1 might serve as a prognostic marker for breast cancer 0cPeng a0et a0al Cancer Cell Int Page of 0cPeng a0et a0al Cancer Cell Int Page of Fig Functional enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a Kyoto Encyclopedia of Genes and Genomes b Disease Ontology c Reactcome DEGs differentially expressed genes CEGs coexpressed genes 0cPeng a0et a0al Cancer Cell Int Page of Fig GO enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a GO analysis b Proteintoprotein internet based on KEGG pathway proteoglycans in cancer ID hsa05205 c Proteintoprotein internet based on KEGG pathway ECMreceptor interaction ID hsa04512 GO Gene Ontology DEGs differentially expressed genes CEGs coexpressed genes KEGG Kyoto Encyclopedia of Genes and GenomesMore importantly our study provides important clues about the role of COL8A1 in breast cancer for the first time The intersected CEGs positively related to COL8A1 and upregulated DEGs were significantly aggregated in several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly we noticed that genes related to COL8A1 WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B 0cPeng a0et a0al Cancer Cell Int Page of Fig Mutation landscapes of COL8A1 and coexpressed genes clustered in two Kyoto Encyclopedia of Genes and Genomes pathways proteoglycans in cancer and ECMreceptor interactionBAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though this pathway is not in | Thyroid_Cancer |
suffer from a high false positive rate because predicted expression of different genes may behighly correlated due to linkage disequilibrium between eQTL We propose a novel statistical method Gene Score Regression GSR to detect causal gene sets for complex traitswhile accounting for genetogene correlations We consider noncausal genes that arehighly correlated with the causal genes will also exhibit a high marginal association with thecomplex trait Consequently by regressing on the marginal associations of complex traitswith the sum of the genetogene correlations in each gene set we can assess the amountof variance of the complex traits explained by the predicted expression of the genes in eachgene set and identify plausible causal gene sets GSR can operate either on GWAS summary statistics or observed gene expression Therefore it may be widely applied to annotateGWAS results and identify the underlying biological pathways We demonstrate the highaccuracy and computational efficiency of GSR compared to stateoftheart methodsthrough simulations and real data applications GSR is ly available at githubcomlilabmcgillGSRIntroductionGenomewide association studies GWAS have been broadly successful in associating geneticvariants with complex traits and estimating trait heritabilities in large populations [] Overthe past decade GWAS have quantified the effects of individual genetic variants on hundredsof polygenic phenotypes [ ] GWAS summary statistics have enabled various downstreamanalyses including partitioning heritability [] inferring causal single nucleotide polymorphisms SNPs using epigenomic annotations [] and gene sets enrichment analysis fora1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Zhang W Li SY Liu T Li Y Partitioning genebased variance of complex traitsby gene score regression e0237657 101371journalpone0237657Editor F Alex Feltus Clemson University UNITEDSTATESReceived March Accepted July Published August Peer Review History PLOS recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s Theeditorial history of this is available here101371journalpone0237657Copyright Zhang This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All of the data used inthis paper are described under subsection Realdata application in the manuscript and pastedbelow as reference We applied our approach toinvestigate pathway enrichment for complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressiontraits Fig 2b using publicly available summarystatistics and genotypeexpression weights basedon GTEx whole blood samples wwwgtexportalhomedatasets2 The GWASsummary statistics were downloaded from publicdatabase databroadinstitutealkesgroupsumstats_formatted We downloadedexpression weights and reference LD structureestimated in Genomes using Europeanindividuals from the TWASFUSION websitehttpgusevlabprojectsfusion Franke labcelltypespecific gene expression dataset wereobtained from databroadinstitutempgdepictdepict_downloadtissue_expression Inaddition we applied GSR to test for gene setenrichment in three wellpowered types of cancerbreast invasive carcinoma BRCA cases and controls thyroid carcinoma THCA casesand controls and prostate adenocarcinomaPRAD cases and controls using geneexpression datasets from The Cancer GenomeAtlas TCGA Uniformly processed normalizedand batcheffect corrected gene expressiondatasets from TCGA and GTEx were obtained fromfigsharecomsData_record_3 Gene expression and phenotype werestandardized before supplying to the GSR softwareStandard GSEA was also performed forcomparison Gene sets were downloaded from theMSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combinedBIOCARTA KEGG and REACTOME to create a totalof gene sets We also downloaded the GO biological process terms as additional genesets as well as the gene sets pertaining tooncogenic signatures for the TCGA data analysisFunding The research is supported by CanadaFirst Research Excellence Fund CFREF HealthyBrains Healthy Life HBHL New Investigator fund at McGill University and Mon trealNeurologic Institute MNI and NSERC DiscoveryGrant RGPIN20190621 The funders had nocomplex traits [] However it remains challenging to link these genetic associations withknown biological mechanisms One main reason is that the majority of the GWAS loci are notlocated in known genic regions of the human genomeTranscriptomewide association studies TWAS [] offer a systematic way to integrateGWAS and the reference genotypegene expression datasets such as the GenotypeTissueExpression project GTEx [] via expression quantitative loci eQTL In TWAS we couldfirst quantify the impact of each genetic variant on expression variability in a population andobtain predicted gene expression levels based on new genotypes Then we could correlate thepredicted gene expression with the phenotype of interest in order to identify pivotal genes[] Moreover when individuallevel genotypes and gene expression levels are not availablewe could still quantify genetophenotype association ie TWAS statistics using only themarginal effect sizes of SNPs on the phenotype and on gene expression respectively []These concepts and implementations have largely facilitated explanation of genetic associationfindings at the gene or the pathway levelHowever as depicted in Fig TWAS are often confounded by the genetogene correlationof the genetically predicted gene expression due to the SNPtoSNP correlation ie linkage disequilibrium LD [] Consequently relying on the TWAS statistics may lead to false positivediscoveries of causal genes and pathways One approach to address this problem is to finemapcausal genes by inferring the posterior probabilities of configurations of each gene being causalin a defined GWAS loci and then test gene set enrichment using the credible gene sets of prioritized genes [] However this approach is computationally expensive restricted to GWASloci and sensitive to the arbitrary thresholds used for determining the credible gene set andthe maximum number of causal genes per locusAnother method called PASCAL [] projects SNP signals onto genes while correcting forLD and then performs pathway enrichments as the aggregated transformed gene scoreswhich asymptotically follows a chisquare distribution However PASCAL does not leverage the eQTL information for each SNP thereby assuming that a priori all SNPs have thesame effect on the gene Stratified LD score regression LDSC offers a principle way to partition the SNP heritability into functional categories defined based on tissue or celltypespecific epigenomic regions [] or eQTL regions of the genes exhibiting a strong tissue specificity [] Although LDSC is able to obtain biologically meaningful tissuespecific enrichments it operates at the SNP level rendering it difficult to assess enrichment of gene setsMoreover neither PASCAL nor LDSC is able to integrate the observed gene expression datameasured in a disease cohort rather than the reference cohort that are broadly availableacross diverse studies of diseases including cancers such as The Cancer Genome AtlasTCGA []role in study design data collection and analysisAlthough expressionbased methods such as gene set enrichment analysis GSEA aredecision to publish or preparation of themanuscript No author received a salary from anyof the fundersCompeting interests The authors have declaredthat no competing interests existoften adopted in combination with the observed gene expression and phenotypes [] theygenerally do not account for the genetogene correlation While this type of correlation is usually caused by shared transcriptional regulatory mechanisms across genes GSEA still likelyproduces false positives in identifying causal pathwaysIn this study we present a novel and powerful genebased heritability partitioning methodthat jointly accounts for genetogene correlation and integrates information captured at eitherthe SNPtophenotype or the SNPtogene level We utilize this method to identify plausiblecausal gene sets or pathways for complex traits We showcase its high accuracy and computational efficiency in various simulated and real scenariosPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Overview of confounding effects on pathway analysis a A hypothetical example illustrates the confounding issue when using the geneticallypredicted transcriptome to assess the pathway enrichments for a target phenotype The causal gene set includes a causal gene which is linked to noncausal gene via their respective causal SNPs and which are in strong linkage disequilibrium b A GWAS locus SNP associations with the targetphenotype are summarized The causal SNP for the causal gene is in red The SNPs that drive noncausal genes are in blue The rest of the SNPs are ingreen SNPs exhibit correlated signals due to the linkage disequilibrium LD as displayed by the upper triangle of the SNPSNP Pearson correlationmatrix c A TWAS locus The genetogene correlation is partly induced by the SNPtoSNP correlation and partly due to intrinsic coregulatoryexpression program d Pathway associations based on averaged gene associations101371journalpone0237657g001MethodsPartitioning genebased variance of complex traitsWe assume gene expression has linearly additive effects on a continuous polygenic trait yyi ¼XAijaj þ �ijð1Ãwhere Aij denotes the expression of the jth gene in the ith individual for i N individuals and j G genes αj denotes the true effect size of the jth gene on the trait andj à �i denotes the residual for the ith individual in this linear model andaj � Nð0 s2�i � Nð0 s2� ÃPiyi ¼ N yy ¼ Here we further assume that both y and A are standardized such that NPiAij ¼ and j Aj ¼ for j GN ANPLOS ONE 101371journalpone0237657 August PLOS ONE 0cWe define the estimated marginal effect size of the jth gene on the trait as ajGene score regressionaj ¼NAj y¼NAj ðXAkak þ �ÃkX¼kNAj Akak þNAj �X¼krjkak þ �ð2Ãð3Ãð4Ãð5Ãwhere � ¼ N Aj � withVarð�0à ¼N Aj Varð�ÃAj ¼N s2�and rjk ¼ and the kth geneN Aj Ak is the estimated Pearson correlation in gene expression between the jth geneWe define w2j ¼ Na2j Then if we further assume α r and � are independent we haveE½w2j � ¼ E½Na2j �X¼ NE½Ã°rjkak þ �0Ã2�k¼ NE½r jk�E½a2k� þ s2�Xkð6Ãð7Ãð8ÃNow consider C gene sets Cc where c C and denote the proportion of total trait Here Cc denotes the numVarðajÃvariance explained by the cth gene set as Ïc with tc ¼ber of genes in the cth gene setPjCcjj2CcConsequentlyE½a2k� ¼ Varðakà ¼Xck2CctcBy approximating E½r N we have thatXjk� with r E½w2jk þ j � ¼ NE½r jk�E½a2k� þ s2�k¼ NXtcXr jk þXtc þ s2�ck2Ccc¼ NXtclðj cà þ cwhere we define gene score as lðj cà ¼tinuous trait is normalizedPr jk and Varðyà ¼k2Ccð9Ãð10Ãð11ÃPctc þ s2� ¼ since the conPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionTherefore if we are able to obtain estimates for w2j and C gene score lj c for j Gand c C we will be able to perform linear regression of the estimated w2j on lj cand derive regression coefficient that is an estimate for each Ïc c C respectivelyThese are available from GWAS summary statistics of SNPtotrait effect sizes eQTL summary statistics of SNPtogene expression effect sizes and a reference LD panel Specifically Suppose we have estimated effect sizes βpà of p SNPs based on a GWAS including Ngwassamples ieb ¼NgwasXywhere XNgwasÃp is the standardized genotype Meanwhile we have the eQTL summary statistics W estimated usingAeQTL ¼ XeQTLWTherefore the predicted gene expression in GWAS is given bySinceA ¼ XWj ¼ Na2w2j¼ NðNAj yÃ2¼ NðNWj XyÃ2¼ NðW j bÃ2the required w2j can be estimated without accessing any individuallevel data Furthermore a reference LD panel SpÃp summarizing SNPtoSNP correlation in thematched population with the GWAS study can provide estimates for rjk asR ¼ ½rjk�¼NAA¼NW XXW¼NW SWPLOS ONE 101371journalpone0237657 August ð12Ãð13Ãð14Ãð15Ãð16Ãð17Ãð18Ãð19à PLOS ONE 0cGene score regressionIt is noteworthy that with individuallevel gene expression data we can also easily obtainthe required w2j and R [rjk] by definitionIn practice many gene sets are not disjoint and share common genes with each otherTherefore we regress one gene set at a time along with a dummy gene set that include theunion of all of the other genes The dummy gene set is used to account for unbalanced genesets and to stabilize estimates of Ïc We also include an intercept in the regression model toalleviate nongeneset biases for example positive correlation between gene scores and truegene effect sizes that could lead to intercept greater than and negative correlation betweengene scores and true gene effect sizes could lead to intercept smaller than Simulation designTo assess the accuracy of our GSR approach we simulated causal SNPs for gene expression aswell as causal gene sets for a continuous trait based on real genotypes and known gene setsfrom existing databases Our simulation included two stages At stage we first simulatedgene expression based on reference genotype panel We then estimated SNPgene effects W gfor each gene g based on the simulated gene expression and genotype which were then used topredict gene expression At stage separately we simulated the a continuous trait using simulated gene expression based on genotype and estimated the marginal SNPphenotype effectsSimulation step simulating gene expression To simulate individual genotype we first partitioned genotype data for individuals ofEuropean ancestry obtained from the Genomes Project [] into independent LD blocks as defined by LDetect [] We then randomly sampled LD blocks and used only those LD blocks for the subsequent simulation We used LD blocks as opposed to whole genome to reduce computational burden required for multiple simulation runs For LD block j j of an individual i i we randomly sampledfrom the available samples for block j and concatenated these sampled LD blocks for this individual We repeated this procedure to simulate genotype Xref forNref individuals as a reference population We standardized the simulated genotype Xref We randomly sampled k incis causal SNPs per gene within ± kb around the genewhere k default We also experimented different number of causal SNPs k allin cis SNPs We sampled SNPgene weights Wg � N ð0 h2g kà where gene expression heritability h2 default which is the variance of gene expression explained by genotype We alsoexperimented different gene heritability h2g ¼ f02 05gg ¼ We then simulated gene expression Agref Xref Wg � where � � N ð0 s2� à and� ¼ s2Nrefk XrefWgk2ð h2g 00 1ÃINrefto match the desired heritability 00 h2gh2g¼s2�kXref Wg k2Nref Finally we applied LASSO regression Agref � �XWg to get Wg for each geneSimulation step simulating phenotypePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regression We simulated another Ngwas GWAS individuals by the predefined LD blocksamong the Europeans in Genome data following the same procedures as decribedabove We then standardized the simulated genotype Xgwas We then sampled a causal pathway Cc from MSigDB such that all of the Gc � jCcj genes inCc were causal genes for the phenotype For each noncausal pathway we removed genes that were also present in the causal pathway We removed noncausal pathways containing fewer than five genes afterwardsdefault Alternatively in more realistic scenarios we allowed for sharing genes with causalpathways by noncausal pathways We sampled genephenotype effect a � N ð0 s2aGcIGcà where the phenotypic varianceexplained by gene expression s2s2a f01 05ga ¼ default We also experimented different We simulated gene expression Ac as in step for the Ngwas individuals and standardized itto obtain �Ac We simulated a continuous trait using causal gene expression y ¼ �Acα þ �y where�y � N ð0 s2à Here s2�yof variance explained 00 s2s2a�ya¼s2�yk �Acαk2Ngwas¼ Ngwask �Acαk2ð s2a 00 1ÃINgwasto match the predefined proportion Lastly we computed GWAS summary SNPtotrait effect size b ¼ N XgwasyWe repeated these simulation procedures times Unless otherwise stated while we wereexperimenting various settings we kept the other settings at their default values k causalSNP per gene gene expression variance explained per causal SNP h2ance explained per gene s2a ¼ one causal pathway Using these obtained summary statistics we were able to perform GSR PASCAL LDSC and FOCUS in each simulated scenarioApplying existing methods PASCAL PASCAL was downloaded from www2g ¼ 01k phenotypic variunilchcbgindexphptitlePascal [] We executed the software using default settings LDSCStratified LD score regression software was downloaded from githubcombulikldsc[] Because LDSC operates on SNP level we considered SNPs located within ± kbaround genes in each pathway to be involved in the corresponding pathway Then for eachpathway we computed the LD scores over all chromosomes We experimented the options ofrunning LDSC with and without the baseline annotations using our simulated data Wefound that LDSC running without the baseline worked better in our case One possible reason is that the baseline annotations cover genomewide SNPs whereas there are much fewerSNPs in the simulated pathways FOCUS We downloaded FOCUS [] from githubcombogdanlabfocus We used FOCUS to infer the posterior probability of each gene beingcausal for the phenotype across all of the LD blocks We then took the credible gene set asfollows We first summed all of the posteriors over all of the genes We then sorted the genesby the decreasing order of their FOCUSposteriors We kept adding the top ranked the geneinto the credible gene until the sum of their posteriors was greater than or equal to the of the total sum of posteriors We used the credible gene set for hypergeometric testfor each pathway to compute the pvalues We also tried other thresholds for credible setsranging from including the fewest genes to including the most genes GSEAGSEA software was obtained from oftwarebroadinstitutegsea [] We used thePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Gene scores correlated with marginal TWAS summary statistics a Gene scores were correlated with marginal TWAS chisquare statistics ofSchizophrenia We grouped genes into bins by their gene scores to reduce noise For each bin we calculated the average gene scores and chisquarestatistics Ï2 An unbinned version is supplied in S1 Fig in S1 File b Summary of Pearson correlation between marginal TWAS summary statisticsand gene scores for traits The negative correlation for T2D indicates that this trait possibly due to complicated genetic architecture andconfounding genetoenvironment interaction and drug effects is not suitable for using our approach101371journalpone0237657g002commandline version of GSEA to test for gene set enrichments using the observed geneexpression and phenotype dataReal data application We applied our approach to investigate pathway enrichment for complex traits Fig 2b using publicly available summary statistics and genotypeexpressionweights based on GTEx whole blood samples The GWAS summary statistics were downloaded from public database databroadinstitutealkesgroupsumstats_formatted[] We downloaded expression weights and reference LD structure estimated in Genomesusing European individuals from the TWASFUSION website httpgusevlabprojectsfusion [ ] Franke lab celltypespecific gene expression dataset were obtainedfrom databroadinstitutempgdepictdepict_downloadtissue_expressionIn addition we applied GSR to test for gene set enrichment in three wellpowered types ofcancer breast invasive carcinoma BRCA cases and controls thyroid carcinomaTHCA cases and controls and prostate adenocarcinoma PRAD cases and controls using gene expression datasets from The Cancer Genome Atlas TCGA Uniformlyprocessed normalized and batcheffect corrected gene expression datasets from TCGA andGTEx were obtained from figsharecomsData_record_35330593 [] Geneexpression and phenotype were standardized before supplying to the GSR software StandardGSEA was also performed for comparisonGene sets were downloaded from the MSigDb website oftwarebroadinstitutegseamsigdbindexjsp Here we combined BIOCARTA KEGG and REACTOME to create atotal of gene sets We also downloaded the GO biological process terms as additional gene sets as well as the gene sets pertaining to oncogenic signatures for the TCGAdata analysisResultsGene scores were correlated with TWAS statistics in polygenic complex traitsOur method GSR is built on the hypothesis that the marginal gene effect sizes on the phenotype should be positively correlated with the sum of correlation with other genes whichPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressioninclude causal genes To validate this hypothesis we defined gene score for each gene as thesum of its squared Pearson correlation with all of the other genes derived from gene expression levels We calculated TWAS marginal statistics as the product of GWAS summary statistics β and eQTL weights W derived from the GTEx whole blood samples Eq To assessthe impact of genetogene correlation on TWAS statistic we correlated the gene scores withthe TWAS marginal statistics for complex traits Overall most traits had Pearson correlation between the gene score and the marginal TWAS statistic above For instance thecorrelation in schizophrenia was InterQuartile Range based on permutations Fig This implies a pervasive confounding impact on the downstream analysisincluding gene set or pathway enrichment analysis causal gene identification etc using theTWAS summary statistics while assuming independence of genes Fig GSR improved pathway enrichment powerIn simulated scenarios with default settings Methods compared to PASCAL and LDSC GSRdemonstrated hugely improved computational efficiency Table superior sensitivity indetecting causal pathways with an improved statistical power as well as competitive specificityin controlling for false positives Fig Specifically in simulations GSR achieved an overall area under the precisionrecall curve AUPRC of and identified the true causal pathway as the most significant one times compared to times by PASCAL which onlyachieved an overall AUPRC of Notably the FOCUSpredicted crediblegene sets were also significantly enriched for causal pathways Fig We then varied four different settings a the number of causal SNP per gene SNPgene heritabilities genephenotype variance explained overlapping causal pathway Wefocused our comparison with PASCAL because it directly tested for pathway enrichment andhas been demonstrated to outperform other relevant enrichment methods [] In all simulation settings GSR demonstrated an improved power in detecting the causal pathways S2 Figin S1 File as it was able to detect causal pathways when multiple SNPs influenced geneexpression when the proportion of variance explained by the gene expression was low orwhen the causal and noncausal pathways were allowed to overlap In contrast a lot of causalpathways were not deemed significant by PASCAL based on a pvalue threshold of which was equivalent to a Bonferronicorrected pvalue threshold of after correcting formultiple testing on approximately pathways tested per simulationImproved power in pathway enrichment leveraging observed geneexpressionOne unique feature of GSR is the ability to run not on only the summary statistics but also onobserved gene expression where the genegene expression correlation is directly estimatedTable Comparison of existing methods with GSRMethodPASCAL []LDSC []FOCUS []GSEA []GSRGWASTWASMeasured expressionRunning timesum stat �sum statsum statsum statsum statindividual expressionindividual expression m h h m min� Summary statistics For custom gene sets the main computation time for LDSC is calculating the LD score for all of the Genome SNPs101371journalpone0237657t001PLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Evaluation of power and robustness of GSR in detecting causal pathways a Precisionrecall curves for GSR and PASCAL summarizingresults from simulations b Summary of pvalues obtained by running GSR along with PASCAL LDSC and FOCUS times For each methodthe enrichment significance for causal pathways and noncausal pathways are displayed We experimented FOCUS with and crediblesets for the pathway enrichments For the ease of comparison we plotted the yaxis on a squareroot negative logarithmic scale Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g003from the insample gene expression To evaluate the accuracy of this application we simulatedgene expression and phenotype for individuals which were provided as input to GSR forpathway enrichment analysis As a comparison we applied GSR to the summary statistics generated from the same datasetAs in the simulation above the SNPexpression weights were estimated from a separate setof reference individuals whereas the SNPphenotype associations were estimated fromonly individuals Notably the sample size for the GWAS cohort is much smaller than theprevious application to mimic the real data where usually fewer than individuals haveboth the RNAseq and phenotype available eg TCGA Additionally we applied standardGSEA [] to the same dataset with the observed gene expression We observed an improvedpower of GSR when using the observed gene expression over GSR using the summary statisticsFig whereas GSEA had a comparable performance as the latter Specifically all causal pathways in the simulated replicates had a pvalue below with the largest pvalue being à as determined by GSR using observed gene expression while no causal pathwayreached this level of significance with the smallest pvalue being à determined byGSEA We also compared the performances of GSR using observed gene expression to GSEAin various simulation settings and obtained consistent conclusions S3 Fig in S1 FileGene set enrichments in complex traitsApplying GSR to complex traits we revealed various pathways where the enriched gene setswere biologically meaningful For example the enriched gene sets for high density lipoproteinHDL predominantly involve lipid metabolism In contrast for Lupus gene sets wereenriched in interferon signalling pathways a known immunological hallmark We listed thetop enrichments over gene sets from MSigDB and Gene Ontology terms for HDL and theautoimmune trait Lupus in S1 Table in S1 FileAdditionally we applied GSR to test celltypespecific enrichments using cell types of which were derived from GTEx and cell types were derived from Franke lab datasets[] We observed biologically meaningful cell typespecific enrichment for the complexPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Comparison of pathway enrichment determined by GSR using or not using observed gene expression information and by GSEA NominalNOM pvalues yielded by GSEA were summarized Red line denotes pvalue threshold of Blue line denotes pvalue threshold of 101371journalpone0237657g004traits Fig In particular central neural system cellspecific gene sets were enriched forschizophrenia immune cellspecific gene sets for lupus immune cellspecific and digestivetract cellspecific gene sets for Crohns disease and cardiac cellspecific gene sets for coronaryartery disease Lastly we correlated traits based on their gene set enrichments and observedmeaningful phenotypic clusters suggesting shared biological mechanisms by the related phenotypes S4 Fig in S1 File For example Crohns disease and ulcerative colitis two subtypes ofinflammatory bowel disease formed a cluster Neurological diseases schizophrenia and bipolardisorder formed a cluster Moreover lipid traits including LDL HDL and Triglyceridesformed their own clusterApplication on observed gene expressionLastly using expression profiles of BRCA THCA and PRAD from TCGA and GTEx [] wetested the enrichments of oncogenic gene sets as well as gene sets from BIOCARTAKEGG and REACTOME in each type of tumor Overall we observed a significantly strongerenrichments for the oncogenic signatures with higher p values compared to the more generalgene sets across all three tumour types ttest pvalue à à and à for BRCA PRAD and THCA respectively S5 Fig in S1 File As a comparison we also ranstandard GSEA and observed qualitatively similar enrichments S5 Fig in S1 FilePLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionFig Celltypespecific enrichment of gene sets for representative complex traits GSR was applied to each complex trait in order toidentify significantly enriched gene sets among predefined celltypespecific gene sets represented by nine different colors Gene setswere indicated by dots and were aligned in the same order on the xaxis Red lines indecate Bonferronicorrected pvalue threshold 101371journalpone0237657g005DiscussionIn this work we describe GSR an efficient method to test for gene set or pathway enrichmentsusing either GWAS summary statistics or observed gene expression and phenotype information We demonstrate robust and powerful detection of causal pathways in extensive simulation using our proposed method compared to several stateoftheart methods Whenapplying to the real data we also obtained biologically meaningful enrichments of relevantgene sets and pathways These features warrant GSR a widely applicable method in variousstudy settings with an aim to interpret association test results and capture the underlying biological mechanismsOur approach has superior computational efficiency In particular GSR took only minutes running on the full summary statistics and less than minutes on the full gene expressiondata with one million SNPs and genes to test for enrichments of over gene sets Inour simulations it is not surprising that FOCUS can accurately finemap causal genes as thesimulation designs followed similar assumptions adopted by FOCUS [] However FOCUSis at least times slower than GSR For the simulated data FOCUS took minutes to finemap all of the genes in GWAS loci whereas GSR took under three minutes to test for pathwayenrichments on the same machine Additionally the computational cost of FOCUS is exponential to the number of causal genes considered within each locus whereas GSR is not affectedPLOS ONE 101371journalpone0237657 August PLOS ONE 0cGene score regressionby the number of causal genes Also because GSR operates at genomewide level no thresholdis needed to decide which genes to be included whereas FOCUS needs userdefined thresholdfor constructing the credible gene set for the subsequent hypergeometric enrichment testGiven these advantages we envision that GSR will be a valuable tool for the bioinformaticcommunity and statistical genetic community as a fast way to investigate the functional implications of complex polygenic traitsIn different simulation settings GSR exhibits improved pathway enrichment power overPASCAL and LDSC two popular methods for partitioning heritability and identifying causalgene sets Since GSR leverages SNPtogene association summaried by eQTL weights whileeither PASCAL or LDSC operate | Thyroid_Cancer |
Neck Tissues A a0Systematic ReviewJerome a0R a0Lechien1234 Stphane a0Hans13 a0· Maria a0R a0Barillari18 a0· Giovanni a0Cammaroto19 a0· Graldine a0Descamps12 a0· Julien a0Hsieh110 a0· Luigi a0Vaira111 a0· Giacomo a0De a0Riu111 a0· Leigh a0Sowerby112 a0· Isabelle a0Gengler113 a0· Justin a0Michel15 a0· Sven a0Saussez124 a0· Thomas a0Radulesco15 a0· Christian a0CalvoHenriquez16 a0· Carlos a0M a0ChiesaEstomba17 a0· Received July Accepted August Springer ScienceBusiness Media LLC part of Springer Nature AbstractTo review the data regarding the expression of angiotensin converting enzyme2 ACE2 and transmembrane protease serine2 TMPRSS2 in head and neck tissue Scopus Cochrane Library Medrxiv Google Scholar and PubMEDMEDLINE were searched by four independent investigators for studies investigating ACE2 or TMPRSS2 expressions in head and neck tissues The following outcomes were considered sample origin animal versus human detection method anatomical location and cell types PRISMA checklist and modified population intervention comparison outcome timing and setting PICOTS framework were used to perform the review Of the identified studies met our inclusion criteria Thirteen studies were conducted during the severe acute respiratory syndrome a0coronavirus2 SARSCoV2 pandemic ACE2 and TMPRSS2 were expressed in oral pharyngeal sinusonasal human mucosa The following cell types expressed ACE2 basal apical goblet minor salivary and endothelial cells TMPRSS2 was found in goblet and apical respiratory cells ACE2 and TMPRSS2 were found in the olfactory region especially in sustentacular nonneural and neural stem cells Animal studies suggested that ACE2 expression may vary regarding age There was an important heterogeneity between studies in the methods used to detect ACE2 and TMPRSS2 leading to a potential identification bias The SARSCoV2 receptors ACE2 and TMPRSS2 are both expressed in many head and neck tissues enabling the viral entry into the host anismKeywords ACE2 a0· TMPRSS2 a0· SARSCoV2 a0· COVID a0· Coronavirus a0· Head NeckIntroductionThe renin angiotensin aldosterone system is one of the most important systems regulating the homeostasis of cardiovascular and pulmonary function this involves many molecules including angiotensin converting enzyme2 ACE2 [] ACE2 is also known to be the functional receptor of some coronavirus species as initially discovered in during the severe acute respiratory syndrome coronavirus SARSCoV epidemic [] The current pandemic of coronavirus disease Jerome R Lechien and Thomas Radulesco have contributed equally to the paper and are joint as cofirst authorsJustin Michel and Sven Saussez equally contributed to the paper and are cosenior authorsjeromelechienumonsacbe Jerome R Lechien Extended author information available on the last page of the COVID19 has brought to light the importance of ACE2 regarding development of infection viral spread and the development of the clinical COVID19 [] At the same time another SARSCoV2 receptor has been identified the transmembrane protease serine2 TMPRSS2 []ACE2 and TMPRSS2 tissue expressions are particularly important to identify viral entry pathways and to better understand the anrelated clinical presentation of the disease [ ] Further evaluation of ACE2 and TMPRSS2 expression in ear nose and throat mucosa is warranted to shed light on the pathophysiology of disease in the head and neck []The aim of this systematic review is to summarize the current data about the expression of ACE2 and TMPRSS2 in head neck tissueVol01234567891 0c MethodsThe review was conducted regarding the Preferred Reporting Items for a Systematic Review and Metaanalysis PRISMA checklist [] A modified population intervention comparison outcome timing and setting PICOTS framework was used to structure the review process [] For this review the PICOTS structure was kept but adapted to experimentalbasic research studies on human and animal tissuesStudiesAnimal and human experimental published studies in Englishlanguage peerreviewed journals were considered Preprint studies were also considered in light of the current pandemic and the significant wealth of knowledge derived over the last few months All studies where investigators assessed ACE2 or TMPRSS2 expressions in head neck tissues through immunochemistry IHC in a0situ hybridization Western Blot RNA sequencing RNAseq or reverse transcription polymerase chain reaction RTPCR were evaluatedParticipants and a0Inclusion CriteriaThe papers had to include either human or animal subjects The authors extracted substantial information about the sample characteristics including species involved and ACE2 and TMPRSS2 identification methodOutcomesThe primary outcome studied was tissue expression of ACE2 and TMPRSS2 The anatomical location the types of cells that expressed both receptors were recorded Particular attention was paid to the method used to detect ACE2TMPRSS2 in tissues Additional useful information such as viral impact on the functioning of the tissuecell that expressed the receptor or interindividual differences were also collectedIntervention and a0ComparisonBecause the aim of the study was to investigate the tissue ACE2 and TMPRSS2 expression we did not consider potential intervention on patient or animal modelsHead and Neck PathologyTiming and a0SettingWe included the studies where the receptor analysis was made on normal subjects andor infected patientsSearch StrategyThe PubMedMEDLINE Google Scholar Medrxiv Scopus and Cochrane search was conducted by independent authors JRL TR CCH GD CMCE to identify papers published between January and April The authors screened publications with database s and available full texts referring to the condition The following keywords were used for the search strategy ACE2² TMPRSS2² COVID19² COVID SARS coronav coronavirus salivary gland Receptor Head Neck Nasal ear nose throat ENT Tissue and Cell The authors investigated papers for number of samplesindividuals study type design inclusion criteria and ACE2TMPRSS2 detection outcomesResultsThe electronic search identified papers of which met our inclusion criteria Table a0 [] A total of studies investigated the expression of ACE2 and TMPRSS2 in human head and neck tissues while five papers focused on mouse and two on monkey samples respectively Table a0 One study focused on ACE2 genetic analysis without reporting sitespecific anatomical expression [] The flow chart of the study process is available in Fig a0 Five studies were preprint [ ]Tissue Expression in a0HumanACE2 ExpressionACE2 was assessed in studies [] The expression of ACE2 was found in all mucosa of the respiratory upper tract including trachea [ ] sinus and nasal cavities [ ] Among the respiratory mucosa ACE2 was expressed in several types of cells including epithelial goblet and endothelial cells [ ] One study reported that ACE2 was expressed on ciliated epithelial cells and not on nonciliated goblet cells [] Butowt et a0al compared the intensity of expression of ACE2 in the upper and lower respiratory tract [] They found that nasal epithelial cells had lower levels of ACE2 expression compared with epithelial cells of the lower respiratory tract [] Among the nasal region two studies investigated the ACE2 expression in the 0cHead and Neck Pathology Table Studies reporting ACE2 or TMPRSS2 head and neck expressionAuthorsDesignVaarala Mixed [] StudyHamming HumanSamples MethodsHuman TMPRSS2MouseHuman ACE2RNAseqACE2 TMPRSS2 expressionSalivary glandsTMPRSS2 humanOral Nasal Nasopharyngeal Epithelium endotheliumACE2 human all mucosa [] StudyIHCHumanJia [] StudyHuman ACE2Tracheal Epithelium endotheliumIHC biotinylation ACE2 humanLiuExperimenal Monkey ACE2 [] StudyIHCNaso Oro Hypopharyngeal Tracheal EpitheliumACE2 MonkeyVirion in SalivaBilinska Animal [] StudyBrannMixed [] StudyButowtMixed [] StudyMouse ACE2 TMPRSS2 Olfactory EpitheliumRNAseq RTPCRACE2 sustentacular cellsIn situ hybridization TMPRSS2 sustentacular cellsWB IHCMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 TMPRSS2 Mouse nonneuronal cellsACE2 TMPRSS2 Human glial and neuronal stem cellsMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 Mouse Human non neuronal cellsCaoHumanHuman ACE2 gene [] StudyGenetic AnalyzisChenHuman [] StudyHikmetHuman [] StudyHuman ACE2RNAseqHuman ACE2IHCLeeHumanHuman ACE2TMPRSS2 Mouse Human neuronal nonneuronal cellsRespiratory EpitheliumACE2 TMPRSS2 Human Lower Airway NasalNo localization providedSalivary glandsACE2 humanNasopharyngeal EpitheliumACE2 human no expression in nasopharynxTracheal Nasal Sinusal EpitheliumFindings TMPRSS2 is expressed in human salivary gland tissues ACE2 was found in endothelial arteries veins and epithelial cells of nasal rhinopharyngeal and oral mucosa Precisely the epithelium expression concerned the basal layer cells ACE2 was more expressed on the apical than the basolateral surface of polarized airway epithelia ACE2 is expressed in salivary gland ducts of the pharyngeal glands ACE2 was expressed in epithelial cells lamina propria respiratory tract Virus was found in saliva of infected monkeys ACE2 TMPRSS2 are expressed in sustentacular cells of the olfactory epithelium but notmuch less in most olfactory receptor neurons Expression of the entry proteins increases in animals of old age In human ACE2 TMPRSS2 were not identified in purified olfactory neurons ACE2 was identified in glial cells olfactory stem cells Nasal epithelial cells have lower levels of ACE2 TMPRSS2 compared with epithelial cells of the lower respiratory tract ACE2 has nonneuronal expression in olfactory epithelium The expression of ACE2 TMPRSS2 mouse were increased in elderly mouse unique expression quantitative trait loci variants were found for ACE2 The genotypes of ACE2 gene polymorphism may be characterized by higher expression levels of ACE2 in East Asian population There would be different susceptibility or response to SARSCoV2 in different populations ACE2 is expressed in human granular cells of salivary glands There was no ACE2 expression in nasopharyngeal cells ACE2 is expressed in ciliated epithelial cells cilia anelle 0c Table continuedAuthorsDesign [] StudyHumanLiHikmetHuman [] Study [] StudySungnak Human [] StudySamples MethodsIHCACE2 TMPRSS2 expressionACE2 humanHuman ACE2Human ACE2IHCRNAseqThyroidNasopharyngeal EpitheliumACE2 human no expression in nasopharynxACE2 humanHuman ACE2 TMPRSS2 Airway Nasal epitheliumRNAseqACE2 humanTMPRSS2 human subset of ACE2 cellsXuHumanHuman ACE2 TMPRSS2 Oral Epithelium [] StudyRNAseqHumanXu [] StudyWuHuman [] StudyHuman ACE2RNAseqHuman ACE2RNAseqACE2 humanACE2 humanOral T cells B cells fibroblastsACE2 humanMinor salivary glandsACE2 humanNasal Oral EpitheliumHead and Neck PathologyFindings2There was no ACE2 expression in the nonciliated goblet cells ACE2 expression is influenced by patient demographics clinical characteristics comorbidities or medication use The use of ACE inhibitor drugs did not increase ACE2 protein expression ACE2 is expressed by thyroid cells There was no ACE2 expression in nasopharyngeal cells ACE2 was expressed in airway epithelial cells ACE2 is more expressed in nasal epithelial cells compared with other respiratory cells goblet ciliated cells TMPRSS2 is only expressed in a subset of ACE2 cells ACE2 is expressed in the oral cavity epithelial cells ACE2 expression was higher in tongue than buccal and gingival tissues ACE2 is expressed in minor salivary glands ACE is expressed in nasal epithelial cells The was a higher virus concentration in the nasalswab comparing with throatswab which is attributed to ACE2expression in nasal epithelial cells ACE2 TMPRSS2 are coexpressed in nasal goblet secretory cellsMixedZiegler [] StudyACE2 Angiotensin Converting Enzyme2 IHC Immunohistochemistry RTPCR reverse transcription polymerase chain reaction SARSCoV2 severe acute respiratory syndrome a0coronavirus2 TMPRSS2 transmembrane protease serine2 WB Western BlottingHuman ACE2 TMPRSS2 Sinusal Nasal goblet epithelial cellsMonkey RNAseqACE2 TMPRSS2 Humanmucosa of the olfactory region including olfactory bulb [ ] The ACE2 receptor was identified in sustentacularnonneuronal cells of the olfactory tissues Moreover ACE2 was found in a low proportion of neuronal stem cells in the olfactory bulb [ ] The expression of ACE2 in olfactory neurons nonstem cells remains uncertain because Butowt et a0al and Brann et a0al observed that ACE2 has only nonneuronal expression pattern in olfactory epithelium [ ]Five studies investigated ACE2 expression in oral and pharyngeal regions including oral and hypo oro and nasopharyngeal spaces [ ] The study that explored ACE2 expression in human nasopharynx [] did not exhibit significant ACE2 immunostaining in nasopharyngeal cells [] ACE2 receptor was identified in oral endothelial [] epithelial [ ] and salivary [] cells Xu et a0al found that ACE2 was also expressed in T and B cells as well as fibroblasts of the oral cavity [] Moreover ACE2 was expressed in major salivary gland tissues [] and thyroid tissue [] In many publications authors reported the type of cells goblet versus epithelial versus stem cells that expressed ACE2 or TMPRSS2 Table a0 Interestingly Xu et a0al almost as much ACE2 expression in the thyroid as in the lungs []The genetic analysis of Cao et a0al reported that there are unique expression quantitative trait loci variants in the East Asian population supporting a gene polymorphism and tissuerelated differences between individuals [] 0cHead and Neck Pathology Fig Flow chart ACE2 Angiotensin Converting Enzyme2 TMPRSS2 transmembrane protease serine2TMPRSS2 ExpressionTMPRSS2 expression was investigated in studies [ ] Similarly to ACE2 TMPRSS2 was identified in nasal [ ] and respiratory mucosa cells [] including both epithelial and goblet cells with higher expression in lower airway compared with upper airway [] Moreover TMPRSS2 receptor was identified in sustentacular and neuronal olfactory cells [ ] but not in olfactory neurons [] TMPRSS2 was also identified in salivary major gland tissue []ACE2 TMPRSS2 Tissue Expression in a0Mouse and a0MonkeySix studies used animal models to assess ACE2 or TMPRSS2 expressions in head and neck tissues [ ] The mouse studies of Butowt et a0al and Bilinska et a0al revealed that elderly mice had higher expression of both ACE2 and TMPRSS2 in nasal mucosa compared with younger mice [ ] In olfactory tissue ACE2 was identified in sustentacularnonneuronal and neural stem cells of mice [] Liu et a0al analyzed ACE2 expression in monkeys [] 0c Head and Neck PathologyTable Summary of Cell Expression of ACE2 and TMPRSS2AuthorsBilinska []Brann []SamplesMouseHuman MouseTissueOlfactoryOlfactoryButowt []Human MouseNasalOlfactoryChen []Hamming []Hikmet []Jia []Lee []Li []Liu []Sungnak []Vaarala []Xu []Xu []Wu []Ziegler []HumanHumanHumanHumanHumanHumanMonkeyHumanMajor Salivary GlandOral Nasal NasopharyngealNasopharyngealTracheal Nasal SinusalThyroidPharyngealTrachealTracheal NasalHuman MouseHumanMajor Salivary GlandOralHumanHumanHuman Mouse MonkeyMinor Salivary GlandNasalOralNasal SinusalCell typesSustentatorialSustentatorialNeuronalStem NeuronalEpithelialSustentatorialNeuronalGranularBasal layerEndothelialEpithelialApical EpithelialEndothelialApical EpithelialGobletUnspecifiedMinor salivary ductalBasal layerGobletApical EpithelialUnspecifiedApical EpithelialFibroblastT and BcellsUnspecifiedUnspecifiedBasal layerGobletACE2NATMPRSS2NANANANANANANANANANANANANANANANANAACE2 Angiotensin Converting Enzyme2 NA not available TMPRSS2 transmembrane protease serine2reporting a higher ACE2 expression in tracheal naso oro and hypopharyngeal tissues as well as in the salivary ducts of the pharyngeal gland and consequently in saliva In this study the cell expression was mainly localized in the lamina propria In the same vein Vaarala et a0al reported TMPRSS2 expression in mouse salivary tissues []Cell Detection MethodsThe following methods have been used for detecting ACE2 and TMPRSS2 in cells of human and animal tissue RNAseq N IHC N RTPCR N in a0situ hybridization ISH N and WB N Different detection approaches were used in studies [ ] One study reported specific genetic analysis [] There were significant differences between studies regarding methods used While Ziegler et a0al and Sungnak et a0al detected ACE2 by RNAseq in goblet cells Lee et a0al did not find any immunohistochemical labeling [ ] However the results reported in sustentacular cells agree in the same direction whatever the technique used whether by RNAseq or by ISH and immunocytochemistry [] The discrepancies are rather observed between studies having performed immunohistochemistry Indeed using two different antibodies Hikmet did not find ACE2 expression in nasopharynx epithelium whereas others demonstrated the staining of the apical surface of epithelia and ciliated epithelial cells [ ] Interestingly all the studies which carried out RNAseq found an expression of ACE2 or TMPRSS2 at the epithelial level which implies that the technique used could generate biases between the studies [ ] 0cHead and Neck Pathology DiscussionThe presentation of COVID19 infection may be in several clinical forms ranging from anosmia in isolation to severe multiple an failure and death The mechanisms underlying the COVID19 polymorphism are still unknown To infect tissues SARSCoV2 needs to entry into the cells which is allowed through ACE2 and TMPRSS2 receptors [] The identification of virus receptor expression in the tissues makes particularly sense to better understand the clinical expression of the disease This systematic review sheds light on many pointsFirst ACE2 and TMPRSS2 receptors are expressed in epithelial and nonepithelial cells throughout the head and neck The head and neck expression may support the otolaryngological clinical picture of the disease which was recently found in European and North American COVID patients [ ] By entering the body via the epithelial cells of the upper aerodigestive tract mucosa the SARSCoV2 virus leads to an inflammatory reaction and the development of otolaryngological symptoms Nasal entrance of the virus through high ACE2 expression was supported in the study of Wu et a0al who found a higher virus concentration in nasal swabs compared with throat swabs []The olfactory cleft is a nasal region that has drawn the attention of many researchers over the past few weeks Indeed recent data supported that more than of COVID19 patients developed subjective olfactory dysfunction especially when patients suffered from mildtomoderate forms of the disease [ ] Because ACE2 and TMPRSS2 are both expressed in the nasal mucosa of the olfactory cleft entrance into the olfactory bulb seems plausible Once in the bulb according to some human studies [ ] the virus could infect cells that express ACE2 or TMPRSS2 namely glial and neuronal stem cells Fig a0Integrating the molecular clinical and radiological characteristics of SARSCOV2 olfactory loss may shed light about its pathophysiological process Taking into account that the loss is often temporary SARSCOV2 may primarily infect the sustentacular cells supporting the olfactory sensory neurons This infection may cause rapid disruption of the olfactory epithelium structure and function with a possible inflammatory response inducing sudden onset smell loss This inflammation is observed in a minority of patient with congested olfactory cleft who underwent CT scan [ ]ACE2 and TMPRSS2 are also found in horizontal basal olfactory stem cells located in the basal layer They are less exposed to the external environment thus less likely to be infected in first line the loss would have been more Fig Epithelium of Olfactory Cleft The figure summarizes the olfactory cleft epithelium 0c Head and Neck Pathologyprogressive However once infected they might slow down recovery time because horizontal basal cells give rise to many cell type in the olfactory epithelium They may also contribute to virus spread to the olfactory bulb vascular pericytes Magnetic resonance imaging MRI studies of the olfactory bulb [ ] in which ACE2 are only expressed in vascular pericytes but not in neurons may show inflammatory signs suggesting that the infection process can extend more centrally and promote inflammatory response [] Inflammatory causes are often quickly reversible for example after a oneweek trial of high dose of corticosteroids or simply days after the resolution of the viral infection suggesting that the olfactory neurons and bulbs are still somewhat intact This seems to be the case for a majority of patients In contrast with more sustained destruction of neuronal olfactory structures the recovery time is much longer and may take a0years given the slow neuroregeneration process [ ]In this systematic review we found that three studies reported high ACE2 expression in major or minor salivary gland human tissues [ ] These data corroborate the literature findings that reported a salivary pattern of SARSCoV2 and related parotitis [ ] Moreover the virus spread into the salivary gland tissues allows us a better understanding the mechanisms underlying salivary transmission Interestingly in Liu et a0al observed that monkeys infected by SARSCoV had a salivary viral spread which was associated with a salivary virion excretion [] These data support the need to conduct future studies investigating the presence of SARSCoV2 in the saliva of infected human and to corroborate the saliva findings with the ACE2 salivary gland expressionThe head and neck expression of ACE2 and TMPRSS2 and the related otolaryngological symptom pattern seems obvious but could vary across individuals and populations In support of this Lee et a0al observed that ACE2 expression is influenced by patient demographics clinical characteristics comorbidities and medication [] As reported in the genetic analysis of Cao et a0al there would be different susceptibilities or responses to SARSCoV2 in different populations [] The polymorphism of ACE2 and TMPRSS2 expression could explain the clinical differences between individuals Indeed many physicians reported in clinical and epidemiological studies different clinical presentation of the disease [ ] which could be associated with virus mutations [ ] The virus mutations and the related impact on receptor binding and infectivity is another point that has to be considered in future studies Otherwise according to the Bgee database https bgee expression of ACE2 and TMPRSS2 evaluated in murine models may increase with age These findings have to be confirmed in humans but could explain more severe clinical pictures in the elderlyThe present study has several limitations First the heterogeneity between studies about the detection method may lead to detection bias as some approaches are more sensitive than others Some studies interrogate gene expression at mRNA level and others at protein levels both types of analysis having their advantages and limitations Compared to transcriptomic analyzes immunohistochemistry brings additional important spatial information in tissue samples but recently Sorokin et a0al demonstrated high and statistically significant correlations between the RNA sequencing and immunohistochemical measurements [] Interestingly they highlighted the complementarity of both techniques for measuring cancer biomarkers in FFPE samples However differences observed across IHC studies suggest the involvement of many parameters The antibody specificity is a big challenge to ensure reproducibility of antibodybased studies and given the high homology between ACE1 and ACE2 cautions must be taken regarding antibody selection Besides a report from the International Working Group for Antibody Validation IWGAV proposed five scientific approaches to validate antibody specificity [] then such strategies must be considered in future investigations to confirm the published observations In addition it seems essential to enlarge and diversify patient cohorts and to combine transcriptomic and proteomic strategies as well as colocalize different markers of SARSCoV2 such as ACE2 and TMPRSS2 to provide an accurate representation of ACE2 expression through all head and neck areas of the whole populationSecond the majority of studies that were conducted during the SARSCoV2 pandemic did not consider many demographic and clinical factors such as the age of patients from who the tissues were extracted or the use of ACE inhibitor medications among others Third some otolaryngological regions remain uninvestigated such as the vocal folds The investigation of these remaining regions may shed further light on some recently reported unusual clinical phenomena such as severe dysphonia []ConclusionACE2 and TMPRSS2 are both expressed in head and neck tissues which may explain the otolaryngological clinical pattern of the disease and the entry of SARSCoV2 into the host anism Future studies considering demographical and clinical characteristics of patients from who the tissues are extracted are needed to better understand the cell entry mechanisms of SARSCoV2Author contributions JR TR SS JM design acquisition of data data analysis interpretation drafting final approval and accountability 0cHead and Neck Pathology for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved CCH CMCE MRB IG design data analysis interpretation revising the manuscript for important intellectual content final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved LS SH GC GD JH LV GR design acquisition of data data analysis interpretation drafting some parts of the manuscript final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolvedCompliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interestReferences Crackower MA Sarao R Oudit GY et a0al Angiotensinconverting enzyme is an essential regulator of heart function Nature Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Somasundaran M Sullivan JL Luzuriaga K Greenough TC Choe H Farzan M Angiotensinconverting enzyme is a functional receptor for the SARS coronavirus Nature Wang Z Xu X scRNAseq profiling of human testes reveals the presence of the 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"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing pr sity score matching In the two groups gastric neoplasms containing NEC components were dividedinto pure NEC and mixed tumors with less than GHMiNEN between and GHMiNEN andmore than GHMiNEN neuroendocrine carcinoma components OS was compared between thesegroups and the gastric adenocarcinoma groupResults The OS of gastric neoplasms containing neuroendocrine NEC components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or Cox multivariable regressionanalysis suggested that tumor category neoplasms containing NEC components or gastric adenocarcinoma wasan independent risk factor for prognosis Interestingly among patients receiving neoadjuvant therapy thedifference was not significantContinued on next page Correspondence buzhaodecjcrcn jijiafuhscpkueducn Jiahui Chen Anqiang Wang and Ke Ji contributed equally to this workDepartment of Gastrointestinal Surgery Key Laboratory of Carcinogenesisand Translational Research Ministry of Education Peking University CancerHospital Institute No Fucheng Road Haidian District Beijing China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of Continued from previous pageConclusions Gastric neoplasms containing any proportion of NEC components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmsKeywords Neuroendocrine carcinoma Gastric adenocarcinoma Overall survivalBackgroundGastric neoplasms containing neuroendocrine carcinomaNEC components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure NECs and mixed tumorscontaining NEC components Every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing NEC components account for approximately of thesecases [ ] Given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []According to the World Health anizationWHO digestive neuroendocrine tumor classificationneuroendocrine neoplasm NEN can be divided intothree categories based on Ki67 levels and mitotic counts HPF Grade G1 Ki67 mitoses Grade G2 Ki67 mitoses Grade G3Ki67 mitoses [] Meanwhile the AmericanJoint Committee on Cancer AJCC defines highly differentiated NEN as a neuroendocrine tumor NET and thepoorly differentiated NEN as a neuroendocrine carcinoma NEC based on the degree of tumor cell differentiation Generally G1 G2 and rare welldifferentiated G3NENs belong to the NETs while poorly differentiatedG3 NENs belong to NECs[ ] Gastric mixedneuroendocrinenonneuroendocrineneoplasm GMiNEN is a special type of gastric NEN that is definedas containing more than of both neuroendocrineand nonneuroendocrine components [] accountingfor approximately of all GNENs and of gastricneuroendocrine carcinomas GNECs [] For thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition Consideringthe heterogeneity ofMiNEN and the malignancy degree of the different components in the tumor La Rosa [ ] proposeddividing MiNEN into three categories highgradeintermediategrade and lowgrade Highgrade MiNENconsists of NEC and carcinomaadenoma intermediategrade MiMEN consists of NET and carcinoma and lowgrade MiNEN consists of NET and adenoma Thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm GHMiNEN was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandGenerally the prognosis of mixed tumors is largely determined by the most malignant component Kim et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components All of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis Therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone We sought tofind studies on the overall survival OS comparison between GHMiNEN and gastric adenocarcinoma butfailed Thus we think that a study of the comparison ofthe OS of GHMiNEN and gastric adenocarcinoma willprovide a valuable supplement to current research on GHMiNEN To overcome the bias caused by the differences between the covariates in the comparison we usedpr sity score matching PSM to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliableMethodsPatient selectionWe retrospectively collected patients diagnosed withgastric NENs and underwent radical resection at PekingUniversity Cancer Hospital Beijing from January to December The inclusion criteria were as follows pathologically confirmed pure NEC or tumorcontaining NEC components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup Patients diagnosedwith cM1 or cT4b before surgery or died from perioperative complications were excluded from the study 0cChen BMC Cancer Page of Patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for PSM analysesperformed The chisquared test and MannWhitney Utest were used to further verify the matching resultsFollowupWe followed the patients at least twice a year Serumtumor markers test gastroscope and computed tomography CT scans were used to reexamine patients aftersurgery Depending on the patients status Magneticresonance imaging MRI and Positron emission tomography computed tomography PETCT were alsoconsidered For patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationDiagnosis and classificationWe reevaluated the diagnosis and classification of GHMiNEN Mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as GHMiNEN and GHMiNENrespectively Atumor consisting of NEC is defined as pure NECAll neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the WHO classification of tumors[] Neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin Syn chromograninA CgA and neuro cell adhesion molecule CD56 orNCAM The tumor staging described in the study wasbased on the AJCC 8th Edition TNM Staging Guidelines[] All possible disagreements were discussed in ourstudy groupDefinition of variables and groupsIn this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery Patients inthe surgery group were assessed by the pTNM stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the ypTNM staging system OSrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egPr sity score matchingTo accurately compare the prognosis of GHMiNENand gastric adenocarcinoma we employed PSM to balance the differences between the two groups PSM wasperformed through the Pamatching plugin in SPSS software Logistic regression models were used toestimate pr sity scores based on gender age tumorlocation tumor size and pathological staging Given a caliper width nearest neighbor matching wasStatistical analysisAll statistical analyses were performed using SPSS statisticalsoftware IBM United States The chisquared test and MannWhitney U test were used forstatistical analysis of categorical variables and continuous variables respectively KaplanMeier method wasused for the comparison of OS The logrank test wasused to compare survival rates Multivariable Cox proportional hazards models were used to identify predictors of survival outcome P was regarded as thethreshold of significanceResultsPatient selection and PSM resultsBetween and among the patients treated atthe Gastrointestinal Cancer Center of Peking UniversityCancer Hospital a total of patients with gastric neoplasms containing NEC components met the inclusioncriteria for the study including cases of pure NECand cases of mixedtype Of these patients a total of patients received neoadjuvant therapy NEC GHMiNEN GHMiNEN GHMiNEN while the remaining patients receivedsurgery directly NEC GHMiNEN GHMiNEN GHMiNEN There were aninsufficient number of patients in group GHMiNEN group to conduct effective statistical analysisso we combined the GHMiNEN group with theNEC group for further analysis We also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery Among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly Fig Immunohistochemical specificity markers were utilizedto identify the neuroendocrine components Fig 2aSyn was expressed in almost all neoplasms containingNEC components while the positive rates ofCgA and CD56 were much lower and respectively No significant difference in the positiverate of Syn and CgA was observed between pure NEC GHMiNEN GHMiNEN and GHMiNENFig 2b c only the positive rate of CD56 was found tobe higher in the pure NEC group than that in the GHMiNEN group Fig 2dTherefore priorto OS comparison PSM wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cChen BMC Cancer Page of Fig Flow chart of patient enrolmentComparison of OS between all patients with NECcomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupBefore PSM we compared the survival curves between all patients with NEC components and patientswith gastric adenocarcinoma by the KaplanMeiermethod Fig Apparently patients with NEC components had a poorer OS than those with gastricadenocarcinoma Fig 3a p in the surgicalgroup In contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy Fig 3b p According to the proportion of NEC components patients were classifiedinto pure NEC GHMiNEN GHMiNENand GHMiNEN The OS was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison Fig 3c dFig Illustrations of immunohistochemical staining patterns in gastric neoplasms containing NEC components a An overview of the expressionof Syn CgA and CD56 in tumors containing NEC components b Syn expression in different NEC component groups c CgA expression indifferent NEC component groups d CD56 expression in different NEC component groups CD56 neuro cell adhesion molecule CgAchromogranin A NEC neuroendocrine carcinoma Syn synaptophysin Pvalue 0cChen BMC Cancer Page of Fig See legend on next page 0cChen BMC Cancer Page of See figure on previous pageFig Comparison of OS between gastric neoplasms containing NEC components and gastric adenocarcinoma a OS comparison betweengastric neoplasms containing NEC components and gastric adenocarcinoma before PSM in the surgical group b OS comparison between gastricneoplasms containing NEC components and gastric adenocarcinoma before PSM in the neoadjuvant group c OS comparison between differentNEC content groups pure NEC GHMiNEN GHMiNEN and GHMiNEN and gastric adenocarcinoma before PSM in the surgicalgroup d OS comparison between the different NEC content groups and gastric adenocarcinoma before PSM in the neoadjuvant group e OScomparison for patients in the surgical group after PSM f OS comparison for patients in the neoadjuvant group after PSM NEC neuroendocrinecarcinoma OS overall survival PSM pr sity score matchingBefore PSM significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group Table Table To balance the clinicopathological differences between the twogroups PSM was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups The detailedclinicopathological characteristics before and after PSMare shown in Table and Table As a result patients with NEC components and patients with gastric adenocarcinoma were matchedin the surgical group Table Patients with NEC components also had a poorer OS than those with gastricadenocarcinoma Fig 3e p Multivariable analysis showed that adjuvant therapy tumor category andTNM stage werefactorsTable independent prognosticTo investigate whether neoadjuvant therapy had an effect on OS patients with NEC components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group Table Interestingly KaplanMeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in OS between the two groups Fig 3f p Comparison of OS between patients with differentproportions of NEC components and patients with gastricadenocarcinomaTo investigate whether the level of NEC componentshad an effect on OS in the surgical group GHMiNEN GHMiNEN pure NEC and pure NEC plus GHMiNEN were compared with gastric adenocarcinoma after PSM The results showed that even thegroup with the lowest proportion of NEC componentsthe GHMiNEN group had a poorer OS thanadenocarcinoma Fig 4a P As expected theGHMiNEN pure NEC and pure NEC plus GHMiNEN groups each with relatively high proportionsof NEC components had worse OS than the gastricadenocarcinoma group Fig 4bd P Detailed clinical information after matching isshown in Additional file Tables S1S4PSM was also performed in the neoadjuvant group Incontrast to the results of the surgery group in the pureNEC group containing the highest proportion ofNEC componentstill no significantdifference in OS from gastric adenocarcinoma Fig5d The other three groups with lower NEC contentwere also notfrom gastricadenocarcinoma in terms of OS Fig 5ac Detailedclinicopathologicaland afterPSM are shown in Additional file Tables S5S8characteristics beforethere wassignificantly differentDiscussionAmong gastric neuroendocrine neoplasms the tumorcontaining NEC components is a special type includingpure NEC and mixed tumor containing NEC components The incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated GNENs [ ]received neoadjuvantIn previous study Kim found that in patientschemotherapywho had notprogressionfree survivalPFS of pure GNEC waspoorer than that of gastric adenocarcinoma while thePFS of mixedtype tumors was not significantly differentIn Kimsfrom that of gastric adenocarcinoma []study the mixed type was defined as NET mixed withgastric cancer rather than NEC NET is much less malignant than NEC [ ] This may be the reason whythere was no significant difference in OS between mixedtype and gastric adenocarcinomas In addition mixed tumors with less than or more than of NEC components were not included in that study which webelieve was a deficit of the study PFS is an important indicator for evaluating prognosis in many cases it can reflect the trend of OS Based on Kims research resultswe regarded tumors containing NEC components as awhole and found that the OS of these tumors was poorerthan that of adenocarcinoma in the surgical group Inthe comparison of OS between mixed tumors with different proportions of NEC components and gastricadenocarcinoma the results for pure NEC cases wassimilar to Kims While the OS of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in Kims study Cox multivariable regression analysis showed thattumor categoryneoplasm with NEC component or adenocarcinoma 0cChen BMC Cancer Page of Table Comparison of clinicopathological characteristics before and after PSM in surgical groupPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n P valueMatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomy ± ± Proximal gastrectomy Surgical procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Table Comparison of clinicopathological characteristics before and after PSM in neoadjuvant groupMatched comparisonPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedure LaparoscopicT stageT0T1T2T3T4N stageN0N1N2N3M stageM0M1ypTNM stageIIIIIIIV ± ± Gastricadenocarcinoman ± ± P valuePatients with NECcomponents n ± ± Page of P valueGastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Page of Table Univariate and multivariate analyses of survival after PSM in surgical groupPatient CharacteristicsUnivariate analysisHR CIMultivariate analysisHR CIP valueAge yearGendermale vs femaleBMIAdjuvant therapyYes vs NoTumor size¥ cm vs cmTumor categoryCarcinoma with NEC component vsGastric adenocarcinoma vsType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedureLaparoscopic vs TNM stageIIIIIIIVP value tumor size and TNM staging were independent risk factors for prognosis This suggests that the prognosis ofgastric neoplasms with NEC components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of NEC components can alsoimpair prognosis which challenges the current cutoffvalue of The proportion of each component that must theoretically be greater than was set in [] Andsince WHO has also adopted this standard to define MiNEN [] This largely avoids the overdiagnosisof MiNEN in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges In additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines [] Nevertheless it is now being questionedby an increasing number of scholars The componentsin mixed tumors are not evenly distributed For large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely []Park compared the OS between tumors with morethan NEC components and gastric adenocarcinomawith or without less than NEC and they found thattumors with an NEC composition of more than hada worse prognosis This suggests that even a small proportion of malignant components can affect prognosis[] While in Parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith NEC components less than with gastricadenocarcinomas directly nor did they compare allNECcontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyIn our study we regarded tumors containing NECcomponents as a whole and found that the OS of thesetumors was poorer than that of adenocarcinoma in thesurgical group In addition we also found that the OS ofmixed tumors with less than between and more than NEC components or pure NEC wasworse than that of gastric adenocarcinoma Analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of Syn and CgAbetween different NEC content groups only the positiverate of CD56 was found to be higher in the pure NECgroup than that in the GHMiNEN group Therole of CD56 in the diagnosis of NEC is still controversial However Syn and CgA are two wellrecognized 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC and gastric adenocarcinoma in the surgical group aOverall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparison between GHMiNEN andgastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastric adenocarcinoma d Overall survivalcomparison between pure NEC alone and gastric adenocarcinomamarkers Therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing NEC componentsStudies on the molecular mechanism of pathogenesisshow that NEC components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity LOH and mutations at multiple loci and key oncogenes such as TP53 APC and RBgenes All these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] Moreoverin the WHO definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid [] no minimumpercentage for either ingredient is established Thereforewe believe that mixed tumors containing NEC components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma We propose considering mixed tumorscontaining NEC components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiesPreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] In a retrospective study involving patientsMa et alfound that neoadjuvant chemotherapy improves the survival of patients with NEC and HMiNENof the stomach [] Van der Veen reported that 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC components and gastric adenocarcinoma in theneoadjuvant group a Overall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparisonbetween GHMiNEN and gastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastricadenocarcinoma d Overall survival comparison between pure NEC and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing NEC components [] However because only eight patients wereincluded in the neoadjuvant group Vans results arequestionable In our study among patients receivingneoadjuvanttherapy no significant difference in OSbetween mixed tumor and gastric adenocarcinoma wasobserved Even for the pure NEC group with the highestNEC contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsAlthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research In addition we proposed treatingall gastric neoplasms containing NEC components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms In the futurewe will conduct more genomics studies to confirmour ideas This study also has its limitations Due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens As a retrospective study despite our performing PSM in advance selection biascannot be completely avoided In addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cChen BMC Cancer Page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with NEC componentless than so we could only treat all mixed tumors withNEC component as a wholeConclusionsOur study demonstrated that gastric neoplasms withNEC components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies Therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients Neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12885020072817Additional file Table S1 Comparison of clinicopathologicalcharacteristics before and after PSM of 30GHMiNEN patients insurgical group Table S2 Comparison of clinicopathologicalcharacteristics before and after PSM of GHMiNEN patients in surgicalgroup Table S3 Comparison of clinicopathological characteristics beforeand after PSM of 70GHMiNEN plus pure NEC patients in surgicalgroup Table S4 Comparison of clinicopathological characteristics beforeand after PSM of pure NEC patients in surgical group Table S5 Comparison of clinicopathological characteristics before and after PSM of 30GHMiNEN patients in neoadjuvant group Table S6 Comparison ofclinicopathological characteristics before and after PSM of GHMiNEN patients in neoadjuvant group Table S7 Comparison of clinicopathologicalcharacteristics before and after PSM of 70GHMiNEN plus pure NECpatients in neoadjuvant group Table S8 Comparison of clinicopathological characteristics before and after PSM of pure NEC patients in neoadjuvant groupAbbreviationsAJCC American Joint Committee on cancer CT Computed tomography GHMiNEN Gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm GNEC Gastric neuroendocrine carcinoma HPF High power fieldMiNEN Mixed neuroendocrinenonneuroendocrine neoplasmNEC Neuroendocrine carcinoma NEN Neuroendocrine neoplasmNET Neuroendocrine tumor MRI Magnetic resonance imaging OS Overallsurvival PETCT Positron emission tomography computed tomographyPFS Progressionfree survival PSM Pr sity score matching WHO WorldHealth anizationAcknowledgmentsThanks to Dr Zhongwu Li of the Department of Pathology Peking UniversityCancer Hospital and his colleagues for their assistance in pathologicaldiagnosis and review Thanks to all colleagues in the Department ofGastrointestinal Surgery of Peking University Cancer Hospital and Dr JiangHong from the Statistics Department for their assistance in this studyAuthors contributionsAll authors contributed to the study conception and design JC performeddata collection and wrote the manuscript AW wrote and t revised hemanuscript KJ helped with statistical analysis and prepared the illustrationsZB edited the manuscript JJ conceived the study and reviewed themanuscript All authors read and approved the final manuscriptFundingThis work was supported by the National Science Foundation for YoungScientists of China Beijing Youth Talent Plan QML20191101 andScience Foundation of Peking University Cancer Hospital Thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the Ethics Committee of Peking UniversityCancer Hospital and the patients written consent was also obtained Writteninformed consent for publication was obtained and stored in PekingUniversity Cancer HospitalConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Matsubayashi H Takagaki S Otsubo T Iiri T Kobayashi Y Yokota T et alAdvanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy Gastric Cancer Park JY Ryu MH Park YS Park HJ Ryoo BY Kim MG Prognosticsignificance of neuroendocrine components in gastric carcinomas Eur JCancer La Rosa S Inzani F Vanoli A Klersy C Dainese L Rindi G Histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms Hum Pathol Ishida M Sekine S Fukagawa T Ohashi M Morita S Taniguchi H et alNeuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis Am J Surg PatholRayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc | Thyroid_Cancer |
Identification and treatment of obstructive sleep apnea by a primarycare team with a subset focus on chronic painmanagement within the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existBackgroundPatients diagnosed with obstructive sleep apnea OSA who also consume prescription opioids have a greater likelihood of morbidity and mortality This study evaluated whether a primary care team focused on chronic pain care management could use a validatedquestionnaire STOPBang and motivational followup to increase identification and treatment of OSAMethodsThis study was a retrospective dual arm prepost controlled study Participants of thisstudy included the complete chronic pain management sub group treated by this primarycare team Participants were � years old and prescribed daily opioids for treatment ofchronic pain All participants had a multifaceted individualized educational meeting thatincluded completing a STOPBang questionnaire Participants who received a score �three were advised to follow up with their primary care physician Participants were seenquarterly throughout the studyResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of months 18month average postintervention vs CPAPuse in the control group months of observation both groupswere chronic opioid users with OSA This was a relative improvement p Asecondary outcome was that of nonprior CPAP users obtained CPAP post intervention a prepost improvement p x2 with degree of freedom Alsoparticipants who were likely to have OSA STOPBang score � or had a positive polysomnography AHI with comorbidities compared to those unlikely to have OSA STOPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamBang score or had a negative polysomnography AHI in this study were more likelyto be male have a higher BMI have hypertension have cardiovascular disease andorhave diabetes all typesConclusionTeam based care management for participants taking prescription opioids where STOPBang questionnaires were completed were associated with an increase in the identificationand treatment of OSAIntroductionStudy rationaleThe objective of this study was to evaluate whether a teambased care management intervention would increase identification and treatment of obstructive sleep apnea in participants taking medication for chronic painCurrent knowledgeObstructive sleep apnea OSA is a chronic sleep disorder characterized by episodes of apneasand hypopneas or the complete or partial collapse of the upper airway [] A diagnosis ismade when a patient has or more of these events in an hour or five of these events if otherpredicting symptoms for OSA are present [] Approximately million American adults havethis sleep disorder Patients with moderate to severe OSA are often treated with a continuouspositive airway pressure device or CPAP Treatment can reduce the risks of hypertension coronary artery disease heart failure arrhythmias sudden cardiac death and stroke [] A validated questionnaire STOPBang score can be used as a screening tool to identify patients atrisk for OSA A STOPBang sleep questionnaire is commonly distributed to patients whoshow signs of having potential risk factors for OSA STOP questions snoring tirednessobserved apnea and high blood pressure and Bang BMI kgm2 Age years Neckcircumference cm and Gender male [ ] A score of three or higher on STOPBangplaces patients at higher risk of OSA requiring further evaluation which includes an overnightpolysomnogram or sleep study to confirm diagnosis []Prescription opioid use for pain management is still a common practice in primary care settings though the rates of overdose due to these drugs in the United States are at an unprecedented high with deaths in [] Patients using opiates for chronic painmanagement have also been found to have a greater likelihood of developing central sleepapnea which worsens the severity of OSA and ataxic breathing [] A relationship existsbetween pain and sleep disruption with over of patients who have chronic pain havingreported difficulty sleeping which increases hyperalgesia [ ] An analgesic effect may bepresent where pain creates sleep disorders and a shortage of sleep increases pain [ ]Patients with OSA who are then prescribed opioids have an increased risk of opioidinducedrespiratory depression OIRD [] Despite the potential morbidity of the combination ofOSA central sleep apnea and Opioid use studies have shown that those that have this combination only remain on definitive treatment for their OSA CPAP of the time [] It istherefore of the utmost importance that patients who chronically use opioids are evaluated forOSA and those diagnosed adhere to CPAPPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIn order to decrease mortality it is necessary to educate patients on the risks and healthconcerns related to taking opioids Brief interventions can influence behavior of patients whoare at high risk for abuse of a substance [] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] These individualized information gathering motivational meetings allow patients to experience empathy as their medical needs are met [ ] Individualized nurseled management educationis a tool that can be used as a brief intervention within a teambased approach [] Instructionand guidance can be coupled with an assessment of a patients potential risk factors for comorbidities such as OSA using a STOPBang questionnaireMaterials methodsDesignThis study was a retrospective dual arm prepost controlled study to evaluate whether anintervention STOPBang questionnaire education and motivational followup wouldimprove OSA diagnosis and treatment compliance The first investigation arm compared thecontrol group daily opioid use with OSA to the part of the intervention group with OSA onCPAP or at risk for OSA STOPBang � This arms purpose was to determine whether theintervention increased treatment compliance with CPAP The second investigation armcompared the study group to itself prepost intervention This arm evaluated whether applyingthe intervention increased diagnosis and treatment of OSA The single independent variablein this study was a participants admission to the chronic pain management subgroup withthe administration of a STOP Bang questionnaire and the dependent variable was CPAP useInclusion criteria consisted of ongoing participation in primary care pain management subgroup daily opioid use and an age � This study occurred between October and January which included monthsfor exposure and then at least months of follow up One of Intermountain Healthcares outpatient facilities in Utah served as the site for this study The total number of patients duringthe duration of the study who entered into the pain management sub group � years oldand were taking a daily dose of opioids was Out of the identified participants participants remained in the clinic during the entirety of this study Fig Ethics approvalIRB Number for this study was obtained from the Institutional Review Board ofIntermountain Healthcare Consent was waived for this retrospective studyControlsA Medline and Pubmed basic search from was used to identify a historical yet contemporary United States based control group that had Obstructive Sleep Apnea and met theinclusion criteria of being � years old and were taking a daily dose of opioids The controlthat was chosen was comparable to this studys intervention group with regard to Race AgeGender Morphine Equivalent Daily Dose and Comorbidities Also the control used camefrom a study that lasted a similar duration []SubjectsEach participant received an information gathering and educational session with a registerednurse in the primary care facility at the time of their admittance into the chronic pain management program between During this session a full pain history was obtained fromparticipants All participants in the study underwent STOPBang screening All participantswho were at high risk for OSA STOPBang � were referred back to their primary carePLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamFig Participant retention101371journalpone0237359g001physician Participants and physicians made joint decisions during a followup appointmentwhether to pursue polysomnography Reasons why participants did not complete polysomnography or attain treatment for OSA are that the participants refused workup there was ashared decision due to participant frailty had cancer end stage or were noncompliant toCPAP Fig Fig participants at high risk for sleep apnea STOPBang score � who did not undergo diagnostic evaluation orcomplete treatment101371journalpone0237359g002PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamEach participant received initial inperson education with the same registered nurse regarding nonpharmacological treatment of pain selfhelp groups risks and benefits of treatment ofpain management compliance with treatment of pain and its comorbidities and informationabout Naloxone an opioid reversal agent This education was provided in a hour sessionusing the FRAMES Feedback Responsibility Advise Menu for Change Empathy andEnhancing Self Efficacy motivational method [] A chronic pain management agreementwas executed Additional information obtained during this session included a Current OpioidMisuse Measure and a STOPBang questionnaire All participants who received a score greaterthan or equal to three on the STOPBang were told to follow up with their primary care physician All primary care providers were made aware they would receive electronic data onSTOPBang questionnaires and participants with scores � would make a follow up appointment with them Upon completion of this session information was recorded electronically andtransmitted to the primary care physician on record Each participant also had quarterly inperson followup appointments with their primary care physician In addition quarterly inperson or phone visits with the pain management nurse were continued Physician visits varied in length and scope Individualized nursing visits that continued to use the FRAMESmethod and lasted for a duration of minutes were focused on remaining compliant with allongoing therapies The nurse would document the interaction and any concerns about compliance with any therapy in the patients EMR Electronic Medical Record at the conclusionof the visits The information was based on the subjective input received from the participantQuarterly followup remained consistent throughout the study Attending all followupappointments both with the primary care physician and the pain management nurse was aprerequisite to remain in the pain management group Remaining in the pain managementwas group was the only avenue to receive controlled pain medication at this facilityData collectionThe baseline biometric parameters of Race Gender BMI Age Tobacco Use Alcohol Use andMorphine Equivalence Daily Dosage MEDD were extracted from each participants electronic medical record EMR Collected data also included information on Chronic DiseaseHypertension Cardiovascular Disease Pulmonary Disease Thyroid Disease Diabetes Gastroesophageal Reflux Disease and Chronic Kidney Disease and specific medication typesBenzodiazepines and Pregabalin STOPBang scores were gathered during the initial chronicpain management encounter when participants joined the study Subjective data on CPAPadherence was found in the EMR documentation of the pain management nurse and primarycare physician follow up reports postinclusion in the studyStatistical methodsMeans and standard deviations were calculated for demographic variables The DAgostinoPearson test was used to confirm normality [] Unequal variance ttesting was used in the statistical analysis of both arms of this study to compare Age BMI and Morphine EquivalentDaily Dosing Two proportion Ztesting was used to compare the control group daily opioiduse with OSA to the participants in the intervention group with OSA on CPAP or at highrisk for OSA STOPBang � with regards to CPAP use Race Gender Diabetes Cardiovascular Disease and Hypertension Two proportion Ztesting was also used to compare the participants not likely to have OSA STOPBang or had a negative polysomnography to thosewith OSA on CPAP or at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP A x contingency table using McNemars test with correction for continuity was used to compare the participants CPAP use prepost intervention []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamIRB approvalIRB Approval was granted from Intermountain Healthcare IRB IRB Number ResultsThe primary outcome of this study was that of participants with likely OSA were usingCPAP for a minimum of months range of months 18month average postintervention vs CPAP use in the control group months of observation which were chronic opioid users with OSA This was a relative improvement p A secondary outcomewas that of nonprior CPAP users obtained CPAP post intervention a prepostimprovement p x2 with degree of freedom Fig This resulted in ofintervention participants being treated with CPAP Comparing the average number of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the control dataset resulted in significant differences for BMI and Male Gender Otherdemographics between these two groups Age p MEDD p Race Caucasianp Hypertension p Cardiovascular Disease p and Diabetes all formsp showed no statistically significant differences between groups Comparing the averagenumber of intervention participants with OSA on CPAP or at risk for OSA STOPBang � to the average number of intervention participants not likely to have OSA STOPBang orhad a negative polysomnography resulted in the following statistically significant differencesBMI p Male Gender p00001 Hypertension p Cardiovascular Diseasep and Diabetes all forms p Other demographics between these two groupsAge p MEDD p Race Caucasian p Benzodiazepine Use p Pregabalin Use p and Active Chronic Disease States Gastroesophageal Reflux p Chronic Kidney Disease Thyroid Disease p and Pulmonary Diseasep showed no statistically significant differences between groups Table DiscussionMain findingsCurrent high death rates compared to healthy subjects of OSA and chronic pain managementwith daily opioid use warrant efforts to improve care [] This studys findings can be anFig CPAP use before and after intervention101371journalpone0237359g003PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamTable Comparison of clinical characteristics S1 AppendixDemographicsAverage Number of Participants withOSA on CPAP or at risk for OSASTOPBang � Control DailyOpioid Use withOSAAgeBMI ���Morphine Equivalent mgdat Conclusion of Study � months Pvalue Average Number of Participants Not Likelyto Have OSA STOPBang or NegPolysomnography PrePostPvalueDemographicsAverage Percentage of Participantswith OSA on CPAP or at risk for OSAControl DailyOpioid Use withPvalueAverage Percentage of Participants NotLikely to Have OSA STOPBang orPrePostPValueSTOPBang � OSANeg PolysomnographyRace CaucasianGender Male��Diabetes���Cardiovascular Disease��Hypertension�Gastroesophageal Reflux DiseaseThyroid Disease All FormsPulmonary Disease All Varieties Active CancerChronic Kidney DiseaseConcurrent Benzodiazepine UseConcurrent Pregabalin Use� p value of �� p value of ��� p value of 101371journalpone0237359t001NANANANANANANA NANANANANANANAimportant step towards improving mortality and morbidity when these two diseases are combined The results suggested that using a STOPBang questionnaire accompanied with motivational education initially as well as in followup improved diagnosis and treatment of OSAThese findings suggest that teambased care can positively impact the outcome of patientswith OSA when they are concurrently on chronic daily opioids and CPAP These findings warrant additional research Diagnosed sleep apnea participants who were using CPAP had a significantly higher rate of being male having hypertension having cardiovascular diseasehaving diabetes all types and having a higher BMI The fact that of participants at riskfor sleep apnea STOPBang � did not receive a full evaluation or end up on definitive treatment highlights the need for continued effort in this fieldLimitationsThe data found in this study regarding CPAP adherence was subject to limitations as wellSome participants that joined this teambased care management approach to treat chronicpain had already been prescribed CPAP and their adherence to the treatment was included inthis studies data set possibly overestimating the conclusions Adherence to CPAP in this studywas based on subjective treatment reconciliation at followup appointments with physiciansand nurses and since many participants received their sleep treatment from outside sourcesCPAP adherence data other than confirmation from the patient was not available If a participant indicated that they had not been using their CPAP � of the time between anyPLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamfollowup appointments after the initiation of treatment they were considered to be nonadherent This type of data collection is subject to significant recall bias and could potentiallyinflate the percentage of participant adherenceThis preliminary research was done in the format of a prepost dual arm retrospectivestudy This type of study has inherent and significant limitations First due to the nature of theintervention team members nurses and physicians were not blinded and continued to evaluate participants throughout the course of this study and could have recommended evaluationand therapy of sleep apnea for alternative reasons This study could have been affected by aherd mentality since the primary care physicians worked together in one group and theircomposite style could have increased or decreased the identification and treatment of sleepapnea These limitations could be mitigated by performing a large prospective multicenterstudyThis study is also limited due to the nature of using a historical control [] Although the control used mimicked the experimental group in most ways daily opioid use MEDD race age andpercentages of diabetes hypertension and cardiovascular disease it varied in both BMI controlgroup had a higher average BMI and gender control group had a greater number of male participants It has been previously demonstrated that a higher BMI favors adherence to CPAP [] This does not detract from the finding that the intervention group in this study which had alower BMI had a higher adherence to CPAP Gender has also been evaluated in previous CPAPcompliance studies Two studies have shown no difference between genders whereas a differentstudy showed an increase in male noncompliance and another where there was an increase inmale compliance [] As there is no definitive determination from these studies it is possiblethat the variance between the intervention and control groups were due to the gender differenceIt would seem from the above studies that there was no strong trend resulting from gender making gender unlikely to be the cause of the preponderance of the varianceThere was a loss of followup with primary care physicians after the administration of theSTOPBang questionnaire in this study Fig Patients may have fotten to or chosen notto schedule a followup appointment with their primary care physician after being determinedhigh risk for sleep apnea The participant received education on six other aspects of opioidmanaged pain during the one hour informational meeting Another limitation was that thedata from the intervention session was transmitted enblock to the primary care physicianelectronically These limitations could have been improved by having the nurse set up a specific appointment to consider sleep disorders in the highrisk group and to either verbally orelectronically deliver this information in an isolated approachComparisonsThis study reaffirmed previous studies that showed STOPBang was a tool that could identifyOSA [ ] Other similarities to previous investigations were found In those studies wheredaily opioid dosing for pain management was present of patients were found to sufferfrom OSA and of patients were found to be compliant to CPAP [ ] The data rangefor previous studies in regard to OSA incidence is consistent with this study in which ofparticipants were either diagnosed with OSA on CPAP or were at high risk for OSA STOPBang � not completing polysomnography or not compliant with CPAP This study was significantly higher with regards to the percentage of remaining on CPAP suggesting thatdiligence in repetitive diagnosis attempts and continued follow with motivational techniquesmay improve care in this type of patient Also this study like others suggested p thathypertension cardiovascular disease diabetes male gender and higher BMI are more common in patients with OSA []PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management teamConclusionsLeft untreated sleep apnea has a high rate of morbidity and mortality The use of prescribedopioids as a form of chronic pain management in a primary care setting can increase apatients likelihood of having sleep apnea A teambased intervention that included theadministration of a STOPBang questionnaire was associated with an increased diagnosis ofOSA prepost improvement level It was also associated with an increased adherenceto CPAP relative improvement The primary reasons CPAP treatment was not receivedincluded refusal of evaluation nonadherence to CPAP or shared physicianpatient decisionbased on agefrailtySupporting informationS1 Appendix Raw study data setXLSXAuthor ContributionsConceptualization Kathleen Whittington Leigh Simpson Dixie HarrisData curation Kathleen Whittington Michael ClayFormal analysis Kathleen Whittington Michael Clay Dixie HarrisInvestigation Joanna TierneyProject administration Joanna TierneySupervision Dixie HarrisWriting original draft Kathleen WhittingtonWriting review editing Kathleen Whittington Leigh Simpson Michael Clay JoannaTierney Dixie HarrisReferencesFarney Robert J The STOPBang equivalent model and prediction of severity of obstructivesleep apnea relation to polysomnographic measurements of the apneahypopnea index Journal ofclinical sleep medicine JCSM official publication of the American Academy of Sleep Medicine vol 65B 105664JCSM1306 PMID Manne Mahesh B Obstructive Sleep Apnea Who Should Be Tested and How ClevelandClinic Journal of Medicine Aug wwwmdedgecomccjmarticle105363cardiologyobstructivesleepapneawhoshouldbetestedandhow Abrishami A Khajehdehi A Chung F A systematic review of screening questionnaires for obstructivesleep apnea Can J Anaesth 101007s126300109280x PMID Vasu TS Doghramji K Cavallazzi R Obstructive sleep apnea syndrome and postoperative complications clinical use of the STOPBANG questionnaire Arch Otolaryngol Head Neck Surg 101001archoto20101020 PMID Kumar S Mcelligott D Goyal A Baugh M Ionita RN Risk of Obstructive Sleep Apnea OSA in Hospitalized Patients Chest 1384779A Ong TH Raudha S FookChong S Lew N Hsu AA Aal H Simplifying STOPBANG use of a simplequestionnaire to screen for OSA in an Asian population Sleep Breath Drug Overdose Deaths Centers for Disease Control and Prevention Centers for Disease Control andPrevention June wwwcdcgovdrugoverdosedatastatedeathshtml Substance Abuse and Mental Health Services Administration Drug Abuse Warning Network National Estimates of DrugRelated Emergency Department Visits HHS Publication No SMA PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team DAWN Series D39 Rockville MD Substance Abuse and Mental Health Services Administration Schneiderhan JMD Clauw DMD Schwenk TMD Primary care of patients with chronic pain J Am MedAssoc Walker James M Chronic Opioid Use Is a Risk Factor for the Development of Central SleepApnea and Ataxic Breathing Journal of Clinical Sleep Medicine JCSM Official Publication of theAmerican Academy of Sleep Medicine American Academy of Sleep Medicine Aug wwwncbinlmnihgovpmcarticlesPMC1978331 PMID Webster LR Choi Y Desai H Webster L Grant BJ Sleepdisordered breathing and chronic opioid therapy Pain Med 101111j15264637200700343x PMID Onen SH Onen F Courpron P Dubray C How pain and analgesics disturb sleep Clin J Pain 10109701ajp000012975731856f7 PMID Cozowicz Crispiana Opioids for Acute Pain Management in Patients with Obstructive SleepApnea Anesthesia Analgesia vol no pp 101213aneJaoude Philippe Lal Ashima Vermont Leaj Porhomayon Jahan Ali A ElSolh Pain Intensity and Opioid Utilization in Response to CPAP Therapy in Veterans with Obstructive Sleep Apnea on Chronic Opioid Treatment Journal of Clinical Sleep Medicine Barbor TF HigginsBiddle JC World Health anization Dept of Mental Health and SubstanceDependence Brief Intervention of Hazardous and Harmful Drinking A Manual for Use in Primary CareGeneva World Health anization Group WBIS A crossnational trial of brief interventions with heavy drinkers Am J Public Health 102105ajph867948 PMID Miller WR Rollinick S Motivational Interviewing Preparing People to Change Addictive Behavior NewYork and London Guilford Press Saunders B Wilkinson C Phillips M The impact of a brief motivational intervention with opiate usersattending a methadone program Addiction 101046j13600443199590341510x PMID Hester RK Miller WR Handbook of Alcoholism Treatment Approaches Vol Boston MA Allyn andBacon DAgostino R Belanger A A Suggestion for Using Powerful and Informative Tests of Normality The American Statistician 1023072684359 Edwards A Note on the Correction for Continuity in testing the significance of differencebetween populations Psychometrika B Young T Finn L Sleep disordered breathing and mortality eighteenyear followup of the Wisconsin sleep cohort Sleep Aug PMID Marshall NS Wong KK Sleep apnea as an independent risk factor for allcause mortality the Busselton Health Study Sleep Aug PMID Liang Y Turner B Assesing Risk for Drug Overdose in a National Cohort Role for Both Daily and TotalOpioid Dose J Pain Apr 101016jjpain201411007 PMID Heejin Kim MinSukim Treatment Outcomes and Compliance According to Obesity in Patientswith OSA European Archives of OtoRhinoLaryngology PelletierFleury N Rakotonanahary D Fleury B The age and other factors in the evaluation of compliance with nasal continuous positive airway pressure for obstructive sleep apnea syndrome A Coxsproportional hazard analysis Sleep Med 101016s1389945700 PMID Anttalainen U Saaresranta T Kalleinen N Aittokllio J Vahlbergy T Polo O CPAP adherence and partial upper airway obstruction during sleep Sleep Breath 101007s1132500701025 PMID Sin DD Mayers I Man GC Pawluk L Longterm compliance rates to continuous positive airway pressure in obstructive sleep apnea a populationbased study Chest 101378chest1212430 PMID Ye Lichun Pien Grace W Ratcliffe Sara J Weaver Terri E Gender Differences in Obstructive SleepApnea and Treatment Response to Continuous Positive Airway Pressure J Clin Sleep Med PMID PLOS ONE 101371journalpone0237359 August PLOS ONE 0cIdentification and treatment of obstructive sleep apnea by a primary care chronic pain management team Mador M Henderson J Effect of Opioids on Sleep and Breathing in Chronic Pain Patients Journal ofClinical Sleep Medicine Aug 105664jcsm3952 PMIDPLOS ONE 101371journalpone0237359 August PLOS ONE 0c' | Thyroid_Cancer |
prevalence of pathogenic variants in DnA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of earlyonset renal cancerTiffiney a0R a0Hartman12 a0Elena a0V a0Demidova345 a0Randy a0W a0Lesh6 a0Lily a0Hoang7 a0Marcy Richardson7 a0Andrea a0Forman8 a0Lisa a0Kessler1 a0Virginia a0Speare7 a0Erica a0A a0Golemis4 a0Michael a0J a0Hall38 a0Mary a0B a0Daly38 Sanjeevani Arora3Pathogenic a0variants a0PVs a0in a0multiple a0genes a0are a0known a0to a0increase a0the a0risk a0of a0earlyonset a0renal a0cancer a0eoRC a0However a0many a0eoRC a0patients a0lack a0PVs a0in a0RCspecific a0genes a0thus a0their a0genetic a0risk a0remains a0undefined a0Here a0we a0determine a0if a0PVs a0in a0DNA a0damage a0response a0and a0repair a0DDRR a0genes a0are a0enriched a0in a0eoRC a0patients a0undergoing a0cancer a0risk a0assessment a0Retrospective a0review a0of a0deidentified a0results a0from a0 a0eoRC a0patients a0undergoing a0testing a0with a0a a0multigene a0panel a0for a0a a0variety a0of a0indications a0by a0Ambry a0Genetics a0PVs a0in a0cancerrisk a0genes a0were a0identified a0in a0 a0of a0patientswith a0 a0in a0RCspecific a0and a0 a0in a0DDRR a0genes a0DDRR a0gene a0PVs a0were a0most a0commonly a0identified a0in a0CHEK2 a0BRCA1 BRCA2 and ATM a0Among a0the a0 a0of a0patients a0with a0a a0BRCA1 or BRCA2 a0PV a0 a0 a0reported a0a a0personal a0history a0of a0hereditary a0breast a0or a0ovarianassociated a0cancer a0No a0association a0between a0age a0of a0RC a0diagnosis a0and a0prevalence a0of a0PVs a0in a0RCspecific a0or a0DDRR a0genes a0was a0observed a0Additionally a0 a0patients a0reported a0at a0least a0one a0additional a0cancer a0breast a0cancer a0being a0the a0most a0common a0 a0of a0females a0 a0of a0males a0Multigene a0testing a0including a0DDRR a0genes a0may a0provide a0a a0more a0comprehensive a0risk a0assessment a0in a0eoRC a0patients a0Further a0validation a0is a0needed a0to a0characterize a0the a0association a0with a0eoRCRenal cancer RC often develops with no signs or symptoms and is referred to as the silent disease While factors including smoking environment obesity and race have been linked to increased risk of RC inherited factors are the most wellvalidated source of increased risk2 Hereditary RC syndromes typically associated with earlyonset disease and a clinically significant family history of cancer result from germline pathogenic variants PV in highpenetrance RCspecific genes including VHL MET FLCN TSC1 TSC2 FH SDH PTEN and BAP15 A previous report of an earlyonset RC eoRC cohort screened with an RCspecific panel found of individuals had a PV in an RCspecific gene7 However for most eoRC patients a PV in an RCspecific gene is not identified leaving many eoRC genetically undefined Thus there is a need to identify additional genes related to eoRC risk Currently there are no National Comprehensive Cancer Network NCCN guidelines for detection prevention or risk reduction in individuals who present with an eoRC but lack a PV in a defined RCspecific gene81Arcadia University Glenside PA USA 2Cancer Biology Program Fox Chase Cancer Center Philadelphia PA USA 3Cancer Prevention and Control Program Fox Chase Cancer Center Cottman Avenue Philadelphia PA USA 4Molecular Therapeutics Program Fox Chase Cancer Center Philadelphia PA USA 5Kazan Federal University Kazan Russian Federation 6Geisinger Commonwealth School of Medicine Scranton PA USA 7Ambry Genetics Konica Minolta Aliso Viejo CA USA 8Department of Clinical Genetics Fox Chase Cancer Center Philadelphia PA USA email SanjeevaniArorafccceduScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cDNA damage response and repair genes DDRR play an important role in maintaining genome integrity and when mutated in the germline can increase cancer risk for several types of cancers including breast colorectal ovarian and others9 Although PVs in DDRR genes are associated with increased risk of a variety of cancer types they are not typically considered risk factors for RC However germline PVs in some DDRR genes have been observed in RC including PVs in the DNA mismatch repair Lynch syndrome genes MSH2 and MLH1 in renal urothelial carcinoma and PVs in CHEK2 in advanced renal cell carcinoma10 To address the hypothesis that PVs in additional DDRR genes may contribute to the missing heritability of eoRC we analyzed germline sequencing data from a cohort of individuals with RCMaterials and methodsAmbry a0Genetics a0eoRC a0study a0cohort a0and a0variant a0determination a0 Deidentified data were requested from RC patients that were tested by Ambry Genetics Konica Minolta Aliso Viejo California using germline cancer testing panels Ambry samples were selected for patients with RC and deidentified data was obtained for all RC patients tested with multigene cancer panels n a0years at diagnosis specimens collected between July December All genetic test results from germline testing of individuals diagnosed with RC at during this time period were used in this studyThere is currently no standard definition specifying the age when RC is considered earlyonset Different models have been used to determine a specific age as a trigger for germline testing in patients with RC who lack family history of RC including ages or a0years For this study we selected individuals a0years or younger as the cutoff for our cohort which is substantially below the median age of RC diagnosis of a0years in the general population as reported in SEER2223 but considerably older than other suggested cutoffs We did so because the main hypothesis of the study was that PVs in DDRR genes might be responsible for increased risk of RC Variants in multiple DDRR genes are associated with earlyonset colorectal cancer2425 which typically manifests in patients at a0years or younger We considered that PVs in DDRR genes were most likely to impact repair of DNA damage induced during cell replication leading to genetic instability and cancer given renal cells turn over much less frequently than colon cells we hypothesized that it may take longer for cancers associated with PVs in DDRR genes to manifest in RC causing us to select a cutoff of a0years old for assessmentDeidentified data included family history of cancer genetic test results personal history of cancers apart from RC presence of multifocal tumors and RCsubtypestage The RC patients had been tested with CancerNext versions and CancerNextExpanded versions and Table a0S1 Deidentified patient information was analyzed for genetic test results and personal and family medical histories Classification of variants by Ambry Genetics is based on ACMG recommendations for standards for interpretation and reporting of sequence variations These variants are also regularly deposited in ClinVar by Ambry Genetics Variant classification was updated through March for all data Gene variants were classified as pathogenic variant PVsee below for criteria variant of uncertain significance VUS or inconclusive or negativeindeterminate Ambry Genetics follows strict criteria when classifying variants as PV Variant Likely Pathogenic VLP VUS Variant Likely Benign VLB and Benign for details see wwwambry gencomclini cianourscien tific excel lence varia ntclass ifica tion Variants reported as PV and VLPs were grouped as PVs All test results were used for this study The analysis of VUS which currently lack clinical significance was beyond the scope of this study Given updating of test panels by Ambry Genetics not all patients were tested for all genes Individuals were provided different versions of the panel over the course of the study see below and also see Table a0S1Any deidentified personal or family history information including sex ethnicityrace age of cancer diagnosis tumor histology history of additional personal cancer and history of family cancer and types was reported first as summarized data and later as deidentified individual case reports For analysis comparing outcomes for RCspecific genes versus genes not typically associated with RC we focused our statistical comparison on only those individuals who had CancerNext Expanded panel version testing which analyzes all genes including the RCspecific genes individuals who had the CancerNext Expanded version test were used for this statistical comparison For additional statistical test comparisons that analyzed the correlations between specific genes and categories such as tumor pathology or age any individual who had been tested for that specific gene was includedThe Western IRB issued a regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus the requirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB provided study oversight and approval protocol number Ambry Genetics provided deidentified patient medical and family history where available and genetic results for the patients All methods were performed in accordance with the relevant guidelines and regulation of the approved studyGenetic a0analysis a0with a0Ambry a0CancerNext a0and a0CancerNext a0Expanded a0panels a0Individuals were provided different versions of the panel by their healthcare provider see Table a0S1 The number of genes in the panels ranged from the smallest CancerNext panel Version which include genes APC ATM BARD1 BRIP1 BMPR1A CDH1 CHEK2 EPCAM MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C SMAD4 STK11 TP53 to the largest CancerNext Expanded Version panel which contained genes APC ATM BAP1 BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM FH FLCN GREM1 MAX MEN1 MET MITF MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE PTEN RAD50 RAD51C RAD51D RET SDHA SDHAF2 SDHB SDHC SDHD SMAD4 SMARCA4 STK11 TMEM127 TP53 TSC1 TSC2 VHL The DDRRs identified in germline testing of this cohort are bolded26Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAmbry Genetics sequenced genomic DNA that was obtained from patient blood or saliva samples DNA was evaluated by next generation sequencing NGS of all coding sequences and ± bases into the ² and ² ends of flanking introns and untranslated regions In addition sequencing of the promoter region was performed for the following genes PTEN c to c MLH1 c to c and MSH2 c to c Additional Sanger sequencing was performed for any regions missing or with insufficient depth of coverage for reliable heterozygous variant detection and on potentially homozygous variants variants in regions with complicated pseudogene interference and when variant calls did not meet allele frequency quality thresholds Additional details on specific testing methods are available at wwwambry gencomclini ciangenet ictesti ng28oncol ogycance rnext expan dedControl a0population a0in a0ExAc a0and a0gnomAD a0 To compare the frequency of DDRR gene PVs found in the study to that in the general population our results were compared to the Exome Aggregation Consortium ExAc dataset of largely unrelated whole exome sequencing results and to the Genome Aggregation database gnomAD dataset consisting of exomes and genomes2728 These datasets are the most commonly used genomic data at the populationlevelClinVar a0analysis a0 ClinVar wwwncbinlmnihgovclinv ar a database of medically relevant gene variants was used to investigate all PVs in this study retrieved on February PVs that were not reported in ClinVar were noted as not reported Associated conditions for each PV were categorized into hereditary cancer predisposing syndromes conditions related to renal cancer and any other conditions To further elucidate any PVs related to renal cancer the search term renal cancer was queried and the results were noted as associated with ClinVar search term Renal CancerStatistical a0 analysis a0 To identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis genes were combined into pathwaysgroups of interest histologys were grouped and cancer types were grouped Each individual was categorized as having a variant in one of the genes within the group or no variant in the group Gene categories were used for comparison of RC diagnosis with a DDRR or an RCspecific geneWe also tested the hypothesis that different gene groups are associated with age at RC diagnosis We used the median age of RC diagnosis in the study cohort a0years and studied PVs in patients a0years or ¥ a0years of age To test the association between the presence of PVs and age of RC diagnosis twosided Fishers exact tests were used and a0pvalues were considered significant Odds ratios OR were calculated to determine the odds that an outcome had occurred given a particular variant compared to the odds of the outcome occurring in the absence of that variant in the population tested Finally we queried the SEER database for patients under a0years old with RC to compare the distribution of their clinical characteristics where available to those in our study cohort22Due to the evolving nature of the panels during the course of this study each version included a different total number of genes and analysis of each gene is based on the number of individuals whose test included that gene Table a0S1 Only data from individuals was considered for comparison of individuals with RCspecific genes compared to those with variants in genes not typically associated with RC as the other individuals did not have all genes analyzed For statistical comparisons analyzing correlations between specific genes with various characteristics all individuals who had been tested for that specific gene were includedTo identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis RCspecific genes other cancerassociated genes and DDRR genes were combined into groups and histologies were grouped The categories for comparison of PVs and patient characteristics are as follows Known RC genes BAP1 FH FLCN MEN1 MET MITF PTEN SDHA SDHAF2 SDHB SDHC SDHD TSC1 TSC2 and VHL versus genes not typically associated with RC APC ATM BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM GREM1 MAX MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D RET SMAD4 SMARCA4 STK11 TEMEM127 TP53 versus DDRR genes alone ATM BARD1 BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D Histology categories combined from the original categories Chromophobe Papillary renal Clear cell Wilms Renal cell likely clear cell but cannot be confirmed Unknown Mixed papillary [clear cell papillary type papillary renalchromophobe renal and sarcomatoidpapillaryclear cell] Mixed chromophobe [chromophobeoncocytoma chromophoberenal cell clear cellchromophobe and clear celloncocytomachromophobe] Oncocytoma Mixed oncocytoma [clear celloncocytoma oncocytomacollecting duct and renal celloncocytoma] and Others [included clear cellsarcomatoid collecting duct mixed epithelial and stromal mucinous tubular and spindle cell multilocular cystic renal neuroendocrine renal cellWilms renal cortical sarcomatoid transitional urothelial and urothelial transitional] Transitional urothelial urothelial and papillary transitional cases were not included in the analysis for counts of pathogenic variants Renal oncocytomas mixed epithelial and stromal tumors are considered benign tumors and were not included in the analysis for counts of pathogenic variants Study a0approval a0 The Western IRB issued a a0regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus a0the Scientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0crequirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB a0provided study oversight and approval protocol number Ambry Genetics a0provided deidentified results for the study All methods were performed in accordance with the relevant guidelines and regulation of the approved studyResultsPatient a0characteristics a0 We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context In the study cohort of cases were between a0years of age and median age of diagnosis was a0years As expected a higher percentage of RC cases were diagnosed between a0years of age as compared to patients diagnosed with RC in the general US population SEER versus Fig a01A The study cohort was female and male Fig a01B Table a0 versus female and male for the general US population prevalence of RC diagnosed Fig a01B Raceethnicities in study cohort were Caucasian African AmericanBlack Ashkenazi Jewish Hispanic other and unknown Table a0The tumor pathologies reported varied Fig a01C and Table a0 Clear cell constitutes of all RCs in SEER and was the most commonly reported histology in the eoRC cohort Renal cell not defined but likely to predominantly reflect clear cell was also common Fig a01C and Table a0 Papillary and chromophobe histology were each identified in of the individuals and respectively Other histologies were identified rarely but included Wilms tumor and oncocytoma For of patients the RC subtype was unknownHigh a0incidence a0of a0other a0cancers a0in a0study a0cohort a0 n of the cases in the study cohort reported at least one additional primary cancer Fig a01D Table a0 Table a0S2 Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database Fig a01D For femalespecific cancers of females also had breast cancer in comparison to the breast cancer rate in women in the general population SEER Fig a01D and Table a0S2 The rate of additional primary cancer in the study cohort is much higher than the rate of each cancer type observed in SEER cases with eoRC Fig a01E Finally patients out of reported a family history of cancer and of these patients specifically reported at least one family member with RC Table a0Multigene a0cancer a0panel a0testing a0identifies a0PVs a0in a0DDRR a0genes a0in a0the a0study a0cohort a0 The most common gene with PVs identified in the eoRC patients was the DDRR gene CHEK2 Fig a02A Table a0S3 and S4 consistent with a recent report by Carlo et a0al16 Of patients with CHEK2 PVs n had a common highly damaging variant c1100delC pThr367Metfs that is known to be associated with an increased risk for breast prostate colorectal and thyroid cancers Table a0S434After CHEK2 PVs were most frequently observed in the DDRR genes BRCA2 ATM and BRCA1 Table a0S3 We compared the overall frequency of PVs in CHEK2 BRCA1 BRCA2 and ATM to the control population in ExAc and gnomAD representing individuals sequenced for diseasespecific and population genetic studies2728 Overall PVs in each of these genes were more common in the study cohort versus the control populations Fig a02BC Table a0S5A An outlier was the moderate risk CHEK2 c470TC p I157T PV38 identified in individuals in the study cohort which was higher in the controls gnomAD OR CI ExAc OR CI We compared the prevalence of all PVs in DDRR genes presented in Table a0S4 from cases to controls from gnomAD23 We found 48fold enrichment of PVs in DDRR genes in the study cohort versus the controls in gnomAD vs respectively Table a0S5B each DDRR gene was corrected for number of patients assessedCancer risk with MUTYH DDRR gene has only been defined for individuals with homozygous or compound heterozygous PVs but not for heterozygous carriers39 We identified individuals with MUTYH PVs out of which were heterozygous carriers and only was compound heterozygous Only the individual with compound heterozygous MUTYH PVs was counted in the full study cohort n Table a0S3 and Fig a02A Similar to MUTYH cancer risk from the FH RCspecific gene c1431_1433dupAAA pK477DUP variant is currently considered to be pathogenic only in the compound heterozygous or homozygous state40 We identified RC patients who were heterozygous carriers of this specific FH variant Tables a0S3 and S4The overall gene variation rate in the full study cohort n is presented in Table a0S3 The full study cohort was not tested for all genes The largest panel was tested in the subcohort of cases and consisted of genes which included RCspecific genes and othercancer associated genes including DDRR genes Table a0S1 Here of cases had PVs PVs were identified in one or more of the genes not typically associated with RC in cases n Table a0S6 versus n with a PV in RCspecific genes Fig a02D Table a0S6 Of the genes not typically associated with RC were in DDRR genes n or n Among the patients patients were found to have PVs in two genes One patient had PVs in two DDRR genes BRCA1 and MUTYH het and the other patient in a RCspecific gene and a DDRR gene SDHB and MUTYH het Table a0S4 There was no MUTYH or FH compound heterozygous or homozygous PV in the subcohort of casesDDRR a0genes a0PVs a0are a0similarly a0enriched a0in a0patients a0diagnosed a0with a0eoRC a0alone a0or a0with a0eoRC a0and a0other a0cancers a0Individuals who were tested for all genes n could be further separated into two subcohorts those with eoRC as their only diagnosis n and those with eoRC and one or more additional types of cancer n To test the hypothesis that DDRR gene PVs might be Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAiega yb ssongad iCsesac AgenrnrWilmsersothal cellnrenrnwonknunramebohpomchroebod hmixepomchroar cellncocytocleodncocytomixeoamalnpillarypillary read pmixeapKey A C D E FSEER cohort n97805Ambry cohort n844Bsesac FemaleMaleSEER cohortAmbry cohortDtear recnac brainstabrectalolorecmiaekuleamonelamnariavoaticcrenapstateproamoarcsyroidthetrialuterinemodneEsesac number of primary cancers reportedFigure a0 Patient characteristics A Age range of individuals diagnosed with RC a0years in SEER cohort compared to the study cohort n of the remaining individuals in the study were diagnosed a0years were diagnosed at a0years and were excluded from the calculations as their age was reported as a wide range of years B Percentage of males and females diagnosed with RC a0years in SEER compared to the study cohort n C The percentages of reported RC histology up to age a0years in the SEER data n compared to the study cohort n not all histological subtypes reported in SEER were reported in the study cohort D The percentage of cancer incidence at a0years in the general SEER population versus the study cohort The SEER data reflect individuals reporting the indicated cancer type not individuals with RC in addition to the indicated cancer type E Percentage of different primary cancers reported a0years in SEER n versus the study cohort n Less than not reported for figure clarityScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cCharacteristicSexMaleFemaleEthnicityAfrican AmericanAshkenazi JewishAsianCaucasianHispanicMiddle EasternMixed EthnicityNative AmericanOtherUnknownMedian age of testingHistologyChromophobeMixed chromophobeClear cellOncocytomaMixed oncocytomaPapillary renalMixed papillaryRenal cellWilmsOthersUnknownPersonal cancer historyRenal cancer onlyRenal cancer plus additional cancer typeFamily history of cancerYesNoNot reportedunknownFamily history of renal cancerYesNoTotalNumber of patients in Ambry study cohort Rate in general population from birth to age SEER of renal cancers of renal cancersnrnrnrnrnrnr a0years Table Demographics and clinical characteristics of RC patients in the Ambry Genetics study cohort Demographics and clinical characteristics of the RC cases in the study cohort were compared to those of RC from birth to age in the SEER data Personal and family history of cancer were reported for the cases in the study cohort For family history of renal cancers numbers include only those who reported on cancer history n nr not reported SEER data included types of renal cancer histologies not all were represented in dataset other based on other category from Ambry cohort Family histories as selfreported on the intake formmedical records and have not been validatedassociated with eoRC we first analyzed PVs in eoRC cases with no additional primary cancer diagnosis Among the patients who only presented with eoRC PVs were identified in of cases n Fig a02E which is approximately twice the reported frequency of PVs in RCspecific genes7 Among this n of PVs were in one of DDRR genes ATM BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A NBN PALB2 RAD51C n were in one of RCspecific genes BAP1 FLCN SDHB VHL and the remaining cases bore PVs in nonDDRR genes associated with cancers other than RC Fig a02ENext we performed similar analysis as described above for patients who presented with eoRC plus one or more additional cancers Among the patients who presented with eoRC and at least one additional cancer Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cPVs were identified in cases Fig a02F Among these of cases PVs in othercancer associated genes including DDRR genes were found in of cases n versus n of cases with PVs in RCspecific genes This population was also enriched for PVs in DDRR genes n ATM BRCA1 BRCA2 CHEK2 MSH6 PALB2 PMS2 versus PVs in RCspecific genes BAP1 FLCN MET MITF PTEN SDHB VHLOverall these data suggest that DDRR gene PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer but that the frequency of PVs in DDRR genes in either group exceeded that in the control populations tested gnomADExAc Fig a0 Table a0S5A The specific PVs identified were similar in frequency to those identified in the full patient cohort n with CHEK2 the most represented DDRR genes Fig a0 To gain additional insight into the prevalence of these PVs in cancer patients we surveyed ClinVar wwwncbinlmnihgovclinv ar and found that multiple PVs from this study Table a0S4 have been reported in hereditary cancer predisposing syndromes HCPS summarized in Table a0S7 HCPS reflects a pattern of cancers in a family characterized by earlier onset with individuals not necessarily having the same tumor andor having more than one primary tumor and having tumors that are more likely to be multicentricRC a0patients a0with a0BRCA1 or BRCA2 a0PVs a0 Notably of the eoRC cases had a BRCA2 PV and RC cases had a BRCA1 PV Table a0 Table a0S3 This included n Table a0 of the cases who presented with only eoRC Interestingly despite the fact that the cohort was female of the detected BRCA1 and BRCA2 PVs were in males Table a0 Of the RC cases with a BRCA1 or BRCA2 PV had an additional cancer associated with hereditary breast and ovarian cancer HBOC syndrome breast ovarian prostate pancreatic or melanoma had an additional nonHBOC cancer and presented with only eoRC Table a0 Family history was reported for cases and of those indicated that at least one family member had an HBOCassociated cancer Of those with a BRCA2 PV reported that at least one family member had RC Table a0No a0correlation a0between a0age a0of a0RC a0diagnosis a0and a0type a0of a0PV a0in a0RC a0 To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort genes were divided into four broad overlapping categories all genes in the panel RCspecific genes nonRC genes including DDRR genes and DDRR genes see Methods The groups were compared to median age at first RC diagnosis of or ¥ a0years Given the invariable earlyonset of Wilms tumor the individuals with this diagnosis were excluded from the analysis Within this eoRC cohort there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above Fig a03ACorrelation a0of a0renal a0histologies a0with a0PVs a0in a0specific a0genes a0 Of the clear cell cases in this cohort had a PV of which were RCassociated PVs Similar findings were observed for the cases described as renal cell carcinoma had a PV of which were RCassociated DDRR gene PVs were found in of clear cell cases and in of renal cell cases Figure a03BC contrast the findings in clear cell and renal cell histology with the other nonclear cell histologiesDiscussionThis study for the first time demonstrates that PVs in multiple DDRR genes occur in patients with eoRC Importantly this study found that DDRR gene PVs were represented both in cases diagnosed with eoRC and additional cancers and also cases diagnosed with eoRC alone Comparison with a large control population indicated that germline PVs in DDRR genes were more common in this study cohort than in the control population although further studies are required to confirm this finding and estimate the penetrance of PVs in DDRR genes for eoRC We also found that germline testing using an RCspecific panel would have identified only of the RC cases with actionable PVs according to the NCCN recommended screening or management guidelines compared to the additional cases identified with the expanded panelsThe most common gene with PVs identified in the patients in this study was the DDRR gene CHEK2 This is consistent with recent reports by Carlo et a0al and Huszno et a0al1516 While evidence is mounting that CHEK2 PVs may increase risk for RC in this study we did not consider CHEK2 as a gene typically associated with RC as it is not currently included on RC panels and would fail to be included in testing in many cases In addition limitations of the previous studies and the analysis reported here together indicate that larger studies with appropriate controls are needed before confirming that CHEK2 indeed confers a risk for RCIdentification of germline DDRR gene PVs can have specific implications for the proband and the family For example of cases diagnosed with eoRC alone had PVs in BRCA1 or BRCA2 but not all of these cases had a family history strongly indicative of HBOC syndrome This is important because identification of a BRCA PV can potentially change medical management for instance PARP inhibitor therapy is effective in tumors with BRCA PVs including nonbreast tumors4142 Also screening and prevention of HBOCsyndrome cancers would likely be increased significantly in the proband and in family members found to have the same PV Further many of the specific PVs identified in this study have been annotated as relevant to various HCPS emphasizing their role in the development of multiple cancer types Our results support broader panel testing as a way to identify unexpected highpenetrant PVs in eoRC patients when there is a personal or family history of additional cancers especially an HBOCsyndrome cancerScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cA stinairav cnegohapt lan deifitneditot BKey A D E FDDRR genesother cancer associated genesrenal cancer associated genesMTADRABACRBACRBPIRBKEHCHLMHSMHSMAERMHYTUMNBNBLAPSMPCDARCPAARPMBANKDCFNPTPABNCLFTEMFTIMNETPAHDSBHDSLHVPathogenic DDRR variants in Ambry cohort vs ExAc populationC Pathogenic DDRR variants in Ambry cohort vs GnomAD populationKey B CATM BRCA1BRCA2CHEK229211GA3576GA8655dupT5712dupA68_69delAG2475delC7558CT9294CG7069_7070delCT3847_3848delGT2339CG4284dupT518delG4441GA4441CT470TC1 | Thyroid_Cancer |
"patients with differentiated thyroid cancer DTC tumor burden of persistent disease PD is avariable that could affect therapy efficiency Our aim was to assess its correlation with the American ThyroidAssociation ATA riskstratification system and its impact on response to initial therapy and outcomeMethods This retrospective cohort study included consecutive DTC patients referred for postoperativeradioiodine RAI treatment Patients were riskstratified using the ATA guidelines according to postoperativedata before RAI treatment Tumor burden of PD was classified into three categories ie very small small andlargevolume PD Very smallvolume PD was defined by the presence of abnormal foci on postRAI scintigraphywith SPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging Small and largevolume PDwere defined by lesions with a largest size or ¥ mm respectivelyResults PD was evidenced in patients Mean followup for patients with PD was ± years Thepercentage of largevolume PD increased with the ATA risk and in low intermediate and highriskpatients respectively p There was a significant trend for a decrease in excellent response rate from thevery small small to largevolume PD groups at months after initial therapy and respectively p and at last followup visit and respectively p On multivariate analysis age ¥ yearsdistant andor thyroid bed disease smallvolume or largevolume tumor burden and 18FDGpositive PD wereindependent risk factors for indeterminate or incomplete response at last followup visitConclusions The tumor burden of PD correlates with the ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATA riskstratification to refine outcome prognostication afterinitial treatmentKeywords Differentiated thyroid cancer Tumor burden Riskstratification Radioiodine 18FDG PETCT Correspondence rciappuccinibaclesseunicancerfr1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France2INSERM ANTICIPE Caen University Caen FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cCiappuccini BMC Cancer Page of BackgroundIn patients with differentiated thyroid cancer DTCthe riskstratification system described in the American Thyroid Association ATA guidelines is auseful tool to predict the likelihood of postoperativepersistent disease PD the response to initial therapyie surgery ± radioiodine [RAI] treatment and thelongterm outcome [] Several features related to PDare likely to influence the response to treatment andthe longterm prognosis This includes the location ofPD neck lymphnodes [LN] or distant metastases the18FFluorodeoxyglucose 18FDGRAIavidity [] oravidity [] of PD the aggressiveness of pathological variants [] and the degree of celldifferentiation [] thepresence of molecular mutations BRAF TERTp []and the tumor doublingtime [] Alone or in combination with previous characteristics notably RAIaviditythe tumor burden of PD is another variable that canaffect treatment efficiency and prognosis This has beenshown in studiessometimes old and using lowresolution imaging methods focusing on patients withdistant metastases [ ] In the daily practice it is wellknown that microscopic RAIavid lesions are morelikely cured than macroscopic ones eg lung miliary vslung macronodules However no studies have specifiedthe prognostic role of tumor burden estimated usinghighresolution imaging techniques both in the settingof distant metastases and lymphnode diseaseThe aim of the study was to assess the correlationof PD tumor burden with the ATA riskstratification system and its impact on response toinitialtherapy and outcome We hypothesized thatpatients presenting postoperatively a low tumor burden of PD would have better response to initial therapy and better clinical outcomes than patients havinghigh tumor burdenMethodsPatientsThe records of consecutive patients with DTC referred to our institution for postoperative RAI treatment between January and February werereviewed For the purpose of the study patients wereriskstratified according to the ATA guidelinesbased on pathological and surgical data available aftertotalthyroidectomy and before postoperative RAItreatment postoperative risk stratification [] Dataavailable in the preoperative period such as imagingstudies showing distant metastases were also used toinform ATA risk stratification In contrast postoperative serum thyroglobulin Tg level was not used todrive RAI treatment in these patients managed before and no diagnostic RAI scintigraphy was performed before RAI treatmentrhTSHPostoperative RAI treatmentAll patients were administered an RAI regimen ± weeks after total thyroidectomy Patients were prepared after either thyroid hormone withdrawal THWinjections of recombinant humanor after two imthyrotropinThyrogen Genzyme CorpCambridge MA USA as previously described [] TSHlevel was measured the day of RAI treatment and was mUIl in all patients The RAI activity or GBq and the preparation modalities were decided byour multidisciplinary committee All patients underwenta postRAI scintigraphy combining wholebody scanWBS and neck and thorax single photon emissioncomputed tomography with computed tomographySPECTCT A complementary SPECTCT such as abdomen andor pelvis acquisition was performed in caseof equivocal or abnormal RAI foci on WBS Patientswere scanned two or file days following or GBqrespectively Initial therapy was defined as surgery iethyroidectomy ± LN dissection plus first RAI treatmentie postoperative RAI treatmentSerum Tg and antiTg antibodies TgAb assayBlood samples for stimulated serum Tg and TgAb measurements were collected immediately before the RAItreatment Serum Tg measurements were obtained withthe Roche Cobas Tg kit Roche Diagnostics Mannheim Germany with a lower detection limit of ngml and a functional sensitivity of ngml until October and with the Roche Elecsys Tg II kit Roche Diagnostics Mannheim Germany with a lower detectionlimit of ngml and a functional sensitivity of ngml thereafter TgAb was measured using quantitativeimmunoassay methods Roche Diagnostics MannheimGermany TgAb positivity was defined by the cutoffsprovided by the manufacturerPathologyPathological variants were defined according to the WorldHealth anization classification [] Poorly differentiated carcinoma widely invasive follicular carcinomaH¼rthle cell carcinoma and among PTC variants tall cellcolumnar cell diffuse sclerosing and solid variants wereconsidered as aggressive pathological subtypes [] Tumorextent was specified according to the TNM []Tumor burden of persistent diseaseAs previously described [] PD was defined as evidenceof tumor in the thyroid bed LN or distant metastasesafter completion ofinitial therapy Confirmation wasachieved either by pathology or by complementary imaging modalities neck ultrasound examination [US]postRAI scintigraphy 18FDG positron emission tomography [PETCT] CT scan or MRI and followup 0cCiappuccini BMC Cancer Page of The tumor burden of PD was classified into three categoriesie very small small and largevolume PDVery smallvolume PD was defined by the presence ofabnormal foci on posttherapeutic RAI scintigraphy withSPECTCT or 18FDG PETCT without identifiable lesions on anatomic imaging neck ultrasound CT scan orMRI Small or largevolume PD were defined by thepresence of metastatic lesions with a largest size or ¥ mm respectively regardless of RAI or 18FDGuptake Examples of patients with very small small orlargevolume PD are presented in Fig RAI and 18FDG uptake in persistent diseaseThe RAI or 18FDG uptake profile was defined at time ofPD diagnosis PD was considered RAIpositive RAI ifat least one metastatic lesion showed RAI uptake andRAInegative RAI otherwise Similarly PD was defined18FDGpositive 18FDG if at least one metastatic lesionpresented significant 18FDG uptake and 18FDGnegative18FDG otherwiseClinical outcome assessmentAs previously described [] clinical assessment ofpatients with a negative postRAI scintigraphy wasscheduled at three months with serum TSH Tg andTgAb measurements while on levothyroxine LT4treatment When the Tg level at three months was ngml in the absence of TgAb the disease status wasassessed at months by serum rhTSHstimulated Tgassay and neck US and in recent years by Tg II assayon LT4 and neck US If there was an excellent responselevel ngmlat months according to the ATA criteria iestimulatedTgor nonstimulatedTglevel ngml without TgAb and negative neck USpatients were followed up on an annual basis For anything other than an excellent response imaging modalities such as CT scan of the neck and thorax 18FDGPETCT or MRI were performed In case of a secondRAI regimen given months after the first RAI therapy for RAIavid PD postRAI scintigraphy with SPECTCT was also used to assess initial treatment responseResponses to initial therapy as assessed at monthsand status at lastvisit were categorized as excellent response indeterminate response biochemical incompleteresponse or structural incomplete response according tothe ATA guidelines []Data analysisQuantitative data are presented in mean ± standard deviation SD except for Tg levels which are presented inmedian range Patients characteristics were comparedusing Chisquare or Fishers exact test the Wilcoxontest or the KruskalWallis test as appropriate TheCochranArmitage trend test was used to examineproportions of excellent response over the differentsubgroups in the following order verysmall small andlargevolume PD The analysis of diseasespecific survival and progressionfree survival was performed usingthe Cox regression model The analysis of prognosticfactors was performed using logistic regression Statistical significance was defined as p All tests wereFig Examples of very small small and large tumor burden in patients with persistent disease PD On the left side a 43yearold female patientwith a 40mm PTC at lowrisk after initial surgery T2NxMx and very smallvolume PD ac posttherapeutic 131I WBS showed a solitary bonyfocus on the right hip a arrow Fused transaxial image of 131I SPECTCT b arrow confirmed the bony uptake and hybrid CT c arrow did notdisplay any bone abnormality On the middle part a 74yearold female patient with a 40mm PTC at lowrisk after initial surgery T2N0Mx andsmallvolume PD df posttherapeutic 131I WBS showed pulmonary metastases d red and black arrows Fused transaxial image e red arrowand hybrid CT scan f red arrow depicted RAIavid lung micronodules ef mm On the right side an 88yearold female patient with a 40mmPTC tall cell variant at highrisk after initial surgery T2N1bM1 and largevolume PD gi no abnormal RAI uptake on posttherapeutic 131I WBSwith SPECTCT whereas 18FDG PETCT showed pulmonary and mediastinal metastases g Maximum intensity image arrows Fused transaxialimage h arrow and hybrid CT scan i arrow showed high 18FDG uptake SUVmax by an 18mm lung nodule 0cCiappuccini BMC Cancer Page of twosided SAS statistical software SAS InstituteInc Cary NC USA was used for data analysisstratification Patients characteristics are reported inTable ResultsCharacteristics of patientsThe study group included papillary thyroid cancers PTC follicular thyroid cancers FTC and poorlydifferentiated thyroid cancers PDTCThere were women and men The mean agewas ± years Three hundred and seventytwo patients were prepared with rhTSH stimulation Eightytwopatients presented positive TgAb at the time of postoperative RAI treatment In the postoperative setting priorto RAI administration patients were at lowriskLR at intermediaterisk IR and athighrisk HRaccording to the ATA riskPersistent disease and tumor burdenOverall PD was detected in patientsTheir characteristics in terms of ATA risk RAI preparation modality PD sites and RAI or 18FDG uptake arepresented in Table Of patients had very smallvolumelargevolume smallvolume and PDFigure shows two points First the rate of PDincreased from in LR patients and in IR to in HR patients p Second the percentage of patients with largevolume PD increased with risk stratification from LRIR to HR patients and respectively p Table Characteristics of patients according to the ATA riskstratification system in the postoperative settingMean age ± SD yrsSex ratio FemaleMean tumor size ± SD mmHistologyPTCFTCPDTCAggressive pathological subtypesNoYesExtrathyroidal extensionMinimalGrossT status TNM T1a T1bT2T3a T3bT4a T4bN status TNM NxN0N1a N1bM status TNM M0M1Positive TgAb levelStimulated Tg level at RAI treatment rangeaaIn patients without positive TgAb levelLRn ± ± IRn ± ± HRn ± ± p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent disease according to the tumor burdenVery smallvolumePD n SmallvolumePD n LargevolumePD n Postoperative ATA riskLRIRHRPreparation modalityTHWrhTSHPD siteLNLN DMDMTB diseaseTB disease DMRAI and 18FDG statusRAI18FDG or NPRAI18FDGRAI18FDGRAI18FDGRAI18FDG NPa21 RAI18FDG NP and one RAI18FDGb15 RAI18FDG NP and two RAI18FDGc10 RAI18FDG NP and six RAI18FDG 22a 17b 16c pFig Tumor burden in patients with persistent disease correlation to the ATA riskstratification system The figure first shows that the rateof PD increased from in LR patients in IR to in HR patients p Second the percentage of patients with largevolume PDincreased with risk stratification from LR IR to HR patients and respectively p 0cCiappuccini BMC Cancer Page of Table Characteristics of patients with persistent diseaseaccording to the ATA riskstratification systemLRn IRn HRn pPD tumor burdenVery smallvolume SmallvolumeLargevolume The distribution of very small small andlargevolume PD in LR IR and HR patients is presented in Table Outcome of patients with persistent diseaseTreatment modalities within the first year of management and during the remaining followup are detailed inTable Mean followup for patients with PD was ± years and was similar between the three groups of tumorburden p Of the patients with PD at months after initial therapy had excellent response indeterminate response biochemical incomplete response and structuralincomplete response At last followup visit the figureswere and respectively The outcome in each of the tumor burden groupsis presented in Table There was a significant trend fora decrease in excellent response rate from the verysmall small to the largevolume PD groups at months after initial therapy and respectivelyp and at last followup visit and respectively p Fig Among the patients died related to DTCduring followup Seven were in the largevolume PDgroup and one in the smallvolume PD group All hadstructural incomplete response at months after initial therapy with 18FDGpositive diseaseFigures and show diseasespecific survival DSSand progressionfree survivalPFS according to theATA riskstratification 18FDG status and tumor burdenSignificant differences in DSS were observed for bothATA riskstratification and 18FDG status but not fortumor burden Patients with 18FDGpositive disease hadshorter PFS Hazard Ratio 95CI thanthose with 18FDGnegative disease Also IR HazardRatio 95CI and HR patients HazardRatio 95CI had shorter PFS than LRpatients Finally patients with small Hazard Ratio 95CI and largevolume PD Hazard Ratio 95CI had shorter PFS than those withverysmall volume PDPrognostic factor analysis in patients with persistentdiseaseMultivariate analysis controlling for age sex postoperative ATA riskstratificationaggressive pathologicalTable Treatment modalities and outcome of patients with PD at months after initial therapy and at last followup visitaccording to tumor burdenVery smallvolumePD n months after initial therapySmallvolume PDn Largevolume PDn pVery smallvolumePD n At last followup visitSmallLargevolume PDvolume PDn n p a Treatment modalities at months after initial therapy treatments given within the first year of followup treatment modalities at last followup visittreatments given after the first year during followupb Local treatment of DM external radiation beam therapy surgery or radiofrequencyAbbreviations PD Persistent disease RAI Radioiodine DM Distant metastasesTreatment modalitiesaRAINeck surgeryNeck external radiationbeam therapyLocal treatment of DMbTyrosinekinase inhibitorsChemotherapyOutcomeExcellent response Indeterminate responseBiochemical incompleteresponseStructural incompleteresponse 0cCiappuccini BMC Cancer Page of Fig Excellent response rate according to tumor burden months after initial therapy a and at last followup visit b in patients withpersistent disease There is a significant trend for a decrease in excellent response rate from the very small small to the largevolume PD groupsat months after initial therapy and respectively p and at last followup visit and respectively p subtypes site of PD tumor burden of PD and RAI or18FDG uptake showed age ¥ years Odds ratio [OR] p distant andor thyroid bed disease OR p smallvolume OR p andlargevolume tumor burden OR p and18FDGpositive disease OR p to be independent risk factors for indeterminate biochemical orstructuralincomplete response at last followup visitTable DiscussionThis study confirms that the incidence of PD aftertotal thyroidectomy and postoperative RAI treatmentis limited in LR patients as compared to IR or HR patients Moreover it demonstrates thatthe tumor burden of PD is correlated to postoperativeriskstratification with very smallvolume lesions preferentially observed in LR patients and small and largevolume in IR or HR patients Most importantly tumorburden of PD is shown as an independent predictor ofresponse to initial therapy and to outcome These findings confirm that tumor burden of PD is a variablewhich might be taken into account to refine outcomeprognosticationTumor burden covers a large range of locoregionalandor distant metastases from a unique microscopic lesion to multiple macroscopic ones sometimes clinicallyevident Also tumor burden encompasses structural egvisible on conventionalfunctionalradiology andorlesions eg visible on RAI scintigraphy or 18FDG PETCT The diagnostic performances of imaging methodsand consequently the concept of tumor burden havedramatically evolved in the last decades The detectionof small LN disease has been improved by the combination of highresolution neck US postRAI SPECTCTand 18FDG PETCT imaging Regarding distant metastases although postRAI WBS still remains the referencefor detecting lung miliary disease the routine use ofdiagnostic CT scan and MRI now enables the detectionof infracentimetric lung bone or brain lesionsIn the past tumor burden of PD as a potential indicator of successful treatment and prognosis was assessedusing different approaches In a study on DTC patients with lung metastases diagnosed from to multivariate analysis showed that lung nodules visible on XRay vs those not visible RAIrefractory lunglesions and multiple metastatic sites were associatedwith poor survival [] In Gustave Roussys experienceoverall survival was reported in DTC patients withdistant metastases lung bone or other sites diagnosedbetween and [] Tumor extent was classifiedinto three categories according to both postRAI planarscintigraphy and Xrays Category consisted in lesionsvisible on postRAIscan but with normal Xraycategory in metastatic lesions cm on Xrays andcategory in lesions cm regardless of RAI avidityOverall metastases were RAIavid in of patientsmore frequently in patients years than 0cCiappuccini BMC Cancer Page of Fig Diseasespecific survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden cyears Multivariate analysis demonstrated that female sex young age years well differentiatedtumor RAI avidity and limited extent category wereindependent predictors ofrecentlyRobenshtok reported the outcome of patientssurvival Morewith RAIavid bone metastasis without structural correlate on CT scan or MRI among DTC patients withbone metastases between and [] After afollowup period of years all patients were alive nonehad evidence of structural bone metastases and none 0cCiappuccini BMC Cancer Page of Fig Progressionfree survival in the patients with PD according to ATA riskstratification a 18FDG status b and tumor burden chad experienced skeletalrelated events confirming theexcellent prognosis after RAI treatmentIn DTC patients with persistent nodal disease there isalso indirect evidence supporting that tumor burden affects treatment response and outcome In a recent retrospective study Lamartina reported the outcome of patients without distant metastases who underwenta first neck reoperation for nodal persistentrecurrentdisease [] Male sex aggressive histology and the presence of more than LN metastases at reoperation wereshown to be independent risk factors of secondary relapse following complete response achieved with first 0cCiappuccini BMC Cancer Page of Table Risk factors for indeterminate biochemical or structural incomplete response at last followup visitVariableAge years ¥ SexFemaleMaleInitial ATA riskstratificationLRIRHRAggressive histological subtypesNoYesSite of PDLN onlyDM andor TB disease with or without LNTumor burden of PDVery smallvolumeSmallvolume mmLargevolume ¥ mmRAI and 18FDG status of PDRAI18FDG or NPRAI18FDG or NPRAI or RAI18FDGPatients at risk nInitial modelOR CIp valueFinal modelOR CIp valuereoperation Conversely the excellent outcome of microscopic nodal involvement detected on SPECTCT at RAIablation was demonstrated by a study from Schmidt [] Of patients with RAIavid LN metastasesat ablation only three still showed nodes with significantuptake on a diagnostic RAI scintigraphy at monthsThe LN successfully treated by RAI were less than cmexcept in one patient whereas those still visible at months were above cm confirming that RAI is highlymore efficientin microscopic than in macroscopiclesionsIn the present study multivariate analysis showed thatage over years distant andor thyroid bed diseasesmall or largevolume tumor burden and 18FDGpositive disease were independent risk factors for indeterminate or incomplete response at last followup visit Incontrast ATA risk stratification and aggressive pathological subtypes did not emerge from multivariate analysis possibly because of the number of patients thenumber of variables tested and confounding variablesHoweverand progressionfreethe diseasespecificsurvival curves confirmed the high prognostic value ofthe ATA riskstratification In practice data supportsthat LR patients have a better outcome than the IR andHR groups not only because PD is uncommon in thosepatients but also because the excellent response rate ishigher in very smallvolume than in small or largevolume lesions We suggest that tumor burden usingthis threeclass discrimination could be implemented inthe assessment of patients with structural incomplete response to help refining the risk prediction This variablecould also be incorporated with the other risk predictorssuch as RAI or 18FDG uptake molecular profile tumorhistology degree of cell differentiation and Tg level andtumor volume doubling time to further improve riskestimatesAlthough retrospective the present study presents several strengths including the large cohort of consecutivepatients and the significant followup Patients diagnosedbetween and were uniformly evaluated usingmodern imaging studiesincluding postRAI scintigraphy with neck and thorax SPECTCT [] and 18FDG 0cCiappuccini BMC Cancer Page of PETCT with a dedicated headandneck acquisition [] Tumor burden was assessed combining functionaland anatomic imaging as adapted from previous papersof our group [ ] One can argue that it would havebeen even more pertinent to assess tumor burden withquantitative values rather than with a threeclass discrimination ie very small small and largevolumeActually a quantitative volumetric assessment is notfeasible because of the RAIavid nodal or metastatic lesions without structural correlate Also a quantitativeassessment based on RAI or 18FDG uptake is notpossible either because of RAIrefractory or nonhypermetabolic lesions Nevertheless we believe that ourdefinition is simple to use in routine practice and easilyreproducibleConclusionsThe tumor burden of PD correlates with the postoperative ATA riskstratification affects the response to initialtherapy and is an independent predictor of residual disease after a mean 7yr followup This variable might betaken into account in addition to the postoperative ATAriskstratification to refine outcome prognostication afterinitial treatmentAbbreviationsATA American thyroid association DM Distant metastasesDTC Differentiated thyroid cancer 18FDG 18FfluorodeoxyglucoseFTC Follicular thyroid cancers HR Highrisk IR Intermediaterisk LN Lymphnodes LR Lowrisk MRI Magnetic resonance imaging NP Not performedOR Odds ratio PD Persistent disease PDTC Poorlydifferentiated thyroidcancers PETCT Positron emission tomography with computed tomographyPTC Papillary thyroid cancers RAI Radioiodine rhTSH Recombinant humanthyrotropin SPECTCT Single photon emission computed tomography withcomputed tomography Tg Thyroglobulin TgAb AntiTg antibodiesTHW Thyroid hormone withdrawal TB Thyroid bed US Ultrasoundexamination WBS Wholebody scanAcknowledgmentsWe are indebted to Gee Knight for the reviewing of the manuscriptAuthors contributionsRC and SB conceived the study and its design RC ALC VSR CL VLH DV EBand SB performed data acquisition and analysis NH performed the statisticalanalysis RC and SB drafted the manuscript All authors read and approvedthe final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used and analysed during the current study are available fromthe corresponding author on reasonable requestEthics approval and consent to participateAll procedures were in accordance with the ethical standards of theinstitutional committee and with the Helsinki declaration and its lateramendments Baclesse Cancer Centre has licensed from the FrenchCommission for Data Protection and Liberties CNIL MR004 ref v0This study was approved by the institutional review board of Baclesse hospital and all subjects gave written informed consentConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nuclear Medicine and Thyroid Unit Fran§ois Baclesse CancerCentre Avenue Gnral Harris F14000 Caen France 2INSERM ANTICIPE Caen University Caen France 3CETAPS EA Rouen UniversityRouen France 4Department of Head and Neck Surgery Fran§ois BaclesseCancer Centre Caen France 5Department of Pathology Fran§ois BaclesseCancer Centre Caen France 6Department of Oncology Fran§ois BaclesseCancer Centre Caen France 7Department of Cancer Biology and GeneticsFran§ois Baclesse Cancer Centre Caen France 8Department of Head andNeck Surgery University Hospital Caen FranceReceived February Accepted August ReferencesHaugen BR Alexander EK Bible KC Doherty GM Mandel SJ Nikiforov YEPacini F Randolph GW Sawka AM Schlumberger M Schuff KG Sherman SISosa JA Steward DL Tuttle RM Wartofsky L American ThyroidAssociation management guidelines for adult patients with thyroid nodulesand differentiated thyroid Cancer the American Thyroid Associationguidelines task force on thyroid nodules and differentiated thyroid CancerThyroid Durante C Haddy N Baudin E Leboulleux S Hartl D Travagli JP Caillou BRicard M Lumbroso JD De Vathaire F Schlumberger M Longtermoutcome of patients with distant metastases from papillary andfollicular thyroid carcinoma benefits and limits of radioiodine therapy J ClinEndocrinol Metab Robbins RJ Wan Q Grewal RK Reibke R Gonen M Strauss HW Tuttle RMDrucker W Larson SM Realtime prognosis for metastatic thyroid carcinomabased on [18F]fluoro2deoxyDglucosepositron emission tomographyscanning J Clin Endocrinol Metab Michels JJ Jacques M HenryAmar M Bardet S Prevalence and prognosticsignificance of tall cell variant of papillary thyroid carcinoma Hum Patholde la Fouchardiere C DecaussinPetrucci M Berthiller J Descotes F Lopez JLifante JC Peix JL Giraud Delahaye A Masson S BournaudSalinas CBorson CF Predictive factors of outcome in poorly differentiated thyroidcarcinomas Eur J Cancer Melo M Gaspar da Rocha A Batista R Vinagre J Martins MJ Costa G RibeiroC Carrilho F Leite V Lobo C CameselleTeijeiro JM Cavadas B Pereira LSobrinhoSimoes M Soares P Gaspar da Rocha A Batista R Vinagre JMartins MJ Costa G Ribeiro C Carrilho F Leite V Lobo C CameselleTeijeiroJM Cavadas B Pereira L SobrinhoSimoes M Soares P TERT BRAF andNRAS in primary thyroid Cancer and metastatic disease J Clin EndocrinolMetab Sabra MM Sherman EJ Tuttle RM Tumor volume doubling time ofpulmonary metastases predicts overall survival and can guide the initiationof multikinase inhibitor therapy in patients with metastatic follicular cellderived thyroid carcinoma Cancer Casara D Rubello D Saladini G Masarotto G Favero A Girelli ME BusnardoB Different features of pulmonary metastases in differentiated thyroidcancer natural history and multivariate statistical analysis of prognosticvariables J Nucl Med Ciappuccini R Hardouin J Heutte N Vaur D Quak E Rame JP Blanchard Dde Raucourt D Bardet S Stimulated thyroglobulin level at ablation indifferentiated thyroid cancer the impact of treatment preparationmodalities and tumor burden Eur J Endocrinol Lloyd RV Osamura RY Kl¶ppel G Rosai J editors WHO classification oftumours of endocrine ans 4th edition Lyon International Agency forResearch on Cancer Brierley JD Gospodarowicz MK Wittekind C TNM classification of malignanttumours 8th edition Oxford Wiley Blackwell Ciappuccini R Heutte N Trzepla G Rame JP Vaur D Aide N BardetS Postablation I scintigraphy with neck and thorax SPECTCTand stimulated serum thyroglobulin level predict the outcome ofpatients with differentiated thyroid cancer Eur J Endocrinol 0cCiappuccini BMC Cancer Page of Robenshtok E Farooki A Grewal RK Tuttle RM Natural history of smallradioiodineavid bone metastases that have no structural correlate onimaging studies Endocrine Lamartina L Bet I Mirghani H Al Ghuzlan A Berdelou A Bidault FDeandreis D Baudin E Travagli JP Schlumberger M Hartl DM Leboulleux SSurgery for neck recurrence of differentiated thyroid Cancer outcomes andrisk factors J Clin Endocrinol Metab Schmidt D Linke R Uder M Kuwert T Five months' followup of patientswith and without iodinepositive lymph node metastases of thyroidcarcinoma as disclosed by 131ISPECTCT at the first radioablation Eur JNucl Med Mol Imaging | Thyroid_Cancer |
Paired box protein8 PAX8 immunohistochemical expression can be used as a diagnostic marker for epithelial cells tumors This study aimed at investigating the immunohistochemical expression of PAX8 among Sudanese females diagnosed with cervical endometrial and ovarian cancers between December and May by studying their Formalinfixed paraffin embedded blocksResults Sixty patients diagnosed with female reproductive tract cancers were included who aged ± years range Cervix was the most common cancer site in patients Regarding cancer stage there was and of the study population had stage 3B and 2B respectively The histopathological diagnosis included and poorly moderately and well differentiated cervical squamous cell carcinoma SCC as well as and endometrial adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma respectively PAX8 was positively expressed in endometrial adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinoma poorly and moderately differentiated SCC All patients diagnosed with well differentiated SCC and metastatic adenocarcinoma showed no expression of PAX8 A statistically significant was seen for PAX8 expression and the different histopathological diagnosis P value Keywords Female reproductive cancer Paired box protein8 Immunohistochemical expressionIntroductionPaired box protein8 PAX8 is a member of the family paired box proteins PAXs [ ] PAX8 consists of amino acids with a molecular weight of approximately kilo Dalton and its molecular properties are located on chromosome 2q13 [] PAX8 is a transcription factor that regulates ans development during the embryonic period as well as to maintain normal cellular functions in some cells after birth [ ] During the embryonic period PAX8 also plays a significant role Correspondence nouh_saadoutlookcom Alfarrabi College for Science and Technology Khartoum SudanFull list of author information is available at the end of the in the development of genital ans derived from the mesonephric and the M¼llerian ducts [] In a previous experiment the deletion of the PAX8 gene resulted in dysfunctional uterus absence of the endometrium and the vaginal ing Also resulted in poor development of the myometrial tissue [] Several studies have described the immunohistochemical utility of PAX8 as a diagnostic marker for epithelial cells neoplasms of many glands and ans such as thyroid thymus and kidney as well as some female reproductive tract tumors [ ]In a healthy female reproductive tract PAX8 shown to be overexpressed in the epithelial cells of the endocervix and the endometrium [] PAX8 was found to be expressed among endometrioid carcinomas transitional The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cAli a0et a0al BMC Res Notes Page of undifferentiated cell carcinomas and the metastatic carcinomas at a range of and [ ] Whereas for the ovarian carcinomas PAX8 was under expressed [] Considering that few studies have investigated the immunohistochemical expression of PAX8 in carcinomas of the endometrium and uterine cervix in the different parts of the world but none from Sudan yet [ ] This study aimed at investigating the immunohistochemical expression of PAX8 among Sudanese females diagnosed with cervical endometrial and ovarian carcinomasMain textMaterials and a0methodsStudy design and a0population characteristicsThis is a descriptive retrospective hospital based study conducted at different histopathology laboratories during the period from December till May in Khartoum State Sudan We retrieved archived formalin fixed paraffin embedded blocks previously collected from female patients with cervical endometrial or ovarian carcinomas The retrieved formalin fixed paraffin blocks represent all the female population admitted at the hospitals for reproductive malignancies diagnosis The participants demographic data was collected including age place of residence The clinical data including site of cancer cancer grade and the histopathological diagnosis were also collectedSections Preparation for a0Immunohistochemistry StainingTwo sections were cut using Rotary microtome Leica Germany from each histopathological block Then one slide was stained by hematoxylin and eosin staining technique The other slide was mounted onto 3aminopropyltriethoxysilane coated slides for immunohistochemistry To retrieve PAX8 tissues antigen we treated the sections with citrate buffer at ° a0C for a0min in a waterbath Then the tissue sections were rinsed first in distilled water and later with Tris buffer saline TBS This was followed by treatment with peroxidase block hydrogen peroxide in methyl alcohol for a0min to quench endogenous peroxidase activity The slides were then placed in a humid chamber Then the slides were drained and rinsed in two successive changes of Tris buffer wash buffer for a0 min each Nonspecific proteinprotein interactions were blocked by incubating and treating the tissue sections in a humid chamber with the power block casein in phosphate buffered saline for a0 min Then the remaining solution was drained from the slides The sections were then incubated in the primary antibody PAX8 antiPAX8 rabbit antihuman monoclonal antibody ab189249 Abcam United Kingdom at room temperature in the humid chamber according to the manufacture instructionsObserving the yellowishbrown or brown appearance of the nucleus was considered a positive result for the PAX8 For the negative control we omitted the incubation with the primary antibody step instead we incubated the section in the phosphate buffer saline PBSResults interpretationsFor the interpretation of the results we depended on the intensity as well as the number of the cells that expressed the marker and the expression was graded into categories Negative No staining less than of the cells were expressing the marker of the cells were expressing the marker more than of the cells were expressing the marker more than of the cells were expressing the marker The slides were interpreted and validated by two expert pathologists blindly of each other results Photomicrographs were taken using Olympus SP350 camera Olympus Imaging America Inc USAStatistical analysisThe statistical analysis of the results was done using IBM SPSS Statistics vs The ChiSquared test was performed to compare the frequencies of categorical variables Statistical significance level was defined as p value at confidence intervalResultsCharacteristics of a0the a0study participantsThe study included patients diagnosed with female genital tract cancer Patients aged ± a0years range a0years Patients were grouped into age groups Those aged a0 years constituted half of the study participants The remaining were and patients distributed across the remaining age groups of a0 years a0 years and a0 years respectively According to patients place of residence patients were originating from the four regions of Sudan Most of the patients were from western part of Sudan followed by from the central part of SudanRegarding the site of cancer the cervix was the most commonly involved patients There were and endometrial and ovarian cancer respectively Based on the International Federation of Gynecology and Obstetrics FIGO cancer grading the majority of the study population was diagnosed with stage 3B and 2B cancer and of the patients respectively The were and stage 4B 3A 2A 1B and 4A respectively 0cAli a0et a0al BMC Res Notes Page of No statistically significant association between FIGO staging and age group was found P value Histologically there were squamous cell carcinoma SCC all of which were cervical cancers and adenocarcinoma SCC and adenocarcinoma were further classified into poorly differentiated SCC moderately differentiated SCC and well differentiated SCC endometrium adenocarcinoma metastatic adenocarcinoma endocervical adenocarcinoma and ovarian mucinous cyst adenocarcinomaBased on age groups age group showed no statistically significant relationship with either patients place of residence cancer site cancer histological type FIGO staging and cancer histopathological type Table a0Immunohistochemical Expression of a0PAXThe immunohistochemical expression of PAX8 was shown as a yellowishbrown or brown staining of the nucleus Fig a0 Based on site of cancer all endometrium carcinoma showed positive expression of PAX8 with P value There were only patients who had positive expression of PAX8 including adenocarcinoma and SCC A statistically significant difference was noted for the PAX8 staining and cancer type with P value The analysis of PAX8 staining results based on the histopathological diagnosis showed that all patients who were diagnosed with well differentiated SCC and metastatic adenocarcinoma had negative results for the PAX8 expression While of the endometrium adenocarcinoma were found positive for the PAX8 expression A statistically significan was t seen for PAX8 expression and the different histopathological diagnosis P value Table a0Table Classification of a0Participants demographic and a0clinical diagnosis based on a0age groupAge group no Total no P value a0years a0years a0years a0yearsResidence of patient Central Sudan East Sudan West Sudan North SudanSite of cancer Cervix Endometrium OvaryCancer histological type SCC AdenocarcinomaFIGO staging Stage Stage 2A Stage 2B Stage 3A Stage 3B Stage 4A Stage 4BHistopathological cancer grades Well differentiated SCC Poorly differentiated SCC Moderately differentiated SCC Endometrium adenocarcinoma Endocervical adenocarcinoma Metastatic adenocarcinoma Ovarian mucinous cyst adenocarcinomaSCC Squamous Cell Carcinoma 0cAli a0et a0al BMC Res Notes Page of Fig Immunohistochemical expression of PAX8 among the different histopathological cancer types and grades The immunohistochemical expression of PAX8 is shown as a yellowishbrown or brown staining of the nucleus a Well differentiated SCC negative b Metastatic adenocarcinoma negative c Poorly differentiated SCC positive d Moderately differentiated SCC positive e Endometrium adenocarcinoma positive f Ovarian mucinous cystadenocarcinoma positive g Endocervical adenocarcinoma positive and h endometroid adenocarcinoma positive 0cAli a0et a0al BMC Res Notes Page of Table Association of a0clinical diagnosis and a0the a0immunohistochemical expression of a0PAX8PAX results no Total no P valuePositiveNegativeCancer histological type SCC AdenocarcinomaCancer site Cervix Endometrium OvaryFIGO staging Stage Stage 2A Stage 2B Stage 3A Stage 3B Stage 4A Stage 4BCancer histopathological grading Well differentiated SCC Poorly differentiated SCC Moderately differentiated SCC Endometrium adenocarcinoma Endocervical adenocarcinoma Metastatic adenocarcinoma Ovarian mucinous cyst adenocarcinomaSCC Squamous Cell Carcinoma DiscussionPrevious studies on the immunohistochemical expression of PAX8 in the normal female reproductive tract showed that PAX8 was expressed in the endometrial endocervical and ovarian epithelial cells as well as in nonciliated epithelial cells of the fallopian tubes [ ] This study investigated the immunohistochemical expression of PAX8 in Sudanese patients who were diagnosed with female reproductive tract cancers Patients on the 5th decade of life were constituting half of the study participants with no statistically significant association between age group and the type of cancer However previous studies had suggested other risk factors which could contribute in the development of certain gynecological cancer [ ]Regarding the place of residence the majority of patients coming from western Sudan This result is in contrary with a previous study in Sudan conducted by Saeed et a0al in which they showed that the percentage of patients suffering from different types of cancers residing in central and northern Sudan were higher compared to the other regions in Sudan [] Nevertheless these findings could suggest the involvement of environmental risk factors however the limited study samples size is insufficient to support this suggestion Therefore further research with a larger samples size investigating the potential environmental risk factors is essential for strategic prevention and protection measuresThe reported number of female patients with cervical cancer was high compared to ovarian and endometrium cancer Similar results were seen previously among Sudanese females [] Also the high frequency of stages 3B and 2B compared to the other stages were comparable to previous study conducted in Sudan [] This similarity underscores a delayed response among Sudanese females in seeking healthcare and urge the need for health promotion and education to encourage young Sudanese females for the early signs detection and seeking healthcare as early as possible for a better treatmentRegarding the classification based on the histopathological diagnosis most of the female diagnosed with SCC This result was also similar to previous study investigated the prevalence of the different gynecologic cancer in Sudan [] However the expression of PAX8 among the studied samples was relatively low compared to previous studies [ ] this could be attributed 0cAli a0et a0al BMC Res Notes Page of to the site of cancer development While agrees with another study where PAX8 was expressed only in patient []Interestingly a high frequency of PAX8 expression was noted among females diagnosed with endometrium cancer compared to SCC this finding is in contrary with a previous report where PAX8 was expressed among only of the studied samples [] Also the result was strongly in accordance with other studies [ ] Besides that the lack of PAX8 expression among those who were diagnosed with well differentiated SCC and metastatic adenocarcinoma could play a significant role in either gynecologic cancer differentiation or in detection of endometrium adenocarcinoma progression to metastatic adenocarcinoma [ ]ConclusionAlthough PAX8 showed a significant expression among adenocarcinomas lesions and negative expression was noted among those with well differentiated SCC and metastatic adenocarcinoma PAX8 might not be beneficial when used alone as a diagnostic marker for the tumors that occur in the female reproductive tractLimitations¢ The small sample size investigated in this study reduced the ability of using the expression of PAX8 as a diagnostic marker Therefore a largescale study is needed and it should include other types of malignant tumors encountered in the female reproductive systemAcknowledgementsThe authors would like to acknowledge the medical staff for their interest and cooperation during the study and thanks to all who participated in completing this studyAuthors contributionsETA NSM and EES provided conceptual framework for the study guidance for interpretation of the data and performed data analysis ETA EES IRS LAH and AMM performed laboratory work NSM EES MSM AAY and AA performed the statistical analysis NSM MSM EES and AA participated in the manuscript preparation revision and coordination All authors read and approved the final manuscriptFundingNot ApplicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Research Ethics Committee of the Faculty of Medical Laboratory Sciences University of Khartoum Sudan Ethical Approval No FMLSREC002042 All participant approved to participate by signing an informed consentConsent for publicationNot ApplicableCompeting interestsNo competing interests to discloseAuthor details Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences University of Khartoum Khartoum Sudan Department of Histopathology and Cytology Faculty of Medical Laboratory Sciences National University Khartoum Sudan Alfarrabi College for Science and Technology Khartoum Sudan Faculty of Medicine Sinnar University Sennar Sudan Molecular Biology Department Faculty of Medical Laboratory Sciences Nile University Khartoum Sudan Faculty of Dentistry Ibn Sina University Khartoum Sudan Department of Neurology Mayo Clinic Jacksonville FL USA Department of Radiology Mayo Clinic Jacksonville FL USA Institute of Endemic Diseases University of Khartoum Khartoum Sudan Mycetoma Research Center University of Khartoum Khartoum Sudan Faculty of Medicine Nile University Khartoum Sudan Received July Accepted August References Gruss P Walther C Pax in development Cell Mansouri A Hallonet M Gruss P Pax genes and their roles in cell differentiation and development Curr Opin Cell Biol Macchia PE Lapi P Krude H Pirro MT Missero C Chiovato L Souabni A Baserga M Tassi V Pinchera A PAX8 mutations associated with congenital hypothyroidism caused by thyroid dysgenesis Nat Genet Vilain C Rydlewski C Duprez L Heinrichs C Abramowicz M Malvaux P Renneboog Bt Parma J Costagliola S Vassart G Autosomal dominant transmission of congenital thyroid hypoplasia due to lossoffunction mutation of PAX8 J Clin Endocrinol Metab Park S VK C Genetics of congenital hypothyroidism J Med Genet Dahl E Koseki H Balling R Pax genes and anogenesis BioEssays Lang D Powell SK Plummer RS Young KP Ruggeri BA PAX genes roles in development pathophysiology and cancer Biochem Pharmacol Stoykova A Gruss P Roles of Paxgenes in developing and adult brain as suggested by expression patterns J Neurosci Mittag J Winterhager E Bauer K Grummer R Congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy Endocrinology Bouchard M de Caprona D Busslinger M Xu P Fritzsch B Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation BMC Dev Biol Mittag J Winterhager E Bauer K Grummer RJE Congenital hypothyroid female pax8deficient mice are infertile despite thyroid hormone replacement therapy Endocrinolog Laury AR Perets R Piao H Krane JF Barletta JA French C Chirieac LR Lis R Loda M Hornick JL A comprehensive analysis of PAX8 expression in human epithelial tumors Am J Surg Pathol Wong S Hong W Hui P Buza N Comprehensive analysis of PAX8 expression in epithelial malignancies of the uterine cervix Int J Gynecol Pathol Ozcan A Shen SS Hamilton C Anjana K Coffey D Krishnan B Truong LD PAX expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study Mod Pathol Bowen NJ Logani S Dickerson EB Kapa LB Akhtar M Benigno BB McDonald JF Emerging roles for PAX8 in ovarian cancer and endosalpingeal development Gynecol Oncol 0cAli a0et a0al BMC Res Notes Page of Ozcan A Liles N Coffey D Shen SS Truong LD PAX2 and PAX8 expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison Am J Surg Pathol distinguishing ovarian mucinous neoplasms from colonic and appendiceal mucinous neoplasm BMC Res Notes Nesrin R KILIC D Risk factors for cervical cancer results from a hospital Ozcan A Liles N Coffey D Shen SS Truong LDJTAjosp PAX2 and PAX8 based casecontrol study Int J Hematol Oncol expression in primary and metastatic m¼llerian epithelial tumors a comprehensive comparison Am J Surg Pathol Nonaka D Tang Y Chiriboga L Rivera M Ghossein R Diagnostic utility of thyroid transcription factors Pax8 and TTF2 FoxE1 in thyroid epithelial neoplasms Mod Pathol Tacha D Zhou D Cheng L Expression of PAX8 in normal and neoplastic tissues a comprehensive immunohistochemical study Appl Immunohistochem Mol Morphol Bowen NJ Logani S Dickerson EB Kapa LB Akhtar M Benigno BB McDonald JFJGo Emerging roles for PAX8 in ovarian cancer and endosalpingeal development Gynecol Oncol K¶bel M Kalloger SE Boyd N McKinney S Mehl E Palmer C Leung S Bowen NJ Ionescu DN Rajput A Ovarian carcinoma subtypes are different diseases implications for biomarker studies PLoS medicine 2008512e232 Nonaka D Chiriboga L Soslow RA Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas Am J Surg Pathol Tong GX Devaraj K HameleBena D Yu WM Turk A Chen X Wright JD Greenebaum E Pax8 a marker for carcinoma of M¼llerian origin in serous effusions Diagn Cytopathol Laury AR Perets R Piao H Krane JF Barletta JA French C Chirieac LR Lis R Loda M Hornick JLJTAjosp A comprehensive analysis of PAX8 expression in human epithelial tumors Am J Surg Pathol Tong GX Devaraj K HameleBena D Yu WM Turk A Chen X Wright JD Greenebaum EJDc Pax8 a marker for carcinoma of M¼llerian origin in serous effusions Diagn Cytopathol Chu PG Chung L Weiss LM Lau SK Determining the site of origin of mucinous adenocarcinoma an immunohistochemical study of cases Am J Surg Pathol Brunner AH Riss P Heinze G Meltzow E Brustmann H Immunoexpression of PAX in endometrial cancer relation to highgrade carcinoma and p53 Int J Gynecol Pathol Ozcan A Shen SS Hamilton C Anjana K Coffey D Krishnan B Truong LDJMP PAX expression in nonneoplastic tissues primary tumors and metastatic tumors a comprehensive immunohistochemical study Mod Pathol Aldaoud N Erashdi M AlKhatib S Abdo N AlMohtaseb A GraboskiBauer A The utility of PAX8 and SATB2 immunohistochemical stains in Saeed ME Cao J Fadul B Kadioglu O Khalid HE Yassin Z Mustafa SM Saeed E Efferth T A fiveyear survey of cancer prevalence in Sudan Anticancer Res Saeed ME Cao J Fadul B Kadioglu O Khalid HE Yassin Z Mustafa SM Saeed E Efferth TJAr A fiveyear survey of cancer prevalence in Sudan Anticancer Res Mohamed KEH Ashmeig AAA Cervical cancer our experience in Sudan Philadelphia AACR Elhasan LME Bansal D Osman OF Enan K Abd Farag EAB Prevalence of human papillomavirus type in Sudanese women diagnosed with cervical carcinoma J Cancer Res Ther Tacha D Zhou D Cheng LJAI Morphology M Expression of PAX8 in normal and neoplastic tissues a comprehensive immunohistochemical study Appl Immunohistochem Mol Morphol Ord³±ez NG Value of PAX immunostaining in tumor diagnosis a review and update Adv Anat Pathol Gailey MP Bellizzi AM Immunohistochemistry for the novel markers glypican PAX8 and p40 ÎNp63 in squamous cell and urothelial carcinoma Am J Clin Pathol Yemelyanova A Gown AM Holmes BJ Ronnett BM Vang R PAX8 expression in uterine adenocarcinomas and mesonephric proliferations Int J Gynecol Pathol Liang L Zheng W Liu J Liang SX Assessment of the utility of PAX8 immunohistochemical stain in diagnosing endocervical glandular lesions Arch Pathol Lab Med Wong S Hong W Hui P Buza NJIJoGP Comprehensive analysis of PAX8 expression in epithelial malignancies of the uterine cervix Int J Gynecol Pathol De Andrade DAP Da Silva VD de Macedo MG De Lima MA de Andrade VM Andrade CEMC Schmidt RL Reis RM Dos Reis R Squamous differentiation portends poor prognosis in low and intermediaterisk endometrioid endometrial cancer PLoS ONE 20191410e0220086Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your ï¬eld¢ rapid 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" The advent of new cancer therapies alongside expected growth and ageing of the population better survival rates and associated costs of care is uncovering aneed to more clearly deï¬ne and integrate supportive care services across the whole spectrum of the disease The current focus of cancer care is on initialdiagnosis and treatment and end of life care The Multinational Association of Supportive Care in Cancer deï¬nes supportive care as the prevention andmanagement of the adverse effects of cancer and its treatment This encompasses the entire cancer journey and necessitates involvement and integration ofmost clinical specialties Optimal supportive care can assist in accurate diagnosis and management and ultimately improve outcomes A national strategy toimplement supportive care is needed to acknowledge evolving oncology practice changing disease patterns and the changing patient demographic The Royal College of Radiologists Published by Elsevier Ltd All rights reservedKey words Beyond cancer chronic cancer deï¬nition living with supportive careStatement of Search Strategies UsedA series of searches were constructed and carried out viaPubMed EMBASE and MEDLINE This generally consisted ofusing phrase searching due to the speciï¬city of the subjectOnce concepts were established the authors used Booleanoperators to combine the concepts together and retrieve themost relevant papers Once a set of results were retrieved theauthors scanned each of the s using and titleï¬elds to identify key papers Fulltext access to papers weresourced via the Christie Library and Knowledge ServiceIntroductionSupportive Care Makes Excellent Cancer Care PossibleMultinational Association of Supportive Care in CancerwwwmasccAdvances in diagnosis surgery radiotherapy and newdrugs have led to improvements in cancer survival PeopleAuthor for correspondence R Berman The Christie NHS FoundationTrust Wilmslow Road Manchester M20 4BX UK Tel ¾447710509402Email address RichardBermanchristienhsuk R Bermannow live nearly six times longer after their cancer diagnosisthan was the case years ago [] Half of people diagnosedwith cancer in England and Wales survive their disease for years or more [] Currently in England around million people are living with a diagnosis of cancer and thisnumber is increasing by over a year The total ï¬gure is setrise to over million by []Many more cancer patients are being treated closer todeath with novel less toxic high efï¬cacy anticancer therapeutic agents developing with increasing pace within thelast decade The advent of molecular targeted agents forexample has brought new beneï¬ts as well as challenges tomodern cancer therapy potentially blurring the distinctionbetween active and palliative interventions []Yet despite this significant progress a large proportion ofpatients with cancer still experience morbidity and symptoms resulting from the cancer andor its treatment []Increases in cancer incidence [] emergency care hospitalisations [] earlier intensive care unit admissions [] andtreatment costs [] have all added to the global burden ofcancer care The disease is becoming a major economicexpenditure for all developed countries [] In the UK andin the USA cancer care costs are substantial and expected to101016jclon20200702009366555 The Royal College of Radiologists Published by Elsevier Ltd All rights reservedPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrise significantly in the future due to growth and aging ofthe population and improvements in survival as well astrends in treatment patterns and costs of care followingcancer diagnosis []cancerandcancerManagingtreatmentrelatedmorbidity is therefore a significant public health andeconomic challenge The coronavirus pandemic has deepened this challenge with many cancer outpatient visitsbeing replaced by telephone consultations and deferral ofsome routine therapy tests and procedures This has placedadditional pressures on an already fragile and vulnerablepopulation [] Patients and carers are experiencing moreuncertainty and anxiety associated with COVID19 A recentstudy found that although patients continue to feel wellsupported by their healthcare teams they have concernsabout the longerterm impact of changes to aspects of theirtreatment Patients and carers are no longer able to accessother support services in the way that they had previouslysuch as hospices and peer support groups []There is a growing body of evidence that timely access tosupportive treatments can lead to improvements in qualityof life and survival as well as beneï¬tting the health economy [15e17] The development of a broad multiprofessionalbasis for the study and expansion of supportive carethrough the Multinational Association of Supportive Care inCancer MASCC has been an important step in fostering thegrowth of an evidence base [] MASCC's success has undoubtedly been underpinned by successful integration ofoncological and nononcological specialties []However variations in the deï¬nition of supportive careallocation of resources and a lack of clarity on who shouldlead onprovide services means that a clinical model forsupportive care in cancer does not yet exist [] Most specialties whilst they overlap other specialties are based on acore of knowledge or skill that is speciï¬c to that specialty[] Supportive care is currently provided by a patchwork ofdifferent medical specialties and is unique because it traverses the entire spectrum of the disease Figure fromdiagnosis through to survivorship or end of life care Theneed for supportive oncology to become a specialty in itsown right is borne out not just by the progress in its development in the UK and abroad but by the unmet supportivecare need [] ampliï¬ed by the rising incidence of cancerworldwide with many patients living longer with incurableillness because of more effective cancer treatments [] Asignificant next step would be to produce an evidencebasednational strategy for supportive care implemented throughappointment of supportive care lead clinicians within eachUK cancer centre This alongside support from the medicalRoyal Colleges and NHS England would be fundamental indeveloping a sustainable clinical modelPerhaps working as a distinct branch of oncology specialists in supportive care medicine should have the skillsand resources to manage a broad range of effects associatedwith longterm cancers and cancer survival This paperexplores areas that are showing promise in this development and identiï¬es key next steps needed to recognisesupportive care as an indispensable component of modernoncologyDeï¬nition of Supportive CareThe Inuit may or may not have words for snow butsupportive care seems to have that number of deï¬nitions orconnotations [] Supportive care has been used as aeuphemism for palliative care and early palliative care[] and research suggests that a change in name frompalliative care to supportive care results in more and earlierreferrals to hospitalbased services [] Palliative care is anintegral component of supportive care but supportive careis much more than palliative care or even early palliativecareMASCC deï¬nes supportive care as the prevention andmanagement of the adverse effects of cancer and its treatment This includes management of physical and psychological symptoms and side effects across the continuum ofthe cancer experience from diagnosis through treatment toposttreatment care Enhancing rehabilitation secondarycancer prevention survivorship and endoflife care areintegral to supportive care []Strategy for Implementation of SupportiveCare Within Cancer CareThe potential beneï¬ts of supportive care includedecreased morbidity improved quality of life and potentially decreased mortality ie secondary to optimal cancertreatment the potential beneï¬ts for healthcare servicesinclude decreased utilisation of healthcare resources andimproved treatment outcomes [] Indeed supportivecare offers patients more than many palliative oncologicaltreatments and should be considered an essential not justan optional extraCurrently many cancer centres in the UK have supportivecare services either as a result of NHS England's EnhancedSupportive Care ESC Programme discussed below andFig The supportive care umbrellaPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxrelated Commissioning for Quality and Innovation CQUIN[] or as a result of local initiatives However the format ofthese teams is variable as is the patient cohort ie restrictedto speciï¬c cancer diagnoses and the interventions offeredie often restricted to symptom controlThus a national strategy is required to standardise supportive care services in relevant settings This needs to beevidencebased and ensure equity of care for all cancer patients irrespective of their cancer diagnosis or stage Thestrategy needs to address the current situation but alsoacknowledge evolving oncology practice ie new treatmentswith new toxicities changing disease patterns ie cancer aschronic disease and changing patient characteristicsIt needs to address education and training discussedbelow and be supported by benchmarking of servicesincluding inspections of clinical services incorporatingpatient feedback Investment will be required to standardise supportive care services and research fundingshould be allocated to determine the optimal model of careas well as the effectivenesscost effectiveness of the individual components of the servicesImplications for TrainingSupportive care encompasses the entire cancer journeyand so necessitates the involvement of most clinical specialties and many nonclinical services Figure Indeedmodern supportive care cannot be provided by a singleclinical specialty alone However as with other cancermultidisciplinary teams a dedicated core team is neededto manage everyday problems with timely input from anextended team if the need arises Importantly the coreteam needs speciï¬congoing education and training inprinciples of supportive careIt is also important to recognise that although manysupportive care services may have evolved from palliativecare services palliative care healthcare professionalsgenerally have limited formal training in supportive careand it is often not appropriate to extrapolate dataexperience from patients with advanced cancer to patientsreceiving anticancer treatment or cancer survivors Forexample the management of nausea and vomiting inadvanced cancer [] is very different from the management of chemotherapyinduced nausea and vomiting []The development of specialist supportive care servicesmust be supported by the educationtraining of the wideroncology workforce in the principles of supportive careand the management of common symptomsproblemsIndeed specialist supportive care services will only ever beable to see the tip of the iceberg and so will need to focuson more complex problems and ones requiring specialistinterventions Moreover for example it is much moreappropriate for the team that gives the oncological intervention to manage the adverse effects of that oncologicalinterventionThus supportive care needs to be incorporated into thecurricula of all healthcare professionals involved in cancercare including primary care physicians AppropriateFig The extended supportive care teamcontinuing professional development opportunities need tobe developed for these groups Patients and their familiesneed access to appropriate educational resources in order tofacilitate rapidsuccessful treatment of the complications ofthe cancer andor the cancer treatmentEnhanced Supportive Care Programme eNHS EnglandNHS England promoted early development of supportivecare within some cancer centres via the ESC CQUIN programme CQUIN is the framework supporting improvements in the quality of services and the creation of newimproved patterns of care [] ESC CQUIN was developedby The Christie NHS Foundation Trust and was based uponsix key principles for the implementation and delivery ofsupportive care Figure [] The programme developedthrough recognition of what specialist palliative care professionals working alongside other cancer care disciplinescould offer across the whole cancer pathway e and throughrecognition of barriers to achieving earlier involvement[] Palliative care and supportive care are often differentlyanised across locations on the basis of resources andtraditions In some centres the two are anised as oneservice whereas in others they are completely separate[] The ESC programme required rebranding a closercollaboration with oncology and referral within weeks ofdiagnosis of incurable cancerNHS England's Specialised Commissioning ImprovingValue Team worked with commissioners and clinical teamsin ESC development Fourteen cancer centres took part inthe ESC CQUIN over a 3year period 2016e2019 Aninterim evaluation of the scheme took place in OctoberPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx The programme was associated with a variety ofpositive outcomes including timelier referral of patientswith supportive care needs improved symptom controlimproved quality of life reduced 30day mortality fromchemotherapyimproved overall survival and reducedhealthcare costs [] ESC's principles of early referral andintervention may have impacted positively on these outcomes by better supporting patients who decide to proceedwith chemotherapy as well as those who decide not toproceedA limitation of the ESC CQUIN related to variation inservice delivery model across the centres Further robustresearch needs to be undertaken to determine the optimalapproach for delivery of supportive care services withincancer centres and in other settingsDeveloping the Research and Evidence inSupportive CareWhen the American Society for Clinical Oncology ASCOcelebrated its 50th anniversaryit listed the ï¬ve topachievements in oncology over that period Prominentlylisted was the development of highly effective antiemetictreatment [] What has been the impact of this keyadvancement in cancer supportive care and how did we getthere Does this progress guide us in improving other areasin supportive careThe impact of preventing emesis is broad and largeNausea and vomiting affect all aspects of daily living thequality of life beneï¬ts of antiemetic prevention have beendocumented Economically this advance allowed nearly allchemotherapy to be given on an outpatient basis ratherthan requiring hospitalisation This also allows people tohave less disruption and to remain with their families whilepursuing normal activitiesThese improvements are the result of thoughtful andlogical research Principles of this research included thefollowing which can be applied to many supportive caresettings i an understanding of appropriate physiology[] ii establishment of good clinical methodology []and iii evidence that affecting speciï¬c neurotransmitterpathways resulted in major clinical beneï¬t [] As aresult of this work 80e90 of patients can be spared emesisin difï¬cult settings as opposed to in the pastAs we enter an era where chemotherapy is progressivelyless used new areas for supportive care emerge Are weprepared to understand in depth unanticipated challengesin supportive care Can we prevent dermatological toxicities with tyrosine kinase inhibitormediated molecularlytargeted approaches through better understanding of themechanisms of these agents and skin physiology Can wepredict who is likely to have autoimmune sideeffects withcheck point inhibitors []Skills in caring for patients with cancer and methods oftreating malignancy continue to improve The advancesmade in preventing chemotherapyinduced nausea andvomiting provide a model that can inï¬uence approaches tomany other aspects of supportive care in cancerInterface with Acute Oncology eAmbulatory Supportive CareAdvances in cancer management continue to improvepatient outcomes This has expectedly been associatedwith an increase in emergency presentations with disease or treatmentrelated complications The challengesof emergency oncology presentations have led to an interest in developing optimal care models for meetingpatients' needs [] Cancer patients seeking emergencycare generally have higher admission rateslongerFig NHS England ESC CQUIN programme principles of ESCPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxlengths of stay and higher mortality than noncancerpatients []Ambulatory care is recognised as a key tenet in ensuringthe safety and sustainability of acute care services Thefundamental basis for ambulatory care is that patientspresenting with acute illnesses can be stratiï¬ed as low riskfor developing complications and therefore do not requiretraditional inpatient care []Individualised management of acute cancer presentations is a key issue for emergency oncology services sothat it can mirror routine cancer care [] There are anincreasing number of acute cancer presentations that can berisk assessed for care in an emergency ambulatory settingThese include lowrisk febrile neutropeniacancerincidental pulmonaryassociated deep vein thrombosisembolism chemotherapyrelated acute kidney injurychemotherapyinduced nausea and vomiting indwellingline infections acute management of pain crises malignanthypercalcaemiaabnormalitiesasymptomatic brain metastases and malignant pleuraleffusion [43e46]and otherelectrolyteAmbulatory models offer the opportunity to integratepalliative care and supportive care with oncology and acuteservices This facilitates improved access for patients toexpertise in cancer care and immediate management of thecomplications of cancer treatment with the goal of preventing downstream complications and future emergencypresentations For example ambulatory enhanced supportive care models have shown utility in the managementof lowrisk febrile neutropenia []Modelling of ambulatory emergency oncology serviceswithin integrated supportive care services is therefore keyin the provision of highquality personalised and sustainable emergency oncology careThe Importance of Supportive Care inExperimental Cancer MedicineExperimentalcancer medicine trialsECMTs arefundamental to the development of novel cancer therapiesThe primary aims of ECMTs are to identify treatmentrelated toxicities and determine the recommended drugdose [] These trials are increasingly complex []intensive with risks of toxicity for patients but there is agrowing recognition that they are a valid therapeutic option []ECMTs have strict eligibility criteria with the need forpatients to have a performance status of or indicatinghigh levels of day to day functioning [] However thesepatients typically have advanced disease multiple previouslines of treatment and therefore a high associated symptom burden [] Hui [] found that patients referredfor ECMTs have a similar symptom burden to those whowere not despite the perception of higher levels of ï¬tness Ahigh symptom burden has also been associated with earlydiscontinuation from trials [] highlighting the potentialrole for supportive care Br 13edart [] suggested thatthis patient group is more likely to accept increased toxicityto facilitate continued access to trial drugs In one studyECMT patients stated that they would still participate in atrial despite the potential risk of serious toxicities and a chance of death []Research suggests that ECMT patients are less inclined toaccept traditional palliative care due to a general andsometimes unrealistic optimism regarding trial participation[] alongside the perception that palliative care is onlyapplicable at the end of life [] However supportive carepractices within the early phase trials setting have the potential to reduce the impact of symptom burden and adverseevents on patients [] potentially increasing trial recruitment and the length of time patients spend on an experimental therapy Evidence in an ongoing study by Ferrell et al[] indicates that additional support can improve thequality of life for this patient group On top of the beneï¬t topatients of access to additional therapies prolonged exposure to trial drugs supports research through increasednumbers of evaluable patients aiding efï¬cient and accurateassessment of novel therapies Thus there is growing evidence for the role of supportive care for ECMT patients withthe need for increased research to assess potential beneï¬tsand identify optimal routes for its deliveryLearning from Other CountriesImplementation of Supportive Care inFranceWith the aim of increasing and improving communityinvestment in supportive care MASCC is promoting severaldifferent approaches to engage countries such as 0f The creation of accreditation for hospitals withdedicated supportive care units 0f Promotion of MASCC and collaboration with localassociations at MASCC meetings 0f Special links with these associations such as jointmembershipsFrance committed to the supportive care approach at theend of the 1990s and as part of its ï¬rst cancer plan in The French Speaking Association for Supportive Care inCancer AFSOS afï¬liated to MASCC was created in with the objectives ofaccompaniment 0f Promoting knowledge and execution of supportivecare in oncology 0f Sharing experience with all professionals involved insymptomsthethroughout all phases of the disease 0f Identifying and understanding the impact of thetransferability and interdependency between disciplines facilitating key aspects obstacles interestsand limitations of work 0f Heightening ethical awareness among medical staffand careofAFSOS has set up a research committee with four strategic priority directions healthcare anisation crossPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxxdisciplinary meetings and departments supportive careunits dedicated teams management of cancer symptomsand treatments health behaviour and human and socialsciencesIts actions are targeted towards institutions eg TheFrench National Cancer Institute Ministry of Health professionals guidance and symposia on speciï¬c topics such asemesis or nutritional disorders as well as patients andtheir speciï¬c associations through a patientfacing website a roadshow truck crisscrossing France and an inventoryof supportive care resources AFSOS has developed nationalmeetings devoted to physicians and nurses physiotherapists or other health caregivers Guideline resources with atoolkit app are discussed during a speciï¬c 2day event andupdated every years AFSOS is involved in promoting international collaboration with other MASCCafï¬liated societies eg Network Italiano Cure di Supporto in Oncologia[NICSO] and the Japanese Association of Supportive Care inCancer [JASCC]This French national mobilisation has led many regionalteams to get involved in cancer safety management projectsfor the beneï¬t of patients and their relatives and can becopied in other countriesof these is poor in the UK [] and there are a number ofreasons why this may be even lower in an oncology setting[] The risk of poor bone health and fracture is increasingly recognised across a number of malignancies forexample a recent large Danish registry study showedincreased risk of fragility fracture in adults with haematological malignancy with the largest risk in the ï¬rst 2e4years following initiation of treatment [] Given thedevastating nature of fractures there is much supportivecare work to be done to identify and treat at risk patientsand manage fragility fractures effectively across the spectrum of the cancer journeyEndocrinologists have had a traditional role in cancersurvivorship [] For example managing the longtermeffects of brain radiotherapy on the pituitary gland inchildhood brain tumour survivors As the prognosis foradult brain tumour survivors improves similar issues mayarise [] More recently endocrine toxicities such ashypophysitis and insulindeï¬cient diabetes caused byimmunotherapy treatments are also keeping endocrinologists busy [] in collaboration with acute oncology []This will be become an even more complex issue asimmunotherapy moves into the adjuvant arena with expertinput into decision making algorithms crucial []Interface with Other Specialities egEndocrinology and DiabetesDiscussionOptimal supportive care of cancer patients requires inputfrom a range of specialties outside of oncology to assistaccurate diagnosis and management and ultimatelyimprove outcomesUp to of inpatients with cancer have diabetes or areat risk of diabetes from the treatments they receive []The importance of this is increasingly recognised patientswith diabetes and cancer have an increased length of hospital stay [] and mortality [] Although there iscurrently a lack of data demonstrating that improving glycaemic control reduces mortality for cancer patients it iscertainly true that effective and timely management ofhyperglycaemia improves quality of life and reduces inpatient length of stay but this requires specialist input from adiabetes teamSimilarly up to of inpatients with cancer experiencehyponatremia commonly secondary to syndrome of inappropriate antidiuretic hormone secretion although in theera of immunotherapy cortisol deï¬ciency is an importantand increasing cause which can be fatal if missed []Untreated hyponatremia can delay oncology treatmentsand extend the length of hospital stay [] Diagnosis andmanagement of hyponatremia is poorly managed in general and the oncology population are no exception [] Weconsider expert supportive care input into the managementof hyponatremia in oncology patients to be essential inimproving this situationFractures particularly those of the hip and spine aredevastating with up to mortality at year following hipfracture and significant ongoing morbidity Vertebral fractures are highly predictive of further fracture but reportingThe current focus of cancer care is on initial diagnosisand treatment and the last year of life end of life care []However a large proportion of patients with cancer experience debilitating morbidity and complex symptomsresulting from cancer andor its treatment across the entirecancer journey Supportive care has been shown to improvequality of life symptom burden and survival as well asbeneï¬tting the health economy [15e17] Thus supportivecare should be an integral component of modern oncologymanagement and should involve input from a range ofspecialties within and outside of oncology Furthermore itscontinued development perhaps most effectively as a subspecialty of oncology is essential in supporting advances inoncology and the changing demographic of the cancerpopulationConï¬icts of interestR Berman is a director of Supportive Care UK Ltd Thisis outside the scope of the submitted workReferences[] Macmillan Cancer Support Living after diagnosis mediancancer survival times Available at wwwmacmillanukdocumentsaboutusnewsroomlivingaftercancermediancancersurvivaltimespdf[] Cancer Research UK Cancer survival statistics Available atwwwcancerresearchukhealthprofessionalcancerstatisticssurvivalheadingZeroPlease cite this as Berman R Supportive Care An Indispensable Component of Modern Oncology Clinical Oncology 101016jclon202007020 0cR Berman Clinical Oncology xxx xxxx xxx[] National Cancer Survivorship Initiative NCSI Living withand beyond cancer taking action to improve outcomesAvailableassetspublishingservicegovukgovernmentuploadssystemuploadsattachment_dataï¬le1810549333TSO2900664NCSI_Report_FINALpdfat[] Clarke G Johnston S Corrie P Kuhn I Barclay S Withdrawal ofanticancer therapy in advanced disease a systematic literature review BMC Cancer [] Klastersky J Supportive care do we need a model Curr OpinOncol [] Global Burden of Disease Cancer Collaboration The GlobalBurden of Cancer JAMA Oncol 20151505e527[] National Audit Ofï¬ce Delivering the cancer reform strategyAvailable at wwwnaoukreportdeliveringthecancerreformstrategy[] Mokart D Pastores SM Darmon M Has survival increased incancer patients admitted to the ICU Yes Intens Care Med2014401570e1572[] National Institutes of Health Cancer trends progress report e update National Institutes of Health [] Sullivan R Peppercorn J Sikora K Zalcberg J Meropol NJAmir E Delivering affordable cancer care in highincomecountries Lancet Oncol 201112933e980[] Yabroff Y Lund J Kepka D Mariotto A Economic burden ofcancer in the US estimates projections and future CancerEpidemiol Biomarkers Prev 201120102006e2014[] Aggarwal A Sullivan R Affordability of cancer care in theUnited Kingdom e is it time to introduce user chargesJ Cancer Policy 2014231e39[] Saini K Heras B Castro J Venkitaraman R Poelman MSrinivasan G Effect of the COVID19 pandemic on cancertreatment and research Lancet Haem 202076e432ee435[] Radcliffe E Khan A Wright D Berman R Demain S RestorickBanks S Understanding the importance of selfmanagement support in people living with cancer reportReport on the impact of COVID19 in press[] Monnery D Benson S Grifï¬ths A Cadwallader C HamptonMatthews J Coackley A Multiprofessionaldeliveredenhanced supportive care improves quality of life for patientswith incurable cancer Int J Palliat Nurs 20182410510e514[] Basch E Deal AM Dueck AC Scher HI Kris MG Hudis C et alOverall survival results of a trial assessing patientreportedoutcomes for symptom monitoring during routine cancertreatment JAMA 20173182197e198[] Cooksley T Campbell G AlSayed T LaMola L Berman RA novel approach to improving ambulatory outpatient management of low risk febrile neutropenia an Enhanced Supportive Care ESC clinic Support Care Cancer 2937e2940[] Klastersky J Christel F Editorial Supportive care in cancerpatients a constantly evolving ï¬eld Curr Opin Oncol 314257e258[] Cooksley T Rice T Emergency oncology development current position and future direction in the USA and UK SupportCare Cancer 2017253e7[] Guly H Preface A history of accident and emergency medicine1948e2004 London Palgrave Macmillan [] Hui D Hannon BL Zimmerman C Bruera E Improving patientand caregiver outcomes in oncology teambased timely andtargeted palliative care CA Cancer J Clin 201868356e376[] Whelan TJ Mohide EA Willan AR Arnold A Tew M Sellick S The supportive care needs of newly diagnosed cancerpatients attending a regional cancer center Cancer 8081518e1524[] Hui D De La Cruz M Mori M Parsons HA Kwon JH TorresVigil I Concepts and deï¬nitions for supportive carebest supportive care palliative care and hospice care inthe published literature dictionaries and textbooks SupportCare Cancer 201321659e685[] Boyd K Moine S Murray SA Bowman D Brun N Shouldpalliative care be rebranded B | Thyroid_Cancer |
"Handling EditorAdrian CovaciKeywordsNuclear accidentsHealth surveillancePreparednessCommunicationStakeholdersRecommendationsSerious accidents at nuclear power plants have been rare but theirstories can teach us how to prevent or mitigate the eï¬ects of futurenuclear catastrophes The accidents at the Fukushima Daiichi nuclearpower plant and Chernobyl nuclear power plant occurred years and years ago respectively and there are still lessons to learn from themregarding numerous issues including radiation exposure assessmentand medical followup of emergency responders evacuees and residents decisions to lift evacuation orders and communication withresponders and stakeholders Bazyka Callen and Homma Lester Soï¬er Some of the lessons from theseaccidents have been extensively reviewed and taken into considerationby national and international anizations such as the InternationalAtomic Energy Agency the International Commission on RadiologicalProtection and the World Health anisation and are reï¬ected inpublished literature Bennett Carr Clarke IAEA 2015a 2015b Nisbet SGDSN This hasallowed the development of various recommendations and guidancedocuments targeting speciï¬c issues of radiation protection training andcommunication and socioeconomic aspects in order to prepare andimprove decision making processes in the early and intermediatephases eg Carr IAEA 2015b Nisbet However the majority of these texts focus on technical issues andare directed towards radiation protection experts rather than for thesupport of aï¬ected populations The traditional approaches of emergency response and recovery including evacuation relocation andhealth surveillance are largely based on dose levels Although manyrecognise the importance of psychosocial or human factors it has beendiï¬cult to adapt the approaches to better address the social economicethical and psychological factors These include the health and welfareeï¬ects that may arise from the accident from the concerns about thepresence of radiation in the environment from the mitigation actionstaken and from the information mixed or absent provided to the population Changes in the ethical and legal requirements for personaldata collection use and storage raise additional challenges particularlyin the area of health surveillance and epidemiologyAbbreviations COVID19 Coronavirus Disease EJP CONCERT European Joint Programme for the Integration of Radiation Protection Research OPERRA Project for the European Radiation Research Area SGDSN Secrtariat gnral de la dfense et de la scurit nationale France SHAMISEN NuclearEmergency Situations Improvement of Medical and Health Surveillance SHAMISEN SINGS SHAMISEN Stakeholder INvolvement in Generating Science UN UnitedNations UNDRR United Nations Oï¬ce for Disaster Risk Reduction101016jenvint2020106000Received July Accepted July Published by Elsevier Ltd This is an access under the CC BYNCND license httpcreativecommonslicensesBYNCND40 0c Main components of the SHAMISEN projectThe SHAMISEN project started in late at a time when somedeleterious eï¬ects of evacuation and ultrasound thyroid screening inFukushima had started to be reported The project therefore aimed toreview the lessons learned from major nuclear accidents in particularfrom experiences of populations aï¬ected by the Chernobyl andFukushima accidents to develop recommendations for medical andhealth surveillance of populations aï¬ected by previous and future radiation accidents The ultimate motivation was to minimise the negative impacts of the accident and improve the health of aï¬ected populations The holistic WHO deï¬nition of health was used in this contextie a state of complete physical mental and social wellbeing and notmerely the absence of disease or rmity WHO The Recommendations were to address in particular the following complementary aspects dose assessment supporting all phases of an accident including emergency response clinical decisionmaking recoveryactions and health surveillance improvement of living conditions ofaï¬ected populations engaging them and responding to their needs andminimising unnecessary anxiety and health surveillance and wherefeasible improvement of estimates of radiationinduced risk for radiation protection and communication with aï¬ected populationsTo achieve this and recognising the need for a holistic approach toaccident management and health surveillance SHAMISEN brought together a team of researchers from institutions including RadiationProtection Authorities Universities Research Centres and Associationsin Europe and Japan with complementary experience and a long trackrecord in post accidental management dosimetry radiation protectionmedical followup and screening population health surveillance healtheconomics epidemiology ethics and sociology of radiation protectionThe project also drew upon additional expertise from Belarus RussiaUkraine Japan and the UK as well as from outside of the radiationresearch ï¬eld and established contacts with major international anizations including the World Health anisation and the NuclearEnergy Agency of the anisation for Economic Cooperation andDevelopmentDetails of the SHAMISEN Project are provided in the paper by Ohbaand collaborators Ohba this issue Brieï¬y the approach involved in particular challengingevaluating the eï¬ectiveness of measures taken after Chernobyl or Fukushima accidents in particularcid129 Systematic thyroid screening with ultrasound for early detection ofpotential thyroid cancer casescid129 Criteria for evacuation and their consequencescid129 Measures taken to contribute to the wellbeing of aï¬ected populations and develop a radiological protection culture and resilience inaï¬ected populationscid129 Challenges and good practice in communication and training withthe objective of regaining the trust of the population and engagingthem in retaking control of their livescid129 Role of ethics in disaster preparedness response and health surveillance autonomy and dignity respect of privacy beneï¬cence¦cid129 Role of health professionals in the diï¬erent phases of the accidentmanagementcid129 Cost eï¬ectiveness of the measures takenAll of this was brought together to develop practical recommendations for preparedness and the diï¬erent phases of the accident SHAMISEN results and the way forwardAlthough the SHAMISEN project was developed during a limitedperiod of months in response to the second call of the EuropeanOPERRA project a series of key results has been achieved The mainresults are several topical reports and a set of recommendations dividedinto ï¬ve main topics eg Evacuation Communication and trainingEnvironment International Dosimetry Health surveillance Epidemiology These topics focusspeciï¬cally on the health surveillance of people following a nuclearaccident combining natural and social sciences values and practice tohelp health professionals decisionmakers and local stakeholders to setup protective actions and health programmes responding to the concernof aï¬ected populations Therefore the SHAMISEN recommendationsare not intended to cover all aspects of emergency and recovery response and preparednessThe formulation of the recommendations is generic enough to beapplied in diï¬erent countries recognising that cultural diï¬erences willbe important The structure describes the general context and the mainreasons for developing each recommendation provides explanations onhow to develop it and indicates who would be involved in the development of the recommendation Depending on the context speciï¬carrangements have to be made for the implementation of these recommendations during the diï¬erent phases eg preparedness earlyand intermediate longterm recoveryThe recommendations provide advice on the values to be consideredwhen addressing the issue at stake and what type of tools and protocolsare needed rather than the tools themselves Due to the duration of theproject it was not manageable to develop them However they providea significant input for further developments for practical tools in different domains and identify the main expectations from stakeholderswith regards to health surveillance in postaccident situationsDeveloped as a research project it was not the intention to specifyabsolute dose criteria for the implementation of actions Of coursediscussions on the feedback experience from the management of theChernobyl and Fukushima accidents point out some challenges associated with the use of speciï¬c dose criteria but the spirit of the recommendations is to provide indications and guidance for the decisionmakers and health professionals with regard to the choice to be madeon the adoption of dose criteria for the diï¬erent actions to be implementedBesides the management of the direct radiation induced health effects the report underlines the need to develop a multidisciplinaryapproach to identify measure assess and alleviate psychological andother indirect health impacts of socioeconomic and social upheavals ofthe consequences of the accident For this purpose it is recommendedto promote the engagement process of local stakeholders since thepreparedness phase targeting the overall wellbeing of populations withdue considerations of the ethical principles of respect for autonomydignity and justiceThis special issue of Environmental International combines a series ofscientiï¬c papers and is an opportunity to emphasize the main analysesdeveloped during the SHAMISEN project combining advanced scientiï¬c research analyses of feedback experience from the Chernobyl andFukushima accidents and applying a multidisciplinary approachAlthough the topics presented in this special issue have already beenaddressed in general in several papers the originality of the approachadopted in the SHAMISEN project provides new insights for healthsurveillance issuesIt is worth mentioning that following the SHAMISEN project otherresearch projects have been launched A ï¬rst series of projects arededicated to the development of Apps with and for citizens as recommended in the SHAMISEN project This has notably been done withthe European research project SHAMISEN SINGS as part of the EJPCONCERT as well as with an ongoing project developed by FukushimaMedical University In addition several projects are currently underdevelopment in diï¬erent countries and at the European level thatpromote a citizen science approach for addressing health and radiological monitoringAs an example of the multidisciplinary approach the SHAMISENproject has identiï¬ed a series of recommendations calling for furthercooperation with diï¬erent European Research Platforms combininglow doses eï¬ects dosimetry radioecology emergency and recoverymanagement social sciences and humanities and medical research 0cEnvironment International and Thierry Schneidera Deborah Oughtonb Elisabeth Cardiscdea CEPN Nuclear Protection Evaluation Centre Rue de la Redoute FontenayauxRoses Franceb Centre for Environmental Radioactivity CERAD NMBU «s Norwayc Barcelona Institute for Global Health ISGlobal Barcelona Spaind Pompeu Fabra University Barcelona Spaine Spanish Consortium for Research and Public Health CIBERESP Institutode Salud Carlos III Madrid SpainEmail address thierryschneidercepnassofr T SchneiderReferencesBazyka D Belyi D Chumak A Lessons from chornobyl considerations forstrengthening radiation emergency preparedness in Ukraine Radiat Prot Dosim 101093rpdncw196Bennett B Repacholi M Carr Z Eds Health eï¬ects of the Chernobyl accidentand special health care programmes Report of the UN Chernobyl Forum expert groupHealth WHO Press GenevaCallen J Homma T Lessons learned in protection of the public for the accident atthe Fukushima Daiichi nuclear power plant Health Phys 101097HP0000000000000666Carr Z Clarke M Akl EA Schneider R Murith C Li C ParrishSprowl J StenkeL CuiPing L Bertrand S Miller C Using the grade approach to supportthe development of recommendations for public health interventions in radiationemergencies Radiat Prot Dosim 101093rpdncw234Clarke R Valentin J International Commission on Radiological Protection Task Group ICRP publication Application of the Commissions Recommendations forthe protection of people in emergency exposure situations Ann ICRP 101016jicrp200905004Croua¯l P Camps J Raskob W Schneider T NERIS Roadmap on medium andlongterm research on preparedness for nuclear and radiological emergency responseand recovery Version eunerisnetlibrarysra259nerisroadmap2020htmlIAEA 2015a The Fukushima Daiichi Accident No Technical Volume IAEAInternational Atomic Energy Agency Vienna Austria wwwiaeapublications10962thefukushimadaiichiaccidentIAEA 2015b Preparedness and Response for a Nuclear or Radiological Emergency NoGSR7 Safety Standards Series IAEA International Atomic Energy AgencyVienna Austria httpwwwpubiaeaMTCDPublicationsPDFP_1708_webpdfImpens N Salomaa S Second joint roadmap for radiation protection researchDeliverable No EJPCONCERT European Joint Programme for the Integrationof Radiation Protection Research H2020 wwwconcerth2020euDocumentashxdtwebï¬leListsDeliverablesAttachments206D37_Second20joint20roadmap_draft_reviewed_20052020_approved03062020pdfguid01b5ac77b2ec4cda9c98917dba396f0fLester MS Public information during a nuclear power plant accident lessonslearned from Three Mile Island Bull N Y Acad Med Nisbet AF Jones A Turcanu C Camps J Andersson KG H¤nninen RRavantaara A Solatie D Kostiainen E Julien T Pupin V Ollagnon HPapachristodoulou C Ioannides K Oughton D Generic Handbook forAssisting in the Management of Contaminated Food Production Systems in Europefollowing a radiological emergency v2 No CAT1TN0901 EURANOS eunerisnetlibraryhandbookshtmlOhba T Liutsko L Schneider T Tanigawa K Fattibene P Laurier D Sarukhan ABarquinero J Kesminiene A Skuterud L Cardis E this issue The SHAMISENProject challenging historical recommendations for preparedness response andfollowup of nuclear accidents lessons learnt from Chernobyl and FukushimaSGDSN National response plan Major nuclear or radiological accident httpwwwgouvernementfrsitesdefaultï¬lesrisquespdfnational_plan_nuclear_radiological_accidentspdfSoï¬er Y Schwartz D Goldberg A Henenfeld M BarDayan Y Populationevacuations in industrial accidents a review of the literature about four major eventsPrehosp Disaster Med UNDRR The Sendai Framework for Disaster Risk Reduction UnitedNations Oï¬ce for Disaster Risk Reduction wwwundrrpublicationsendaiframeworkdisasterriskreduction20152030WHO WHO Deï¬nition of Health World Health anisation Geneva httpwwwwhointaboutdeï¬nitionenprinthtmlThese recommendations have already been considered in the development of the European joint roadmap for radiation protection researchImpens and Salomaa and of the strategic research agenda ofdiï¬erent European platforms notably NERIS on emergency and recovery Croua¯l The results of the SHAMISEN project have been presented and discussed in several national and international workshops and meetingsRecommendations are being disseminated to decision makers and radiation protection authorities for translation into strategy and policy aswell as to scientiï¬c medical and nonexpert audiences They are nowreferred and used as basis of the reï¬ections and the initiatives of national and international anizations for both preparedness NuclearEnergy Agency World Health anisation International Commissionon Radiological Protection ICRP National committee for postaccident management CODIRPA in France and the management of theFukushima situation with a key role of the Japanese partners involvedin the SHAMISEN project Fukushima Medical University NagasakiUniversity Hiroshima UniversityMore broadly the approach adopted in the SHAMISEN project andits results may contribute to address other hazards including naturaldisasters industrial accidents or even pandemic crisis Similarities canbe emphasized with the Sendai Framework for Disaster Risk Reduction UNDRR adopted at the 3rd UN World Conference in This framework underlines the importance of improving theunderstanding of disaster risk better addressing vulnerability and hazard characteristics strengthening risk governance reinforcing accountability for risk management with development of preparednessinvolvement of stakeholders and due considerations of resilience ofhealth infrastructureFinally the pandemic crisis of COVID19 highlights a series of issuesquite similar to those addressed in the SHAMISEN project conï¬nementversus evacuation psychosocial aspects communication and dialogueanisation of the transition phases and of course the preparation ofhealth surveillance strategies and structures of epidemiological studiesThese diï¬erent issues would beneï¬t from crosscomparison analysisand the s presented in this special issue could certainly contributeto the reï¬ectionDeclaration of Competing InterestThe authors declare that they have no known competing ï¬nancialinterests or personal relationships that could have appeared to uence the work reported in this paperAcknowledgmentsSHAMISEN was supported by the EURATOM European AtomicEnergy Community program of the European Commission in the framework of the OPERRA Project for the European RadiationResearch Area project FP7 grant agreement No The authors are grateful to all partners experts and stakeholderscontributed to theandorwho participated in the projectRecommendationsISGlobal acknowledges supportfrom the Spanish Ministry ofScience Innovation and Universities through the Centro de ExcelenciaSevero Ochoa Program CEX2018000806S and supportfrom the Generalitat de Catalunya through the CERCA Program NMBUacknowledges the support of the Research Council of Norway RCNthrough its Centres of Excellence funding scheme project number Corresponding author 0c" | Thyroid_Cancer |
Hepatocellular carcinoma HCC is a high mortality disease the fifth most general cancer worldwide and the second leading to cancerrelated deaths with more than new patients diagnosed each year First the high expression of centromere M CENPM in mammary gland tissue of bcatenin transformed mice was identifiedMaterials and methods In our study we evaluated the expression of CENPM in hepatocellular carcinoma based on data obtained from an online database Multivariate analysis showed that the expression of CENPM and M classification was an independent prognostic factor for patients with hepatocellular carcinomaResults Survival analysis showed that patients with high CENPM had a worse prognosis than patients with low CENPM P A multivariate Cox regression hazard model showed that B cells CD8 T cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer P Using the network the most frequently changed neighbor genes of CENPM were shown and the most common change was RAD21 Conclusion Our study found that the expression of CENPM was significantly increased in patients with hepatocellular carcinoma and it was related to a variety of clinical characteristics its correlation with the level of immune infiltration and poor prognosis so CENPM can be used as a useful prognosis for patients markers and HCCKeywords Hepatocellular carcinoma Centromere protein M Data mining PrognosisBackgroundHepatocellular carcinoma HCC a high mortality disease which is the fifth most general cancer in the world and the second most common lead to cancerrelated deaths with over new patients diagnosed each year [ ] Viral hepatitis and nonalcoholic steatohepatitis are the most common causes of cirrhosis and approximately of cases develop to HCC [] Due to the recurrence of HCC the prognosis of HCC remains discouraging and the 5year overall survival rate which Correspondence wawang123soutlookcomDepartment of Infectious Diseases Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Chinais only to [] Despite the rapid development of advanced medical technology there are still no useful curable strategies for HCC patients [] Byeno et a0al [] reported that based on longterm survival data serum OPN and DKK1 levels in patients with liver cancer can be deemed as novel biomarkers that show prognostic useful for liver cancer Other serum markers such as alphafetoprotein AFP and alkaline phosphatase ALP or AKP are proverbially used in clinical but they lack sufficient sensitivity and specificity [] Therefore finding useful biomarkers is indispensable for diagnosis and treatment for HCC patientsPosttranscriptional modifications are essential for tumorigenesis and development Centromere protein M CENPM otherwise called PANE1 CENPM and The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWu a0and Yang Cancer Cell Int Page of C22orf18 which encodes a kinetic protein binds to spindle microtubules to regulate chromosomal separation during cell division [] Expression of the PANE1 gene was found preferentially in immune cells involving tumor tissues and tumor derived cell lines and leukemias and lymphomas [] Brickner et a0 al [] found highly expressed in B lineage chronic lymphocytic leukemia BCLL cells and resting CD19 B cells may be a potential therapeutic target for BCLL Bierie et a0al [] also demonstrated that human CENPM transcript cRNA was detected only in a0vivo or in a0vitro in activated B cells and T cells These studies suggested CENPM may play critical role in tumor immune response and may be deemed to therapeutic target for immunotherapy However the role of CENPM in HCC prognostic remains unclear In our study we evaluated the expression of CENPM in HCC based on data from an online database to further understand the biological pathway of CENPM related to the pathogenesis of HCC In addition we also analyzed the connection between CENPM expression and clinical features as well as the correlation of its expression with immune infiltration level in HCC comes an online tumor infiltrating immune cells analysis toolMaterials and a0methodsData collectionInformation on RNAsequencing data tissues workflow type HTSeqCounts and comparative clinical data patients data format BCR XML were identified and got from the level standardized FPKM of the TCGAHCC cohort Use boxplots to imagine expression differences for discrete variables [] The clinical factors included gender stage age grade Tphase Mphase Nphase survival status and number of days of survival Data analysis were checked by R version and R Bioconductor software packagesGSEA enrichmentGene Set Enrichment Analysis GSEA created a list of all gene permutations related to CENPM expression The samples were then divided into a high CENPM group and a low CENPM group as training sets to distinguish potential functions and use GSEA to clarify significant survival differences Genome replacement is performed multiple times with each exam The degree of expression of CENPM was used as a phenotypic marker Normalized enrichment scores NES and nominal Pvalues have been used to classify the pathways of enrichment in each phenotypeImmune infiltrates analysisTIMER [] is a comprehensive database for the systematic study of immune infiltration in various malignant tumor types The abundance of immune infiltrates CD8 T cells B cells CD4 T cells macrophages neutrophils and dendritic cells was evaluated by our statistical methods and has been estimated using pathology Methods evaluated it The network also enables users to explore the clinical relevance of one or more tumor immune subpopulations and has the flexibility to correct multiple covariates in a multivariate Cox proportional hazard model Meanwhile we contrast the differential level of CENPM between tumors and normal on all TCGA tumorsUALCAN and a0cBioPortal analysisUALCAN [] is a userfriendly intelligent network asset for analyzing discovering cancer data and indepth analysis of TCGA gene expression information One of the highlights of the portal is that it allows users to found between biomarkers or computer approval of potential genes of interest and to evaluate genes in different clinical subgroups such as gender age race tumor grade etc expression cBioPortal [] is an online free asset that can visualize analyze and download largescale cancer transcription datasets The portal included cancer studies The tab biological interaction network of CENPM and its coexpressed genes was got and neighboring genes with altered frequencies were containedTargetScan analysisTargetScan [] is a web for predicting potential biological targets of miRNAs TargetScanHuman deems that the match to human ²UTR and its orthologs is estimate by a UCSC genomewide adjustment As an alternative they are ranked according to their predicted conservative positioning possibilities FunRich [] is a tool designed to process varieties of geneprotein datasets in spite of the anism and used for functional enrichment analysis We used Funrich tools for miRNA enrichment analysis including analysis of biological pathways biological processes BP cellular components CC and molecular functions MFStatistical analysisScatter plots and paired plots visualize the differences between normal and tumor samples Use delete ways to handle disappeared data and if any individual value is disappeared the data will exclude the full sample The relationship between clinical factors and CENPM was used by logistic regression Wilcoxon rank sum test and Kruskal test Multivariate Cox analysis was used to assess the effect of CENPM expression on survival and other clinical factors such as age gender stage distant metastasis BenjaminiHochbergs means of converting P values to FDR 0cWu a0and Yang Cancer Cell Int Page of ResultsPatients characteristicsThe TCGA database contains patients The clinical and pathological properties of these samples are shown in Table a0 The middle age at diagnosis in TCGA was a0 years old range a0 years and median finally contact for subjects was a0 months range a0months Meanwhile followup for subjects conformed alive and death patients Our study cohort included female and Table TCGA hepatic carcinoma patient characteristicsClinical characteristicsAge at diagnosis yearFutime monthGender Female MaleStage I II III IV NAGrade G1 G2 G3 G4 NATclassification T1 T2 T3 T4 TX NAMclassification M0 M1 MXNclassification N0 N1 NX NAStatus Alive DeathData express as mean minmaxTotal male patients Stage I was located in patients stage II in stage III in and stage IV in Tumor stage was found T1 in patients T2 in T3 in and T4 in Node stage contained N0 in and N1 in of cases had distant metastases All the subjects were adenomas or adenocarcinomasCENPM expression and a0clinical factorsScatter plot showing difference in CENPM expression among normal and tumor samples P we then use paired plot to demonstrated the CENPM expression between normal and tumor from the same patients and the results was significant difference P Fig a01a b The outcomes suggested that the expression of CENPM was significant difference The expression of CENPM correlated significantly with the patient grade P clinical stage P and Tclassification P Fig a01df Univariate analysis utilizing logistic regression uncovered that CENPM expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors Table a0 CENPM expression in HCC as appreciably connected with grade OR CI G1 vs G3 stage OR CI I vs III and Tclassification OR CI T1 vs T3 indicated that patients with low CENPM expression are inclined to advance to a further advanced stage than those with high CENPM expressionSurvival and a0multivariate analysisSurvival analysis found that HCC with CENPMhigh had a worse outcome than that with CENPMlow P Fig a0 1c The univariate analysis suggested that CENPM linked essentially to stage HR CI P and Tclassification HR CI P Table a0 Multivariate analysis showed that the expression of CENPM HR P and M classification HR P were independent prognostic factors for patients with HCC Table a0GSEA analysisTo identify useful pathways that may be differentially initiated in liver cancer we performed a GSEA analysis between low and high CENPM expression datasets We chose the most abundant signaling pathway depending on the standardized enrichment score NES Table a0 The results showed that CENPM high expression differentially enriched cell cycle DNA replication RNA degradation certain cancers phagocytosis P53 signaling pathway and purine metabolism Fig a0 0cWu a0and Yang Cancer Cell Int Page of Fig CENPM expression and the association among clinicopathologic factors a The scatter plot showed the difference of CENPM expression between normal and tumor samples P b paired plot to demonstrated the CENPM expression between normal and tumor from the same patients and the results was significant difference P c Survival analysis P d Grade e Stage f TstageTable CENPM expression associated with a0pathological characteristics logistic regressionclinical Clinical characteristicsTotal NAge vs ¤ Gender female vs maleGrade G1 vs G3Stage I vs IIITstage T1 vs T3Odds ratio in a0CENPM expression PvalueCategorical dependent variable greater or less than the median expression levelImmune infiltrates related to a0CENPM in a0HCCThe correlation between CENPM liver cancer in expression and the abundance of immune infiltrates was statistically significant P Fig a0 3a A multivariate Cox proportional hazard model showed that Bcells CD8 T cells macrophages and dendritic cells infiltrated by immune cells were statistically significant in liver cancer P indicating that these immune cells significantly affect the prognosis it is worth further research and exploration Table a0 At the same time the expression of CENPM was also statistically significant P Finally we compared CENPM expression between various tumors and normal tissues The results showed that CENPM was overexpressed in various cancers P Fig a03bAssociations survival Table a and a0 clinicopathologic characteristics in a0 TCGA patients using Cox regression b Multivariate survival model after a0variable selectionwith a0overall Clinicopathologic variableHR CIPvaluea Age continuous Gender female vs male Stage IIIIIIIV Grade G1G2G3G4 Tclassification T1T2T3T4 Distant metastasis M0M1MX Lymph nodes N0N1NX CENPM expression high vs lowb Distant metastasis M0M1MX CENPM expression high vs low UALCAN and a0cBioPortal analysis in a0HCCIn the age subgroup normal age a0years normal age a0years normal age a0years and normal age a0 years among patients with liver cancer CENPM has substantially higher transcription levels than healthy individuals Analysis in the weight subgroup gender subgroup ethnicity subgroup tumor grade subgroups analysis also showed significantly higher CENPM in HCC patients Fig a0 In order to determine the 0cWu a0and Yang Cancer Cell Int Page of Table Gene sets enriched in a0phenotype highGene set nameKEGG_CELL_CYCLEKEGG_DNA_REPLICATIONKEGG_RNA_DEGRADATIONKEGG_BLADDER_CANCERKEGG_NON_SMALL_CELL_LUNG_CANCERKEGG_THYROID_CANCERKEGG_FC_GAMMA_R_MEDIATED_PHAGOCYTOSISKEGG_P53_SIGNALING_PATHWAY KEGG_PURINE_METABOLISMSizeNESNOM PvalFDR qvalNES normalized enrichment score NOM nominal FDR false discovery rate Gene sets with NOM Pval and FDR qval are considered as significantbiological interaction network of CENPM in liver cancer we used the network in the Network tab in cBioPortal showing the most frequently changed neighbor genes in CENPM and the most common change was RAD21 Fig a0 and Table a0miRNAs related to a0CENPMAccording to the online database the top of the miRNA families are hsamiR13075p hsamiR449b3p and hsamiR67785p related to the gene CENPM The conserved sites of the miRNA family that are widely conserved in vertebrates Fig a06a Using the Funrich database to explore the function of the identified miRNAs BP are significantly enriched in the regulation of nucleobases signal transduction cell communication transport regulation of gene expression and anogenesis CC are mainly concentrated in the nucleus cytoplasm Golgi apparatus endosome actin cytoskeleton and early endosome The MF are mainly transcription factor activity transcription regulation activity protein serine GTPase activity and ubiquitinspecific protease activity rich biological pathways in the ErbB receptor signaling network TRAIL signaling pathway Glypican pathway and syndecan1 mediated signaling events and signal transduction events mediated by hepatocyte growth factor receptor cMet Fig a06beDiscussionIn this work we performed a detailed assessment of CENPM expression in hepatocellular carcinoma based on the TCGA database and explored its relationship with clinicopathological features survival function immune infiltration and expression differences Understanding whether higher expression biomarkers in tumors are directly related to hepatocellular carcinoma can help us understand the mechanism of the observed clinical survival patterns In our findings the significant expression of CENPM suggests that CENPM may play an important CENPM is an role in regulating cancer progression This should draw attention to current views on the improvement of liver cancer and may reveal potential biomarkers or indicators to determine prognosisindispensable centromere protein involved in centromere assembly which regulates mitochondrial protein assembly and chromosome segregation [] Huang et a0 al [] cloned and identified the cDNA sequence of porcine PANE1 and found that porcine PANE1 gene was expressed differently in seven different tissues with the highest expression in lymph nodes and the lowest expression in kidney Until now the expression of CENPM and its potential prognostic effect on hepatocellular carcinoma has not yet been investigated our outcomes showed that the expression of CENPM in hepatocellular carcinoma was related to advanced clinical pathologic factors grade clinical stage Tclassification survival time and poor prognosis Univariate analysis uncovered that CENPM expression as a clearcut ward variable was related to poor prognostic clinicopathologic factors and Mclassification may play an indispensable role in the inclined to advance to a further advanced stage The univariate and multivariate analysis also suggested CENPM still remained freely connected with OS and recommended that CENPM may act as a potential prognostic biomarker of prognosis and therapeutic target in hepatocellular carcinoma but more researches needed to conduct for further study In addition we further analyzed various clinicopathological features of HCC samples using the UALCAN database and all of them showed high transcription of CENPMTo identify differential signaling pathways in liver cancer GSEA analysis results show that cell cycle DNA replication RNA degradation some cancers phagocytosis P53 signaling pathway and purine metabolism are differentially enriched in CENPM high expression phenotype CENPM may influence cell cycle DNA replication RNA degradation then controls the begins and development 0cWu a0and Yang Cancer Cell Int Page of Fig Enrichment plots from gene set enrichment analysis GSEAof cancer cells Kim et a0al [] was identified CENPM as a key gene that mediates the anticancer effect of garlic and cisplatin on bladder cancer and showed that patients with low CENPM expressed better progressionfree survival than patients without high expression Studies also found the CENPM genes encode a human minor histocompatibility antigen expressed by tumor cells [ ] Yu et a0al [] found CENPM could as AFPrelated diagnostic biomarkers in HCC and validate the results using quantitative realtime PCR Our study for the first time investigated the CENPM mRNA expression and its prognostic significance in hepatocellular carcinoma Chen et a0al [] demonstrated that LHX6 can inhibit the proliferation invasion and migration P53 signaling pathways during hepatocarcinogenesis Qin et a0 al [] found that P53stabilizing and activating RNA can strengthen the interaction between hnRNP K and P53 which ultimately leads to the accumulation and transactivation of P53 So CENPM may play a role via P53 signaling pathway and more researches needed to conduct in the future 0cWu a0and Yang Cancer Cell Int Page of Fig Immune infiltrates correlation with CENPM in HCC a Correlation between CENPM in HCC expression and abundance of immune infiltrates P b CENPM expression between various tumor and normal tissueTable Multivariate survival model analysis based on a0TIMER online toolClinicopathologic variableCoefHR CIPvalue SigAgeGender MaleRace BlackRace WhiteStage IIStage IIIStage IVPurityB cellsCD cellCD4 T cellsMacrophages NeutrphilsDendriticCENPMPvalue significant codes ¤ ¤ ¤ ¤ · ·Previous studies demonstrated that human CENPM transcript cRNA was only detected in activated B and Tcells either in a0vivo or in a0vitro These studies suggested CENPM may play important role in tumor immune response so we used an online tool to analysis immune infiltrates correlation with CENPM in HCC Multivariable Cox proportional hazard model showed that B cells CD8 T cells macrophages and dendritic cells of immune infiltrates statistically significant P in HCC indicating that these immune cells significantly affecting the prognosis A latest study showed CD8 CD68 and FoxP3 immune cells were associated with HCC particularly in the invasive margin [] Macrophages not only promote the proliferation colony formation and migration of HCC cells but also maintain tumor growth and metastasis by secreting hepatocyte growth factor HGF [] Pang et a0 al [] proposed that fusion of dendritic cells DC with tumor cells can effectively activate antitumor immunity in the body and affect tumor progression [] These studies indicate that CENPM may play an important role in tumor immune response and can be a good therapeutic target for immunotherapy 0cWu a0and Yang Cancer Cell Int Page of Fig Boxplot showing relative expression of CENPM in subgroups of patients with HCC UALCANTo determine the biological interaction network of CENPM in liver cancer we applied the most frequently changed neighbor genes of CENPM on the Network tab in cBioPortal and the most frequent change was RAD21 RAD21 is a nuclear phosphoprotein which becomes hyperphosphorylated in cell cycle M phase One study found that depletion of RAD21 resulted in reduced levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters resulting in enhanced selfrenewal of hematopoietic stem and progenitor cells HSPC [] Recent studies have shown that removing RAD21 in a background lacking Pds5 can rescue the phenotype observed only in the absence of Pds5 [] Our study may provide information on adhesion kinetics in replication fork studies in patients with liver cancer Our study also used the Targetscan online tool to distinguish CENPMrelated miRNAs To check the function of the identified miRNAs bioenrichment was performed through the Funrich database It is rich in ErbB receptor signaling network TRAIL signaling pathway Glypican pathway syndecan1 mediated signaling events and biological pathways of hepatocyte growth factor receptor cMet signaling events Studies have reported that selective cMet inhibitors have antitumor activity in HCC and have acceptable safety and tolerability in ChildPugh A liver function patients [] A recent study found that abnormal HGFcMet upregulation and activation are often observed in bladder cancer [] Studies have also found that metastasis associated with colon cancer MACC1 regulates PDL1 expression and tumor immunity in gastric cancer GC cells through the cMetAKTmTOR pathway [] We hypothesized that CENPM may regulate the expression of cMet leading to the occurrence of HCC and more related research Fig The network for CENPM and the most frequently altered neighbor genesTable The type and a0frequency of a0CENPM neighbor gene alterations in a0HCC cBioPortalGene symbolRAD21RPS27AHCTF1NUF2PMF1Amplification Homozygous deletionMutation Total alteration 0cWu a0and Yang Cancer Cell Int Page of Fig Enrichment analysis of the miRNA altered in the CENPM in HCC Funrich and Targetscan a Conserved sites for miRNA families broadly conserved among vertebrates b Cellular components c KEGG pathway analysis d Biological processes e Molecular functionsis needed To date this study demonstrates for the first time the important role of CENPM in the prognosis of hepatocellular carcinoma However future clinical trials are needed to validate these results and promote the use of CENPM in the prognostic evaluation of hepatocellular carcinomaConclusionsOur study found that the expression of CENPM was significantly increased in patients with hepatocellular carcinoma and was related to a variety of clinical features its correlation with the level of immune infiltration and poor prognosis so CENPM may become a useful biomarker for the prognosis of patients with liver cancerKEGG Kyoto encyclopedia of genes and genomes BP Biological processes CC Cellular components MF Molecular functions OS Over survivalAcknowledgementsNot applicableAuthors contributionsWZH designed and analyzed the research study WZH wrote and revised the manuscript YDL and WZH collected the data and all authors contributed to final manuscript All authors read and approved the final manuscriptFundingThis work is not supported by grantsAvailability of data and materialsRNAseq data and corresponding clinical data were acquired from the data portal for TCGA https porta lgdccance rgovEthics approval and consent to participateNot applicableAbbreviationsHCC Hepatocellular carcinoma CENPM Centromere protein M GSEA Gene set enrichment analysis TCGA Cancer genome atlas GO Gene ontology Consent for publicationNot applicable 0cWu a0and Yang Cancer Cell Int Page of Competing interestsThe authors declare that they have no competing interestsReceived January Accepted August References Torre LA Bray F Siegel RL Ferlay J LortetTieulent J Jemal A Global cancer statistics CA Cancer J Clin Tang Y Wang H Ma L et al Diffusionweighted imaging of hepatocellular carcinomas a retrospective analysis of correlation between apparent diffusion coefficients and histological grade Abdominal Radiol Coskun M Hepatocellular carcinoma in the cirrhotic liver evaluation using computed tomography and magnetic resonance imaging Exp Clin Transplant 201715Suppl Lang H Sotiropoulos GC Brokalaki EI et al Survival and recurrence rates after resection for hepatocellular carcinoma in noncirrhotic livers J Am Coll Surg Jiao Y Fu Z Li Y Meng L Liu Y High EIF2B5 mRNA expression and its prognostic significance in liver cancer a study based on the TCGA and GEO database Cancer Manag Res Byeon H Lee SD Hong EK et al Longterm prognostic impact of osteo pontin and Dickkopfrelated protein in patients with hepatocellular carcinoma after hepatectomy Pathol Res Pract Shen Y Bu L Li R et al Screening effective differential expression genes for hepatic carcinoma with metastasis in the peripheral blood mononuclear cells by RNAseq Oncotarget Renou JP Bierie B Miyoshi K Cui Y Djiane J Reichenstein M Shani M Hennighausen L Identification of genes differentially expressed in mouse mammary epithelium transformed by an activated betacatenin Oncogene Bierie B Edwin M Melenhorst J et al The proliferation associated nuclear element PANE1 is conserved between mammals and fish and preferentially expressed in activated lymphoid cells Gene Expr Patterns Brickner AG The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in Blymphoid cells and BCLL Blood Kruppa J Jung K Automated multigroup outlier identification in molecular highthroughput data using bagplots and gemplots BMC Bioinf Li T Fan J Wang B et al TIMER a web server for comprehensive analysis of tumorinfiltrating immune cells Cancer Res 20177721e108e110110 https doi10115800085472CAN Chandrashekar DS Bashel B Balasubramanya SAH Creighton CJ Rodriguez IP Chakravarthi BVSK Varambally S UALCAN a portal for facilitating tumor subgroup gene expression and survival analyses Neoplasia https doi101016jneo201705002 Gao et al Sci Signal Cerami et al Cancer Discov when publishing results based on cBioPortal https doi1011582159 Agarwal V Bell GW Nam J Bartel DP Predicting effective microRNA target sites in mammalian mRNAs eLife 20154e05005 https doi107554eLife Pathan M Keerthikumar S Ang CS Gangoda L Quek CY Williamson NA Mouradov D Sieber OM Simpson RJ Salim A Bacic A FunRich an open access standalone functional enrichment and interaction network analysis tool Proteomics https doi101002pmic20140 Foltz DR Jansen LE Black BE Bailey AO Yates JR III Cleveland DW The human CENPA centromeric nucleosomeassociated complex Nat Cell Biol Huang H Deng H Yang Y et al Molecular characterization and association analysis of porcine PANE1 gene Mol Biol Rep Kim WT Seo SP Byun YJ et al The anticancer effects of garlic extracts on bladder cancer compared to cisplatin a common mechanism of action via centromere protein M Am J Chin Med Yu Z Wang R Chen F et al Five novel oncogenic signatures could be utilized as AFPrelated diagnostic biomarkers for hepatocellular carcinoma based on nextgeneration sequencing Dig Dis Sci Chen HQ Zhao J Li Y et al Epigenetic inactivation of LHX6 mediated microcystinLR induced hepatocarcinogenesis via the Wntβcatenin and P53 signaling pathways Environ Pollut 2019252Pt A216 Qin G Tu X Li H et al lncRNA PSTAR promotes p53 signaling by inhibiting hnRNP K deSUMOylation and suppresses hepatocellular carcinoma Hepatology https doi101002hep30793 Ihling C Naughton B Zhang Y et al Observational study of PDL1 TGFβ and immune cell infiltrates in hepatocellular carcinoma Front Med Lausanne Dong N Shi X Wang S et al M2 macrophages mediate sorafenib resistance by secreting HGF in a feedforward manner in hepatocellular carcinoma Br J Cancer Pang YB He J Cui BY et al a potential antitumor effect of dendritic cells fused with cancer stem cells in hepatocellular carcinoma Stem Cells Int Janco JMT Lamichhane P Karyampudi L Knutson KL Tumorinfiltrating dendritic cells in cancer pathogenesis J Immunol Fisher JB Peterson J Reimer M et al The cohesin subunit Rad21 is a negative regulator of hematopoietic selfrenewal through epigenetic repression of HoxA7 and HoxA9 Leukemia CarvajalMaldonado D Byrum AK Jackson J et al Perturbing cohesin dynamics drives MRE11 nucleasedependent replication fork slowing Nucleic Acids Res Bouattour M Raymond E Qin S et al Recent developments of cmet as a therapeutic target in hepatocellular carcinoma Hepatology Sim WJ Iyengar PV Lama D et al cMet activation leads to the establishment of a TGFβreceptor regulatory network in bladder cancer progression Nat Commun Tong G Cheng B Li J et al MACC1 regulates PDL1 expression and tumor immunity through the cMetAKTmTOR pathway in gastric cancer cells Cancer Med Publishers NoteSpringer Nature 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Decreased expression of the thyroid hormoneinactivating enzyme type deiodinase is associated with lower survival rates in breast canceriuri Martin Goemann1 Vicente Rodrigues Marczyk15 Mariana RecamondeMendoza23 Simone Magagnin Wajner15 Marcia Silveira Graudenz45 Ana Luiza Maia thyroid hormones tHs are critical regulators of cellular processes while changes in their levels impact all the hallmarks of cancer Disturbed expression of type deiodinase DIO3 the main tHinactivating enzyme occurs in several human neoplasms and has been associated with adverse outcomes Here we investigated the patterns of DIO3 expression and its prognostic significance in breast cancer DIO3 expression was evaluated by immunohistochemistry in a primary cohort of patients with breast cancer and validated in a second cohort using RnA sequencing data from the TCGA database DNA methylation data were obtained from the same database DIO3 expression was present in normal and tumoral breast tissue Low levels of DIO3 expression were associated with increased mortality in the primary cohort Accordingly low DIO3 mRnA levels were associated with an increased risk of death in a multivariate model in the validation cohort DnA methylation analysis revealed that the DIO3 gene promoter is hypermethylated in tumors when compared to normal tissue In DIO3 is expressed in normal and tumoral breast tissue while decreased expression relates to poor overall survival in breast cancer patients Finally loss of DIO3 expression is associated with hypermethylation of the gene promoter and might have therapeutic implicationsBreast cancer is the most common cancer in women worldwide accounting for more than two million new cancer cases and of all cancerrelated deaths in women in Despite remarkable advances in the treatment of breast cancer in recent decades not all patients benefit from current therapeutic options and thus will experience relapse23 Genomic tests improve the clinical prediction of patient outcomes and determine the necessity of adjuvant chemotherapy with endocrine therapy34 However it is a highly heterogeneous disease that is diverse in its behavior and responsiveness to the different modalities of treatment56 Breast cancer is characterized based on receptor and gene expression profiles that together with the classic clinicopathological variables guide the treatment and estimate the risk of recurrence34 Gene expression profiling studies have established at least four molecularly distinct types of breast cancer that can be expanded to the intrinsic subtypes luminal A LumA luminal B LumB HER2enriched basallike and normallike7Numerous studies have established thyroid hormones THs as critical regulators of multiple cellular processes in normal and tumor cells10 They contribute to cellular proliferation and differentiation during development and adulthood and are finetuned for tissuespecific control1011 Clinical studies associate TH levels with breast 1Thyroid Unit Endocrine Division Hospital de Clnicas de Porto Alegre Rua Ramiro Barcelos Porto Alegre RS CEP Brasil 2Institute of Informatics Universidade Federal Do Rio Grande Do Sul Porto Alegre Brazil 3Bioinformatics Core Hospital de Clnicas de Porto Alegre Porto Alegre Brazil 4Department of Pathology Hospital de Clnicas de Porto Alegre Porto Alegre Brazil 5Faculdade de Medicina Universidade Federal Do Rio Grande Do Sul Porto Alegre Brazil email almaiaufrgsbrScientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Patterns of expression of DIO3 in breast samples Immunostaining was performed as described in Materials and Methods From left to right A normal glandular breast tissue B breast carcinoma with low expression overall intensity C breast carcinoma with moderate expression overall intensity and D breast cancinoma with high expression overall intensity of DIO3 protein evaluated through immunohistochemistry The staining intensity level is used to calculate the Hscore combined with the percentage of positive cells see Methodscancer risk and mortality1213 while in a0vitro models demonstrate the effect of THs on breast cancer cell proliferation apoptosis and migration14 T4 promotes cell proliferation through the αv3 integrin receptor14 while the proliferative effects of T3 depend at least partially on the presence of estrogen receptors in breast cancer cells1718 Clinically however the effects of THs on specific histopathological and molecular subtypes of breast cancer are still unclear1920Modulation of THs concentrations is orchestrated by a group of selenoproteins called iodothyronine deiodinases which can activate and inactivate thyroid hormones21 Briefly the type deiodinase DIO1 catalyzes both activation and inactivation of thyroxine T4 generating triiodothyronine T3 and reverse triiodothyronine rT3 respectively22 Type deiodinase DIO2 acts locally converting the prohormone T4 into the active T3 Meanwhile type deiodinase DIO3 is the main THinactivating enzyme by degrading T4 and T3 to inactive metabolites rT3 and diiodothyronine respectively21 The DIO3 gene is found in the DLK1DIO3 genomic region which is located on human chromosome 14q3223 DIO3 gene is subject to genomic imprinting an uncommon epigenetic phenomenon that results in the preferential expression of one of the alleles paternal allele in the case2425 DIO3 gene expression is increased in several tissues during embryogenesis but it decreases in most tissues in adulthood2627 Notably DIO3 is expressed in normal and pathological hyperproliferative conditions where it has been implicated in cell proliferation and differentiation20252628 In particular studies have demonstrated that the local control of THs signaling provided by the regulation of DIO3 activity is associated with cancer development progression and recurrence28 We have previously reported that DIO3 mRNA and activity levels are increased in papillary thyroid cancer PTC which are associated with larger tumor size and the presence of lymph node and distant metastasis at diagnosis30 Others have described hyperexpression of this enzyme in basal cell carcinoma BCC where it modulates intracellular T3 concentrations and thus contributes to the cell tumorigenic potential31 DIO3 exerts a similar function in colon cancer which suggests that attenuation of the TH signal is part of the oncogenic process at least in some types of cancer28Considering the implied role of the DIO3 gene in human neoplasms and the potential effect of TH in breast carcinogenesis13 we investigated the expression patterns of DIO3 in normal breast tissue and breast cancer Here we demonstrate that DIO3 is expressed in normal breast tissue and breast cancer tissue In breast cancer reduced DIO3 expression is associated with decreased overall survival Interestingly loss of DIO3 expression might be explained at least partially by gene promoter hypermethylationResultsDIO3 in normal breast and fibroadenoma DIO3 immunohistochemistry staining was detected in all samples of normal breast tissue N at an overall moderate intensity Hscore ± DIO3 staining was predominantly cytoplasmatic and more pronounced in the apical extremity in luminal cells in both ducts and acini of the breast Fig a01A DIO3 was markedly positive in myoepithelial cells Fig a01A bottom Benign fibroadenoma lesions N were also positive for DIO3 staining with an intensity comparable to healthy tissue Hscore ± vs ± P Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cCharacteristicMedian age at diagnosis rangeyearsTumor size in the largest dimensionmmMedian IQRMean ± SDEstrogen receptorno PositiveNegativeMissingProgesterone receptorno PositiveNegativeMissingHER2 statusno PositiveNegativeMissingHistological type of tumorno Invasive Ductal Carcinoma IDCInvasive Lobular Carcinoma ILCDuctal Carcinoma in a0situ DCISClinicalpathological subtypeno Luminal ALuminal BHER2Triple NegativeNon classifiedLymph node metastasisno YesNoDistant metastasisno YesNoTumor stagingno Stage IIIStage IIIIVMissingPretreatment hypothyroidismno Posttreatment hypothyroidismno Followup mean ± SDmonthsAllcause mortalityno Mean survival months CIPrimary cohort N Validation cohort N ± AJCC NANA PAM50 NANA Table Baseline characteristics of patients with breast cancer included in the primary cohort and in the validation cohort NA not available IQR interquatile range SD standard deviation HER2 human epidermal growth factor receptor2 AJCC American Joint Committee on Cancer Classified by the AJCC staging system Classified by PAM50 data available for patientsDIO3 protein in breast cancer the primary cohort To study DIO3 expression in breast cancer we analyzed a cohort of patients who had been seen at our institution primary cohort N and validated the results in the TCGABRCA cohort validation cohort N The clinicopathological characteristics of the patients from both cohorts are summarized in Table a0Patterns of DIO3 staining evaluated through immunohistochemistry in breast cancer samples are shown in Fig a01BD DIO3 staining in FFPE breast cancer tissues was positive in samples of invasive ductal carcinoma IDC with a mean Hscore of ± When evaluating invasive lobular carcinoma ILC only of samples was positive for DIO3 Hscore A sample of ductal carcinoma in a0situ DCIS was also positive for DIO3 expression Hscore A graph comparing the Hscore for DIO3 in nonmalignant tissues and malignant breast cancer types is presented in Fig a02A Mean DIO3 Hscores of primary tumors were similar to the nontumoral tissues with a marginal decrease in DIO3 seen in invasive lobular carcinoma ILC P Scientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cType of tissuetumorANormalbreastDFibroadenomaIDCILCLymph node statusPNSerocSHerocSHEstrogen receptor statusHER2 statusCPNSpositivenegativeTNM stagingFPNSPNSpositivenegativeDistant metastasisPNSBEerocSHerocSHerocSHerocSHnegativepositiveabsencepresenceIIIIIIIVGLog Rank p0012liavvrusfoytilibaborPDIO3 positiveDIO3 negativePatients at riskDIO3 posDIO3 negMonthsFigure a0 DIO3 staining and clinicopathological characteristics of patients with breast cancer in the primary cohort AF Box plots of DIO3 staining in breast tissue samples evaluated through immunohistochemistry and quantified by HScore Samples were divided according to clinicopathological data as follows A type of tissue analyzed B ER status C HER2 status D lymph node status E distant metastasis and F TNM anatomic staging G KaplanMeier plot of overall survival in patients with the presence gray or absence black of DIO3 staining in breast cancer evaluated through immunohistochemistry ER estrogen receptor HER2 human epidermal growth factor receptor2 IDC invasive ductal carcinoma ILC invasive lobular carcinoma NS not significant P The mean Hscore of invasive ductal carcinoma was similar to that of normal tissue P No differences were observed between the molecular subtypes of breast cancer P data not shown There was no difference in the Hscore between tumors with ERpositive and ERnegative status P Fig a02B or between tumors with HER2positive and HER2negative status P Fig a02C Among the primary tumors there was no significant correlation between Hscore and Ki67 levels P or between Hscore and histological tumor grade P We found no association of DIO3 positivity negative or positive with tumor size P The mean Hscore in primary tumors of patients without nodal metastases was similar to that observed in patients with lymph node metastasis P Similarly Hscores of primary tumors of patients with distant metastasis did no differ from those without distant metastasis P Fig a02DE There were no differences on DIO3 Hscores when comparing patients with stage III vs stage IIIIV disease P Fig a02F We obtained both primary and lymph node tissues from patients In this subset of patients DIO3 staining was comparable between paired primary tumor and lymph node metastasis P Table a0 shows the variables associated with an increased risk of death in the primary cohort univariate analysis We observed that negative DIO3 staining was associated with poor prognosis HR CI to P Therefore additional studies were performed using KaplanMeier analysis and the logrank Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cVariableAge at diagnosis yearsTumor size mmLymph node metastasis pos vs negDistant metastasis pos vs negER status pos vs negP status pos vs negHER2 positivity pos vs negTNM staging IIIIV vs IIIDIO3 status neg vs posHR CI P valueTable Univariate Cox regression analysis of overall survival in breast cancer patients in the primary cohort HR hazard ratio CI confidence interval ER estrogen receptor P progesterone HER2 human epidermal growth factor receptor2test Patients with negative DIO3 staining had a worse overall survival than those with positive DIO3 staining The mean overall survival was a0months CI to in the DIO3negative group and a0months CI to in the DIO3positive group Fig a02G logrank P DIO3 mRNA in breast cancer patients validation cohort It has been previously demonstrated that DIO3 protein levels and activity correlate with DIO3 mRNA levels in different contexts303233 Therefore to validate differences of DIO3 expression among patients with breast cancer we analyzed DIO3 mRNA expression in a second cohort using available gene expression data from the TCGABRCA study In this second population DIO3 expression was found to be reduced in primary solid tumors N compared to that observed in normal breast samples N logFC adjusted P value Fig a03A even when the comparison was made only with matched normal tissues logFC adjusted P value Fig a03B The majority of tumor subtypes with the exception of normallike tumors classified according to PAM50 classification system showed reduced DIO3 expression compared to normal tissue Fig a03C On the other hand DIO3 expression was increased in ERpositive samples compared to that in ERnegative samples logFC P Fig a03D There was no significant difference when comparing DIO3 expression between patients with or without lymph node disease logFC adjusted P value or distant metastasis logFC adjusted P value Fig a03E Decreased DIO3 mRNA expression was observed in all tumor stages compared to that seen in normal tissue P However no differences were found between the different tumor stages Fig a03F Interestingly lower DIO3 expression was associated with greater tumor size P and ER negativity P We then evaluated the prognostic value of DIO3 mRNA expression for patient survival We considered patients as having high DIO3 expression when their logCPM values were above the median and as having low DIO3 expression when their logCPM values were below the median Low DIO3 expression was associated with reduced survival with an HR of CI to P in the univariate model Table a0 Additional analysis using a multivariate model adjusted for all variables with a P in the univariate analysis demonstrated that low DIO3 was an independent prognostic factor for death HR IC to P Table a0 Fig a04A The estimated overall survival rate at five years in the KaplanMeier analysis was CI to in the high DIO3 group and CI to in the low DIO3 group Fig a04AIn the subgroup analysis of patients with advanced disease stage IV those with low DIO3 expression had reduced overall survival compared to patients with high DIO3 expression P Fig a04B Notably low DIO3 expression was associated with worse overall survival among patients with ERpositive tumors P but not among those with ERnegative tumors P Supplementary Fig a0Methylation of DIO3 gene promoter To further investigate possible factors that could lead to decreased DIO3 expression in breast cancer we performed DNA methylation analysis of a subgroup of patients from TCGABRCA database from whom DNA methylation data were available N Our analysis demonstrated that global DNA methylation levels of breast cancer samples were similar to those of healthy breast tissues Fig a05A However the methylation levels of CpG sites in the DIO3 gene region were increased compared to those from healthy tissue Fig a05B P Figure a0 details the CpG sites that are hypermethylated within the DIO3 gene region The first kbp of ² flanking region red are known to be extremely G C rich of the sequence and this region is highly conserved between mouse and human genome34 Promoter region a0bp of the ² flanking region is composed of several promoter elements Fig a05C enhanced including a TATA box two CAAT boxes and CG rich regions35 We observed a significant increase in DNA methylation levels in CpG sites that are located both at the promoter region and in the ² flanking kbp conserved region of the gene Fig a05CDScientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The relationship between DIO3 mRNA expression and clinicopathological parameters in breast cancer samples of patients from the TCGABRCA cohort expressed in Log2 counts per million voomtransformed Comparative expression demonstrates that DIO3 mRNA is decreased in tumoral tissue when compared to normal tissue when analyzing A all samples or B only matched samples C All tumor subtypes have decreased expression of DIO3 mRNA when compared to normal tissue with the exception of normallike tumors compared to normal tissue DIO3 mRNA levels were also reduced in basallike tumors when compared to luminal A logFC adjusted P value and in luminal B when compared to luminal A subtypes logFC adjusted P value and D DIO3 expression is increased in ERpositive samples when compared to ERnegative samples E DIO3 expression is similar in patients with or without metastasis F When samples were separated according to tumor staging all tumor stages had decreased DIO3 expression when compared to normal tissue but there was no difference in expression between the stages ER estrogen receptor Adjusted P value in comparison to normal tissueVariablesAge at diagnosis yearsTumor size ¥ a0cm vs ¤ a0cmLymph node pos vs negDistant metastasis pos vs negE2 status pos vs negP status pos vs negHER2 positivity pos vs negTNM staging IIIIV vs IIIDIO3 status low vs highUnivariate analysisHR CI P value Multivariate analysisHR CI P value Table Univariate and multivariate Cox regression and for overall survival in the validation cohort HR hazard ratio CI confidence interval ER estrogen receptor P progesterone HER2 human epidermal growth factor receptor2 All variables with P were included in the multivariate model TNM is not included as it is derived from variables already present in the modelDiscussionDisruption of the iodothyronine deiodinases expression leads to changes in TH concentrations which might contribute to cancer development and progression by impacting virtually all the hallmarks of cancer10 Here we demonstrate that the THinactivating enzyme DIO3 is expressed in normal breast tissue and that its expression is highly prevalent in breast cancer More interestingly our results demonstrated that low DIO3 expression Scientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cAOverall SurvivalP BOverall Survival Stage IV patientsHighLowDIO3DIO3groupgroupP0011liavvrusfoytilibaborPliavvrusHighLowDIO3DIO3groupgroupHR for death IC to foytilibaborPPatients at riskHigh DIO3Low DIO3MonthsPatients at riskHigh DIO3Low DIO3MonthsFigure a0 KaplanMeier estimates of overall survival in patients of the TCGABRCA cohort according to DIO3 mRNA expression Patients were grouped according to the median of DIO3 expression in the population as presenting high DIO3 expression gray lines or low DIO3 expression black lines Plot A shows the overall survival in the entire cohort Plot B refers only to patients with stage IV disease HR hazard ratio CI confidence intervalwas an independent prognostic factor for reduced overall survival in two different populations of patients with breast cancerData on the expression of iodothyronine deiodinases in human breast tissue are scarce Low levels of DIO1 were reported in normal and lactating tissues but DIO2 and DIO3 have not been analyzed thus far36 Here we show that DIO3 is expressed at both the mRNA and protein levels in normal human breast tissue Expression of DIO3 mRNA has been previously described in breast cancer cell lines MCF7 and MDAMB231 cells DIO3 mRNA was found to be upregulated in MCF7 cells and downregulated in MDAMB231 cells when compared to the nontumoral cell line MCF10A cells DIO3mediated T3 deiodination also occurs in MCF7 cells In these cells DIO3 expression is to regulated by retinoids but not by estradiol37 These findings are consistent with the presence of DIO3 in other tissues of ectodermal origin such as the skin and the nervous system4041The role of thyroid hormone metabolism on human tumorigenesis has been largely debated10 In breast cancer previous studies showed that higher levels of the thyroid hormone receptor alpha were an independent prognostic factor for increased overall survival42 More recently high levels of the thyroid hormone receptor beta in breast tumors were also associated with increased breast cancerspecific survival43In basal cell carcinomas BCC for instance a DIO3mediated decrease in T3 levels relates to increased cell proliferation31 Similarly in colon cancer cells DIO3 knockdown and consequent increases in T3 levels are associated with reduced cell proliferation and induction of differentiation44 High levels of DIO3 expression in primary PTC tumors were associated with advanced disease at the diagnosis30 Some data indeed suggest that T3 can contribute to tumor growth in breast cancer cells in a0vitro17 while a microenvironment with low T3 levels could facilitate invasiveness and dedifferentiation However in agreement with our data in breast cancer similar levels of DIO3 mRNA are observed in glioblastoma and liver carcinomas as compared to respective normal tissues45 These differences could be attributed to the tissue embryological origin since the tissues of ectodermal origin seem to maintain DIO3 expression during adulthood while DIO3 gene is subject to imprinting in other tissues Loss of DIO3 expression was associated with tumor aggressiveness in colon cancer and also in thyroid cancer DIO3 expression is present in papillary and follicular subtypes but not in the most aggressive and dedifferentiated anaplastic subtype30 Taken together these results indicate that although expression of the enzyme is often upregulated in the neoplastic tissue compared to normal tissue loss of DIO3 expression is a common hallmark of dedifferentiation in the neoplastic process which might confer its prognostic significance Alternatively the distinct pattern of expression could be the result of DIO3 regulation or related to the cancertype specific methylation signatureAlthough this was an exploratory study our results point to a prognostic role for DIO3 expression in breast cancer In a primary cohort of patients with breast cancer negative DIO3 staining in the primary tumor was associated with significantly worse prognosis HR CI to P when compared to patients who were DIO3positive More interesting in the second cohort low DIO3 expression was an independent prognostic factor for death in a model adjusted for age tumor size lymph node and distant metastasis estrogen and progesterone status HR IC P The prognostic role of DIO3 expression was particularly relevant in the subgroup of patients with advanced diseaseIntriguingly the difference in survival between groups with distinct DIO3 expression was limited to ERpositive patients Previous studies indicate the existence of a crosstalk between estrogen and THdependent regulatory pathways in breast cancer14174647 which might be a potential explanation T3 regulates cell cycle progression and proliferation in breast cancer cells in a0vitro by a common mechanism involving ER and T3 receptormediated pathways46 Moreover T4 can phosphorylate nuclear ERalpha in MCF7 cells via a MAPKdependent pathway promoting proliferation14 Therefore loss of DIO3 expression and the consequent increase in intracellular T3 levels could be specifically detrimental to tumors that express ER as our results suggest Contributing to this Scientific RepoRtS 101038s41598020708924Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Panel A demonstrates mean global DNA methylation levels values in breast cancer tissue compared to healthy breast tissue Panel B demonstrates that the mean DNA methylation of DIO3 gene region is increased in tumor tissue when compared to normal tissue P Panel C is a schematic representation of the location of DIO3 gene in chromosome and the regions that were evaluated by CpG probes The promoter region is composed by several promoter elements including a TATA box two CAAT boxes and CG rich regions C enhanced Significant hypermethylation in several CpG sites is observed in the promoter region of the gene Panel D presents mean values of CpG sites mapped in DIO3 gene region comparing normal and tumoral tissueinterplay previous studies have demonstrated that estrogen progesterone and their receptors regulate DIO3 activity in rat uteri and decidua4849 Therefore we cannot rule out that in the breast DIO3 expression depends partially on the presence of functional estrogen and progesterone receptorsScientific RepoRtS 101038s41598020708924Vol1234567890wwwnaturecomscientificreports 0cThe DIO3 gene is subject to genomic imprinting an uncommon epigenetic phenomenon that results in the preferential expression of one allele the paternal allele in this case2425 The disturbed expression of genes and miRNAs or altered hypermethylation patterns of the DLK1DIO3 genomic region is involved in the pathogenesis of different types of cancer50 Thus we hypothesized that the loss of DIO3 expression in breast tumors could be a consequence of gene hypermethylation in the tumoral context Indeed our results show that while the mean global methylation in breast tumors is comparable to that of normal tissue the DIO3 genomic region especially its promoter region is significantly hypermethylated in tumors Fig a05C enhanced These findings might explain at least in part the reduced DIO3 expression in breast cancer Of interest the DIO3 gene was also found to be hypermethylated in Bcell Tcell and myeloid malignancies and lung cancer5152Our study has some limitations The absence of data on DIO3 enzymatic activity limits the assumption that the decreases of DIO3 levels cause alterations in intracellular TH homeostasis Alternatively changes in DIO3 expression could simply represent a consequence of broader epigenetic modifications in the tumoral context It is also important to consider that complete clinical data on patient thyroid status was not available which could interfere with deiodinase expression54 Therefore the complex changes on deiodinases and the overall effect on intracellular TH status are still unclear in breast cancer Additionally our analysis is limited to two populations using two different methodology and despite robust supporting data results should be confirmed in other cohortsIn the results of this study demonstrate DIO3 expression in breast tissue and breast cancer Importantly low DIO3 expression is associated with reduced overall survival suggesting that DIO3 might have a prognostic role in this disease Reduced DIO3 expression in breast cancer can be explained at least in part by gene hypermethylation Due to its potential to modulate thyroid hormone intracellular levels and interplay with estrogen metabolism in breast cancer the DIO3 expression might have therapeutic implicationsMethodsPatients and tissues primary cohort Neoplastic tissue from patients diagnosed with breast cancer was retrospectively collected from a consecutive series of unselected patients in the pathology department of Hospital de Clnicas de Porto Alegre Tissue samples of the normal breast N and fibroadenomas N were also obtained Histopathological reports containing information on tumor type grade and immunohistochemistry were retrieved clinical data were retrospectively reviewed in medical records Tumors were histologically classified according to the 8th edition of the American Joint Committee on Cancer AJCC staging system56 All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional andor national research committee The study was reviewed and approved by the Institutional Review Board and Research Ethics Committee from the Hospital de Clnicas de Porto Alegre with a waiver of informed consent Protocol number Immunohistochemistry studies and DIO3 staining assessment DIO3 protein expression was evaluated by immunohistochemical studies on 6mm sections of formalinfixed paraffinembedded FFPE tissue blocks from normal breast tissues fibroadenomas and primary breast cancers The immunohistochemical technique consists of tissue deparaffinization and rehydration antigenic recovery inactivation of endogenous peroxidase and blockage of unspecific reactions Samples were incubated overnight at a temperature of a0°C with an antiDIO3 rabbit polyclonal antibody Abcam Cambridge UK at a dilution of followed by subsequent incubation with a biotinylated secondary antibody a streptavidinHRP conjugate LSAB Dako Carpinteria CA USA and diaminobenzidine tetrahydrochloride Kit DAB Dako The slides were examined using an Olympus BX51 microscope The QCapture Pro software Qimaging Surrey BC Canada was used to capture the images DIO3 staining was evaluated by an experienced pathologist blinded to the molecular profile and TNM staging The immunohistochemical results of DIO3 staining were assessed dichotomously negative or positive and semiquantitatively using the Hscore method as described previously5758 The Hscore combines the percentage of positive cells and staining intensity level weak moderate strong and is calculated using the following formula [ cells cells cells ] with results ranging from to Positive epidermis and placenta and epidermal nevus and negative connective and adipose tissue internal controls were assessed for all the evaluated cases Samples from the primary cohort were classified concerning the presence or absence of these receptors and the level of Ki67 expression into the following groups Luminal A LumA luminal B LumB triple negative and HER2 A Ki67 index cut point of was defined to distinguish HER2 negative lumB from lumA tumors5960Differential gene expression and methylation analysis For the validation cohort RNA sequencing RNASeq RSEM gene expression data from The Cancer Genome Atlas TCGA breast cancer BRCA study were obtained from the Genomic Data Commons GDC Data Portal gdcporta lcninihgov using the TCGAbiolinks RBioconductor package61 Raw expression signals for primary solid tumor samples N and solid normal tissue samples N were normalized and analyzed for differential expression of DIO3 using the limmavoom pipeline from the limma RBioconductor package62 P values were adjusted for multiple comparisons using the false discovery rate FDR procedure of Benjamini and Hochberg63 Clinicopathological information for TCGABRCA samples was downloaded through TCGAbiolinks and the Broad GDAC Firehose gdacbread insti tute merged level clinical data For tumors of the TCGABRCA cohort data retrieved from PAM50 classification were used to define tumor subtype classification64 Overall survival OS was estimated by the KaplanMeier method and compared by the logrank test using functions provided by TCGABiolinks For the methylation analysis we used the TCGAbiolinks RBioconductor package30 to obtain and analyze Illumina a0K methylation and clinical data for samples from the TCGABRCA study includScientific RepoRtS 101038s41598020708924V | Thyroid_Cancer |
Despite the proven benefits of iron chelation therapy ICT in the management of chronic iron overload and related complications compliance to longterm ICT is challenging Results from the ECLIPSE study an label randomized multicenter 2arm phase study evaluated the safety of deferasirox dispersible tablet and filmcoated tablet FCT formulations in patients with transfusiondependent thalassemia TDT or very low low or intermediate risk myelodysplastic syndrome MDS treated over weeksMethods The aim of the current study a 2year label multicenter singlearm phase study is to evaluate the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or lowerintermediaterisk MDS treated for years after the completion of weeks of treatment with deferasirox in the ECLIPSE phase studyResults Of patients enrolled completed treatment and study Adverse events AEs reported in most patients were of mild to moderate severity Headache and diarrhea were the most frequently reported AEs None of the serious AEs including death were considered treatment related No new safety signal was identified and longterm safety of deferasirox FCT was consistent with the known safety profile of deferasirox No major concerns associated with gastrointestinal tolerability renal safety or hematological abnormalities thrombocyt ianeutr ia were reported during the years Patients receiving deferasirox FCT had a treatment compliance by pill count of and persistence continuous use for ¥ days of Reduction in serum ferritin level was almost consistent starting from week across all postbaseline time points relative reduction month month Correspondence giovanbattistaruffoarnascivicoit UOC Ematologia e Talassemia AO CivicoDi CristinaBenfratelli Piazza Nicola Leotta Palermo ItalyFull list of author information is available at the end of the The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTartaglione a0et a0al Exp Hematol Oncol Page of Conclusions The results from this 2year interventional study suggest that the recommended dosing of deferasirox FCT with better tolerability palatability and compliance offers a favorable option of ICT for longterm management of iron overload and associated complications in TDTTrial registration ClinicalTrialsgov NCT02720536 Registered March wwwclini caltr ialsgovct2showNCT02 Keywords Deferasirox Chelation Filmcoated tablet Iron overload Longterm Safety Transfusiondependent thalassemia Myelodysplastic syndromeIntroductionIron overload in chronic anemias represents a serious consequence of impaired hematopoiesis and repeated blood transfusions leading to endan damage reduced quality of life and decreased survival Iron chelation therapy ICT can be a lifelong requirement in chronic transfusiondependent refractory anemias including thalassemia sicklecell disease and myelodysplastic syndrome MDS [ ] Despite the proven benefit of ICT patient compliance to longterm ICT is challenging [] Compliance with ICT is reported to influence the frequency and severity of iron overloadrelated complications [] and a substantial increase in morbidity mortality and treatment cost has been seen among patients who are noncompliant to ICT []Currently main iron chelators are available for clinical use deferoxamine deferiprone and deferasirox Parenterally administered deferoxamine was the mainstay of chelation therapy until the availability of oral chelators in [] Oncedaily oral administration of deferasirox dispersible tablet DT formulation presented a better option with greater compliance and quality of life over parenteral deferoxamine [] Currently there are no direct comparison studies for the oral chelators however oncedaily simpler use of deferasirox has been projected to be a more costeffective option than the thricedaily administration of oral deferiprone in the management of longterm complications [ ] The deferasirox filmcoated tablet FCT formulation with a simpler oral administration and improved palatability and gastrointestinal GI tolerability compared with deferasirox DT offers a better option for optimal patient acceptance and improved compliance to longterm therapy [ ] Thus an appropriate choice of ICT plays an important role in patient compliance to chelation therapyThe deferasirox FCT used in this study contained the same active substance of the iron chelator deferasirox and was strengthadjusted to achieve comparable exposure to the currently approved deferasirox DT Deferasirox FCT is available in dose strengths a0mg a0mg and a0mg and is dosed based on body weight Unlike the DT deferasirox FCT can be administered once daily orally without any need for dispersion either on an empty stomach or with a light meal [ ]Results from the ECLIPSE study an label randomized multicenter 2arm phase study that evaluated the safety of deferasirox DT and FCT formulations in patients with transfusiondependent thalassemia TDT or MDS very low low or intermediate risk treated over a0weeks have been published previously [] The purpose of the current study was to collect data on the longterm safety and efficacy of deferasirox FCT in a subset of patients with TDT or very low low or intermediaterisk MDS who had the possibility to continue treatment with deferasirox FCT after completion of a0 weeks of treatment in the ECLIPSE study The study also aimed to collect efficacy data for deferasirox FCT in the reduction or maintenance of iron burden as measured by the serum ferritin levelMethodsKey inclusion and a0exclusion criteriaPatients recruited at European sites [Austria n Greece n Italy n ] who had completed the 24week treatment in the ECLIPSE study with tolerance to deferasirox and fulfilled all eligibility criteria were included in this study Patients were male or female aged ¥ a0 years with TDT or very low low or intermediaterisk MDS who had received deferasirox DT at doses ¥ a0mgkgday or ¥ a0mgkgday respectively as per clinical judgement Patients had a transfusion history of ¥ packed red blood cell units were anticipated to be transfused with ¥ a0unitsyear during the study and had serum ferritin a0ngmL at screening Key exclusion criteria in the study included patients with creatinine clearance CrCl below contraindication limit as per local label serum creatinine SCr upper limit of normal ULN alanine aminotransferase ALT ULN unless liver iron concentration was confirmed as a0mg Fedry weight within a0months prior to screening urine protein to creatinine ratio UPCR a0mgmg impaired GI function that may significantly alter the absorption of oral deferasirox or clinicallaboratory evidence of active hepatitis Bhepatitis C infection 0cTartaglione a0et a0al Exp Hematol Oncol Page of Study designThis was a 2year label multicenter singlearm phase study NCT02720536 aimed to provide additional efficacy safety and drug exposure data following the ECLIPSE study []Patients who were assigned to either the deferasirox DT or the deferasirox FCT arm and had completed the study period of a0weeks with tolerance to deferasirox in the ECLIPSE study were allowed to participate in this study It was planned that any patient continuing directly from the ECLIPSE study would receive the same dose of deferasirox FCT or an equivalent FCT dose at the start of this study corresponding to their DT dose at the end of the ECLIPSE study As all patients had a lag period between the completion of the ECLIPSE study and the start of this study ie patients who completed the ECLIPSE study switched to commercially available deferasirox DT or another ICT they entered the current study following a washout period with a starting dose that was based on clinical judgment For each patient the daily dose was calculated based on the patients actual body weight and then rounded up or down to the nearest whole tablet according to available strengths of deferasirox FCT tablets a0mg a0mg and a0mg Dose adjustments were allowed every a0months based on serum ferritin levels and clinical judgment with ± to a0mgkgday up to a maximum dose of a0mgkgdayThe study was conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki and was approved by independent ethics committees at participating sites The study is registered at wwwclini caltr ialsgovct2showNCT02 Patients or parentsguardians provided written informed consent or assent prior to enrollmentOutcomesThe primary objective of the study was to evaluate the overall safety of the deferasirox FCT formulation measured by the frequency and severity of adverse events AEs and changes in laboratory values of interest such as SCr and CrCl The secondary objective was to evaluate the efficacy of deferasirox FCT with respect to serum ferritin levels decreased or maintained according to individual therapeutic goal measured by the absolute and relative changes in serum ferritin levels over timeStatistical evaluationsNo formal sample size was calculated A maximum of patients aged ¥ a0years were originally planned to be enrolled from the ECLIPSE study patients were enrolled into this study There were no screening failuresData from all centers participating in this study were for analyses The statistical software SAS® version collected using electronic case record forms and pooled was used for analysis The analyses were descriptive in nature no hypothesis was tested Data were summarized with respect to demographic and baseline characteristics primary and secondary assessments along with safety observations All analyses were based on data collected as per protocolscheduled assessments according to or including clinical judgment of the investigatorPatient compliance to deferasirox FCT was evaluated using the count of deferasirox FCT by the relative consumed tablet count Descriptive statistics including confidence intervals CIs for the mean relative consumed tablet counts were provided Persistence defined as continuous use of deferasirox FCT without a gap for ¥ or ¥ a0days over a fixed time interval of interest was summarized at month a0 month month and month The incidence of any treatmentemergent AEs ie AEs from the start of study treatment to a0days after the last intake of study drug overall and by maximum grade severity mild moderate or severe as reported by the clinician were summarized using frequency counts and percentages of patients For each of the laboratory parameters of interest SCr CrCl ALT and aspartate aminotransferase [AST] the worst postbaseline values were summarized as shift tables based on notableextended ranges For serum ferritin and hematological parameters red blood cells [RBCs] platelets total white blood cells hemoglobin and hematocrit the absolute mean and the relative mean changes from baseline were summarized at each postbaseline visitResultsOverall patients were enrolled from countries across Europe of whom discontinued early from treatment Fig a0Of the patients enrolled the majority patients [] including patients aged a0 years had TDT and patients had MDS all aged ¥ a0years Most patients were Caucasians and females comprised of the study population All patients had a history of prior ICT Demographics and other baseline characteristics are described in Table a0In most patients deferasirox monotherapy was the last ICT before study treatment The majority of patients had received prior medication or therapy The most common prior medications by the Anatomical Therapeutic Chemical classification system class included vitamin D and vitamin D analogues patients [] proton pump inhibitors PPIs patients [] sequential preparations of progestogens 0cTartaglione a0et a0al Exp Hematol Oncol Page of Patients enrolled N53Countries Austria Greece Italy Disease TDT MDS Completed treatment and studyaDiscontinued from treatment Withdrewconsent Pregnancy Unsatisfactorytherapeuticeffect Death Adverseevents Abnormallaboratoryvalues Unwillingnessto comply withprocedures Fig Patient disposition MDS myelodysplastic syndrome TDT transfusiondependent thalassemia aThe patients who completed the study had received treatment for at least monthsand estrogens patients [] fixed combinations of progestogens and estrogens patients [] and thyroid hormones patients []Almost all patients [] received concomitant medication or significant nondrug therapies on or after the start of study treatment The most common concomitant medications by ATC class included vitamin D and vitamin D analogues patients [] anilides patients [] antibiotics patients [] other agents for local oral treatment patients [] PPIs patients [ and other antibiotics for topical use patients []All patients received at least transfusions during the study with a maximum of transfusions received by patient The majority of patients [] including the MDS patients received to transfusions Additional file a0 Table a0S1Exposure to a0treatment and a0complianceThe mean standard deviation [SD] duration of exposure to treatment was days with most patients [] being treated for at least a0weeks The majority of patients receiving deferasirox FCT were in the longest exposure category ¥ a0weeks The sum of each patients treatment exposure to deferasirox FCT was patientyears Almost all patients [] received deferasirox FCT at an average dose of ¥ a0 mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose received Table a0 MDS patients n received an average dose of ¥ a0mgkgday with mgkgday as the mean SD average actual deferasirox FCT dose receivedPatients had a mean relative consumed tablet count of CI The proportions of patients with continuous use of deferasirox FCT with no interruption for ¥ a0days and ¥ a0days were n and n respectively at a0monthsEfficacyA decrease in mean serum ferritin levels was observed from week a0 onward except for month a0 though the median serum ferritin levels showed a consistent decrease across all postbaseline time points The mean SD actual decrease in serum ferritin level was µgL from baseline to month a0 µgL from baseline to month a0 and µgL from baseline to month a0 Fig a0 The decrease relative change in [SD] was higher at month a0 than at month a0 and month a0 [] vs [] vs [] The mean SD actual decrease in serum ferritin level from baseline to month a0 was reported as a0µgL relative change in percentage in patient at month a0 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Demographics and a0other baseline characteristicsDemographic variableAge years mean SDAge category years n ¥ to ¥ to ¥ 65aSex n Male FemaleBMI kgm2 mean SDPredominant race n Caucasian Asian OtherbMain underlying disease n MDS IPSSR risk stratification Very low risk Low risk Intermediate risk TDTTime since diagnosis years mean SD MDS TDTPrior ICT received during the ECLIPSE study n Deferasirox DT Deferasirox FCTLast ICT received before deferasirox FCT in the current study n Deferiprone Deferoxamine and deferiprone Deferasirox monotherapyTransfusion history Total number of transfusions received over the past months mean SD Time since last blood transfusion days mean SDBaseline serum ferritin µgL mean SDDeferasirox FCT N BMI body mass index DT dispersible tablet FCT filmcoated tablet ICT iron chelation therapy IPSSR Revised International Prognostic Scoring System MDS myelodysplastic syndrome SD standard deviation TDT transfusiondependent thalassemiaa The oldest patient was years oldb Other included two patients who selfidentified as whiteSafetyAdverse eventsOf the patients almost all [] reported at least AE regardless of study drug relationship Additional file a0 Table a0S2 provides an overview of all AEsThe most common AEs by preferred term were headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain The most common AEs reported in of patients by system an class SOC during the treatment with deferasirox FCT were related to infections and infestations [] influenza rhinitis gastroenteritis pharyngitis and urinary tract infection occurred in of patients by preferred term followed by GI disorders [] diarrhea nausea vomiting upper abdominal pain and abdominal pain occurred in of patients by preferred term Table a0 None of the AEs in the infections and infestations SOC were treatment related Of the AEs that were suspected to be treatment related in patients increased UPCR 0cTartaglione a0et a0al Exp Hematol Oncol Page of Table Exposure to a0treatment and a0complianceDeferasirox FCT N Duration of exposure days mean SDDuration of exposure categories weeks n to to to ¥ Patient treatment yearsAverage actual dose mgkgday mean SDAverage actual dose category mgkgday n to to ¥ Compliance Relative consumed tablet count mean SD Persistence Continuous use of deferasirox FCT with no interruption for ¥ days n Up to months n Up to months n Up to months n Up to months n Continuous use of deferasirox FCT with no interruption for ¥ days n Up to months n Up to months n Up to months n Up to months n FCT filmcoated tablet SD standard deviation diarrhea increased blood creatinine gastritis and proteinuria were the most common reported in of patients AEs by preferred term Of these suspected treatmentrelated AEs increased blood creatinine and diarrhea both of mild severity were reported in MDS patientsThe maximum grade of severity of AEs was reported as mild moderate and severe in and of patients respectively Table a0 Severegrade AEs reported by preferred term irrespective of study drug relationship included increased UPCR splenomegaly atrial fibrillation cardiac failure goiter cholestasis hepatic failure gastroenteritis lower respiratory tract infection lymph gland infection urosepsis femur fracture spinal fracture lumbar vertebral fracture rib fracture ulna fracture transfusion reaction arthralgia back pain malignant melanoma papillary thyroid cancer headache device failure renal colic calculus urinary hydronephrosis and ureterolithiasis each None of the patients had severe GI AEs Moderate and severegrade AEs by maximum severity grade were reported in patients and patients with deferasirox as prior chelation therapy and patients and patients with other ICT as prior chelation therapy respectively Of the patients moderategrade AEs were reported in patients in the to a0mgkgday dailydose group and patients in the ¥ a0 mgkgday dailydose group while severegrade AEs were reported in patients each in the to a0 mgkgday and ¥ a0 mgkgday dailydose groups respectivelySerious AEs SAEs regardless of study drug relationship were reported in patients MDS n TDT n and none of these were considered treatment related SAEs reported in patients with MDS included cardiac failure device failure femur fracture cholestasis hepatic failure and malignant melanoma reported in patient who died lumbar vertebral fracture in patient and urosepsis in patient Other SAEs reported in patients with TDT included spontaneous abortion atrial fibrillation biliary colic urinary calculus cholecystitis diverticulitis fracture goiter hydronephrosis lower respiratory tract infection lymph gland infection ovarian adenoma panic attack papillary thyroid cancer renal colic rib fracture ulna fracture and ureterolithiasis None of these SAEs were reported in more than patient each Death not suspected to be treatment related occurred in a 72yearold male patient with very lowrisk MDS as per the Revised International Prognostic Scoring System [IPSSR] The cause of this ontreatment death according to clinical judgment was malignant melanoma with multiple metastasis in the liver and spleen with unknown primary origin Another contributing factor for the death was liver failure and intrahepatic cholestasisAEs leading to discontinuation of study treatment were reported in patients of which drug ineffective AE in patient and serum ferritin abnormal AE in patient were considered treatment related Treatment was discontinued due to an AE moderate in severity not suspected to be treatment related in MDS patient AEs that led to dose adjustmentinterruption occurred in patients with the most frequently reported being increased UPCR patients [] and vomiting patients [] Treatmentrelated AEs that led to dose adjustmenttemporary interruption were reported in patients and included increased UPCR patients [] increased blood creatinine patients [] gastritis glycosuria proteinuria upper abdominal pain patients [] each and diarrhea gastric ulcer increased serum 0cTartaglione a0et a0al Exp Hematol Oncol Page of morfnitirrefmuresni egnahcLgµenilesabDSnaeMW2n44 M1n51M2n48M3n50 M4n43M5n46 M6n44 M7n40M8n37M9n42M10n36M11n41M12n36M13n43M14n39 M15n28M16n39 M17n32M18n31M19n31M20n33M21n26M23n28 M24n25M22n33M25n21M27n1M26n22Fig Change in serum ferritin from baseline µgL by time point M month SD standard deviation W week Error bars represent the ± SD values for the respective mean valuesNumber of observations at each timepointTime pointTable Common adverse events reported by a0maximum grade of a0severityAEs N a0System an class a0 a0Preferred termOverall n Mild n Moderate n Severe n Number of patients with at least one event Infections and infestations Rhinitis Gastroenteritis Pharyngitis Urinary tract infection Gastrointestinal disorders Diarrhea Nausea Vomiting Upper abdominal pain Abdominal pain General disorders and administration site conditions Asthenia Influenza Pyrexia Respiratory thoracic and mediastinal disorders Cough Oropharyngeal pain Investigationsa Urine proteincreatinine ratio increased Musculoskeletal and connective tissue disorders Musculoskeletal pain Nervous system disorders Headache Proportion of patients with AEs reported by preferred term and grouped by system an classAE adverse eventa Abnormal laboratory values reported as AEs 0cTartaglione a0et a0al Exp Hematol Oncol Page of ferritin hypertransaminasemia and increased transaminases patient [] each AEs required additional treatment in patients with TDT macular edema and skin ulcer in patient [] diarrhea radius fracture and breast discomfort in patient [] each and none were suspected to be treatment relatedAdverse events of a0special interestOverall patients reported AEs of special interest of which AEs suspected to be treatment related were reported in patients Common AEs of special interest incidence included increased UPCR patients [] severe and suspected to be treatment related patients [] proteinuria patients [] increased blood creatinine patients [] MDS n and hypertransaminasemia patients [] Hepatic failure due to metastatic liver disease MDS n and transfusion reaction TDT n of severe grade but not suspected to be treatment related were reported in patient each One patient with MDS discontinued the treatment due to decreased creatinine renal clearance moderate in severity but not suspected to be treatment related Additional file a0 Table a0S3Laboratory parametersWorst postbaseline elevations in SCr of ULN at consecutive measurements at least a0days apart occurred in patients MDS n TDT n One patient with MDS had worst postbaseline UPCR a0 mgmmol at consecutive measurements at least a0 days apart notable range Two patients with MDS had worst postbaseline CrCl value within the notable range a0 mLmin at consecutive measurements at least a0days apart and patients had worst postbaseline CrCl a0mLmin value Two patients with TDT had a worst postbaseline ALT level in the notable range ULN and baseline value Elevations of transaminases AST or ALT ULN were Table Shift tables of a0laboratory values based on a0notableextended rangesALT UL¤ ULN ULN to ¤ ULNTotalAST UL¤ ULN ULN to ¤ ULNTotalSerum creatinine¤ ULNTotalCreatinine clearance¥ ¥ to MissingTotalUrinary proteinurinary creatinine ratio mgmmol¤ MissingTotalBaselinen n n n n ¤ ULN n ¤ ULN n ¥ n ¤ n Worst postbaseline value¤ ULN n ULN to a0¤ ULN n ULN to a0¤ ULN n ULN n ULN n Notable range n Extended range n Notable range n Extended range n Two consecutive ULN n Notable range n One value ¥ to a0 n Notable range n Missing n One value n Extended range n One value n Two values n Notable range n Missing n ALT alanine aminotransferase AST aspartate aminotransferase ULN upper limit of normal 0cTartaglione a0et a0al Exp Hematol Oncol Page of uncommon only patient with TDT had a postbaseline increase in AST and ALT values ULN and baseline value Table a0Hematological parametersThe majority of patients had low RBC of hematocrit of and hemoglobin of values at baseline Among patients with a baseline platelet count ¥ 109L only patients had worst postbaseline values in the notable range ¥ to 109L Similarly among patients with a baseline absolute neutrophil count ¥ 109L the worst postbaseline values were found to be in the notable range ¥ to 109L in patient and extended range 109L in patient Mean ± SD change mean relative percentage change from baseline to month and month in key hematological parameters is represented in Additional file a0 Table a0S4DiscussionThis 2year phase interventional study evaluated the efficacy and safety of deferasirox FCT in adult and pediatric patients mean age a0 years with MDS n and TDT n who had previously completed a0weeks of deferasirox treatment in the ECLIPSE study over a mean duration of further a0days Almost of patients received an average actual deferasirox FCT dose of ¥ a0mgkgday during the study The average actual dose in MDS patients was comparatively less than that in TDT patients reflecting the notion of physicians using lower doses to treat MDS patients [] None of the patients were administered with higher than the maximum recommended doses of deferasirox FCT a0mgkgday []Various studies have demonstrated that compliance with ICT significantly improves morbidity and mortality [ ] Compliance mean relative consumed tablet count and persistence rates with deferasirox FCT during this study were found to be on the higher side Persistence rates proportion of patients with continuous use of deferasirox FCT with no interruption for ¥ a0days or ¥ a0days in this study are comparable to the realworld data on persistence in patients who switched from deferasirox DT to deferasirox FCT []Common AEs reported with deferasirox FCT in this study headache diarrhea pyrexia nausea vomiting cough and upper abdominal pain were consistent with the known profile of deferasirox [ ] AEs reported in of the patients were of mild to moderate in severity none of the GI AEs were of severe grade nor led to discontinuation of treatment These results were in line with those of previous studies which reported that GI tolerability profile may be improved with the FCT compared with the DT Lack of excipients that cause GI irritation and ease of administration along with a light meal could have contributed to the better GI tolerability of FCT [ ] It is noted that the concomitant use of PPIs during the study was higher than reported prior use This study was not designed to investigate the reasons behind the increased use of PPIs but we assume that they might have been prescribed as a general prophylaxis to avoid GI complications from other concomitant therapies analgesics antibiotics etc prescribed in comorbid conditions infections or proceduresSAEs regardless of study drug relationship were reported in of patients and none including the death due to malignant melanoma with multiple metastasis in the liver and spleen were considered treatment related Notably of SAEs reported in patients were reported in MDS patients aged ¥ a0 years reflecting the considerable burden of the underlying hematological disorder together with the comorbidities of the affected elderly patients [] Discontinuation of treatment due to treatmentrelated AEs drug ineffective and abnormal serum ferritin during the a0years was observed in only TDT patients The low rate of SAEs and absence of treatmentrelated deaths in this study could have been a result of the greater compliance and persistence rates with deferasirox FCTThe ECLIPSE study reported that abnormal renal parametersrenal AEs were more common in patients receiving deferasirox FCT than in those receiving deferasirox DT due to an intake of higher than the recommended dose [] Most of the renal abnormalitiesAEs reported during this 2year study were mild and reversible with dosage adjustment or temporary interruption of deferasirox FCT Only patient discontinued the treatment due to decreased creatinine renal clearance which was moderate in severity and not suspected to be treatment related Increases in SCr and liver function tests in the notableextended ranges were observed in some patients and those increases were consistent with the known safety profile of deferasirox FCT [] No substantial difference in severity of all AEs was noted based on dosing groups to a0mgkgday and ¥ a0mgkgday as well Administration of the recommended doses of deferasirox FCT with constant dosage adjustment as per serum ferritin levels might have resulted in fewer renal and liver abnormaliti | Thyroid_Cancer |
Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [] A majority of adult patients with t816p112p133 are therapy related [] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturers instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Childrens Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patients DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturers protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patients DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent à a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilents CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of à 109L with blasts a hemoglobin count of a0 gL and a platelet count of à 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [] Among them cases showed a gain by 1q of variable sizes [ ] As an uncommon entity t816 accounts for of all cases of AML [] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB FrenchAmericanBritishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin Peoples Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr Luppi M Fabbiano F Visani G Martinelli G Venditti A Novel agents for acute myeloid leukemia Cancers Basel Cancer Research UK Acute myeloid leukaemia AML incidence statistics https wwwcance rrese archu khealt hprofe ssion alcance rstati stics stati stics bycance rtypeleuka emiaamlincid ence Accessed Aug Jung J Cho BS Kim HJ Han E Jang W Han K Lee JW Chung NG Cho B Kim M Kim Y Reclassification of acute myeloid leukemia according to the WHO classification Ann Lab Med Murphy T Yee KWL Cytarabine and daunorubicin for the treatment of acute myeloid leukemia Expert Opin Pharmacother Byrd JC Mrózek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B CALGB Blood Lindsley RC Mar BG Mazzola E et al Acute myeloid leukemia ontogeny is defined by distinct somatic mutations Blood Vardiman JW Thiele J Arber DA et al The revision of the World Health anization WHO classification of myeloid neoplasms and acute leukemia rationale and important changes Blood Saultz JN Garzon R Acute myeloid leukemia a concise review J Clin Med Döhner H Weisdorf DJ Bloomfield CD Acute myeloid leukemia N Engl J Med Strickland SA Shaver AC Byrne M et al Genotypic and clinical heterogeneity within NCCN favorablerisk acute myeloid leukemia Leuk Res Yang JJ Park TS Wan TSK Recurrent cytogenetic abnormalities in acute myeloid leukemia Methods Mol Biol Coenen EA Zwaan CM Reinhardt D et al Pediatric acute myeloid leukemia with t816p11p13 a distinct clinical and biological entity a collaborative study by the InternationalBerlinFrankfurtMunster AMLstudy group Blood Xie W Hu S Xu J Chen Z Medeiros LJ Tang G Acute myeloid leukemia with t816p112p133KAT6ACREBBP in adults Ann Hematol Haferlach T Kohlmann A Klein HU et al AML with translocation t816p11p13 demonstrates unique cytomorphological cytogenetic molecular and prognostic features Leukemia Gervais C Murati A Helias C et al Acute myeloid leukaemia with 8p11 MYST3 rearrangement An integrated cytologic cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique Leukemia Diab A Zickl L AbdelWahab O et al Acute myeloid leukemia with translocation t816 presents with features which mimic acute promyelocytic leukemia and is associated with poor prognosis Leuk Res Schouten TJ Hustinx TW Scheres JM Holland R de Vaan GA Malignant histiocytosis Clinical and cytogenetic studies in a newborn and a child Cancer Brown T Swansbury J Taj MM Prognosis of patients with t816p11p13 acute myeloid leukemia Leuk Lymphoma Barrett R Morash B Roback D et al FISH identifies a KAT6ACREBBP fusion caused by a cryptic insertional t816 in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype Pediatr Blood Cancer Schumacher J Szankasi P Kelley TW Detection and quantification of acute myeloid leukemiaassociated fusion transcripts Methods Mol Biol DÃazBeyá M Navarro A Ferrer G et al Acute myeloid leukemia with translocation 816p11p13 and MYST3CREBBP rearrangement harbors a distinctive microRNA signature targeting RET protooncogene Leukemia Veigaard C Nørgaard JM Kjeldsen E Genomic profiling in high hyperdiploid acute myeloid leukemia a retrospective study of cases Cancer Genet Yasar D Karadogan I Alanoglu G et al Array comparative genomic hybridization analysis of adult acute leukemia patients Cancer Genet Cytogenet van der Veken LT Buijs A Array CGH in human leukemia from somatics to genetics Cytogenet Genome Res Laskowska J Szczepanek J StyczyÅski J Tretyn A Array comparative genomic hybridization in pediatric acute leukemias Pediatr Hematol Oncol Mehrotra M Luthra R Ravandi F et al Identification of clinically important chromosomal aberrations in acute myeloid leukemia by arraybased comparative genomic hybridization Leuk Lymphoma Kulikowski LD Bellucco FTS Nogueira SI et al Pure duplication 1q41qter further delineation of trisomy 1q syndromes Am J Med Genet A 2008146A2663 Nowaczyk MJM Bayani J Freeman V Watts J Squire J Xu J De novo 1q32q44 duplication and distal 1q trisomy syndrome Am J Med Genet A 2003120A229 Cone EB Dalton SS Van Noord M Tracy ET Rice HE Routh JC Biomarkers for wilms tumor a systematic review J Urol Xu B Ghossein R Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma Endocr Pathol Fournier A Florin A Lefebvre C Solly F Leroux D Callanan MB Genetics and epigenetics of 1q rearrangements in hematological malignancies Cytogenet Genome Res Lancman G Tremblay D Barley K et al The effect of novel therapies in highmolecularrisk multiple myeloma Clin Adv Hematol Oncol Bacher U Schnittger S Grüneisen A Haferlach T Kern W Haferlach C Inverted duplication dup1q32q21 as sole aberration in lymphoid and myeloid malignancies Cancer Genet Cytogenet Marcellino BK Hoffman R Tripodi J et al Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53 Blood Adv Beach DF Barnoski BL Aviv H et al Duplication of chromosome [dup1q21q32] as the sole cytogenetic abnormality in a patient previously treated for AML Cancer Genet Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | Thyroid_Cancer |
This study was performed to explore the effective management of bleeding associated with radiofrequency ablation RFA of benign thyroid nodulesMethods Thirtyfive patients with benign thyroid nodules who were treated with ultrasoundguided RFA from July to December at the Third Affiliated Hospital of Sun YatsenUniversity were retrospectively reviewed The technique efficacy bleeding and other complications were assessed during the followup periodResults The mean technique efficacy was 06 at month and 06 at months after the procedure One case of an intranodular haematoma and two cases of voicechange month were observed All patients recovered with corresponding treatmentConclusion Although the incidence of haemorrhage is low serious haematomas are lifethreatening Therefore having a comprehensive understanding of the potential complicationsan accurate clinical strategy and adequate technical skills may prevent or help to properly managethese complicationsKeywordsRadiofrequency ablation benign thyroid nodules haemorrhage management haematomaultrasoundDate received January accepted June 1Department of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Guangzhou China2General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsBo Liu General Surgery Department The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province China Jie RenDepartment of Medical Ultrasound The Third AffiliatedHospital of Sun Yatsen University Tianhe RoadGuangzhou City Guangdong Province ChinaEmails renjieguangzhou126com liubojake126comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cIntroductionAssociationfor AdultThyroid nodules are extremely commonand the associated morbidity rate rangesfrom to according to highresolution ultrasound US ï¬ndings12Mostthyroid nodules are benign andrequire no intervention other than clinicalfollowup According to the AmericanThyroidManagementGuidelinesPatients withThyroid Nodulesand DifferentiatedThyroid Cancer thyroidstimulating hormone suppression therapy for benign thyroid nodules BTNs is not recommendedbecause the potential harm outweighs thebeneï¬t3 Radioiodine therapy was historically an effective treatment for thyroid hotnodules and a possible alternative to surgery Howeverthis technique has beenproven to have uncertain efï¬cacy andsome adverse effects such as hypothyroidrecurrence4 Surgery may beism orconsideredgrowing BTNs withpressurerelated symptoms neck discomfort cosmetic concerns or decreased quality of life3 At present partialtotal thyroidsurgery is considered the gold standardtreatment Surgeryassociated withnumerous complications such as nerveinjury anaesthesiarelated problemslonghospital stays conspicuous scars haemorrhage and lesions ofthe parathyroidglands78 In addition hypothyroidism isinevitable after totalthyroidectomy andrequires lifelong hormone supplementationHenceincreasingly minimally invasivetherapeutic strategies are currently used totreat BTNs In most cases several thermalablation techniques such as laser ablationmicrowave ablation radiofrequency ablation RFA and highintensity focused UShave been shown to be effective in BTNsAmong these thermal ablation techniquesRFA is the most widely applied910forisRFA of thyroid diseases ï¬rst reported in is considered efï¬cacious and safeJournal of International Medical Researchfor treatment of BTNs1415 To date no lifethreatening complications related to RFAhave been reported Howeverseveralcases of haemorrhagerelated to ï¬neneedle aspiration FNA or core needlebiopsy CNB have been reported16Although a microinvasive procedure suchas FNA can result in massive uncontrolledbleeding resulting in upper airway respiratoryuncontrolledbleeding is a rare but lifethreatening complication of RFA Thus management ofbleeding associated with RFA of BTNs isof vitalimportance This study was performed to explore the effective managementof bleeding associated with RFA of BTNsobstructionsuchMaterials and methodsofThis study was approved by the EthicsCommitteethe Third Afï¬liatedHospital of Sun Yatsen University andwritten informed consent was obtainedfrom all patients prior to the performanceof USguided FNA or CNB and RFA Therequirement to obtain informed consent forpublication was waived because of the retrospective nature of the studyPatientsAll consecutive patients who underwentRFA of BTNs at our institution from July to December were analysed Thefollowing inclusion criteria were appliedconï¬rmation of benignancyBethesdaClass II by FNA cytology or CNB complaints of pressure symptoms compressivesymptoms neck discomfort orforeignbody sensation or cosmetic problems a2cm maximum diameter of the indexnodule anxiety about a malignancy unsuitability for surgery or unwillingness toundergo surgery and a normal serum thyrotropin concentration normal completeblood counts and normal blood coagulation test results The exclusion criteria 0cHu et alwere nodules showing malignant featuresie taller than wide spiculated marginmarked hypoechoic appearance or microcalciï¬cations on US imaging19 abnormalthyroid function performance of othertreatments for the thyroid nodules within months before the procedure pregnancyand age of years For the presentstudy only patients with 15 months offollowup after the procedure were included Thirtyï¬ve patients met the inclusioncriteriaPretreatment assessmentBefore the procedure conventional USï¬ndings USguidedFNA ï¬ndingscontrastenhanced US CEUS ï¬ndingsand laboratory and clinical results wereevaluated Two radiologists TW and JR with and years of thyroid US experiencerespectively performed the USUSguided FNA and CEUS examinationsusing a Logiq E9 US device GE MedicalSystems Milwaukee WI USA equippedwithtransducerwith a MHzfrequency range MHz The USexamination included characterisation ofthe location shape size margins solidcystic proportions echogenicity calciï¬cation status and internal vascularity ofeach nodulefrequency ofan ML615centrelinearLaboratory tests included the levels ofthyroidstimulating hormone free triiodothyronine free thyroxin and thyrotropina complete blood cell count and a coagulation test prothrombin time and activatedpartial thromboplastin time The nodulevolume was calculated using the followingvolume¼ length 02 width 02equationdepth 02 In addition all patientsunderwent vocal cord function assessmentsby an experienced laryngologist before theablation procedure Atenrolment allpatients were asked to rate their pressuresymptoms on a 10cm visual analoguescale grade cm and the cosmeticgrading score was assessed by the physicianas described in the consensus statement20Procedures and equipmentpreventsignificantAll RFA procedures were performed by oneradiologist JR with years of experienceperforming RFA in an outpatient clinic Weused an RF generator VIVA RF SystemVR STARmed Gyeonggisi South Korea andan internally cooled 18gauge 70mmlength or 10mm activetip electrodeStar RF ElectrodeVR STARmed Localanaesthesia with lidocaine was appliedto the puncture site The hydrodissectiontechnique was used under US guidance glucose and norepinephrine weremixed and injected into the surroundingthyroid capsule which provided a safe distance between the needle tip and adjacentcritical structures During the procedurewe paid special attention to the preservation of surrounding important structurestocomplicationsTherefore two essential techniques werethe transisthmic approach andappliedtechnique2122 Ablationthe movingshotwas suspended when the index nodule wascovered by hyperechoic zones The technique efï¬cacy TE was then evaluated byCEUS at to minutes after RFA untilthedisappearedTechnicalthechange of an entire nodule to a noenhancement zone on realtime CEUSFor nodules with an enhancement zonean additional ablation was performed todestroy the nodule as much as possibleComplications were monitored immediatelyafter the procedure and during the followup period Major and minor complicationsand adverse effects were deï¬ned accordingto the criteria established by the Society ofInterventional Radiology2324success was deï¬ned ashyperechoiczones 0cJournal of International Medical ResearchFollowup evaluationatandperformedserum thyroidAny speciï¬c complaints or concerns wererecorded for month Postproceduralfollowup wasand months after treatment At each followup visit a US examination CEUS examinationhormonemeasurements were performed pressuresymptoms and the cosmetic grading scorewere evaluated and the volume ofthetreated nodule was calculated The TE wascalculated using the following equationTE¼ ï¬nal nodule volumeinitial nodule volume 02 Statistical analysisstatistical analyses were performedAllusing SPSS software version IBMCorp Armonk NY USA Continuousvariables are expressed as mean 06 standarddeviation Quantitative data for volumeand TE were analysed using a pairedttest A P value of 14 was consideredstatistically significantResultsThe patients characteristics are summarised in Table Thirtyï¬ve patients underwent RFA including male and femalepatients mean age years The meanlargest BTN dimension was 06 mmrange mm and the mean BTNvolume was 06 mL Twentytwototal complications minor and majorcomplications were observed among thetreated patients None of these complications was lifethreatening and all occurredwithout sequelaeNodule volumeAfter treatment the overall volume of thesignificantly decreased 06nodules 06 mL at mL at month and 06 mL at monthsbaselineTable Patients baseline characteristics n¼ Characteristics 06 06 06 06 06 06 06 06 Age at treatment yearsMalefemale ratioBody weight kgBody height cmBody mass index kgm2Symptom score Cosmetic score Cosmetic score of Cosmetic score of Cosmetic score of Preablation serum FT4 level pmolLPreablation serum TSH level mIULIndex nodule on ultrasoundRight sideLeft sideLargest dimension mm to to 15Data are presented as mean 06 standard deviation ornumber of patientsFT4 free thyroxin TSH thyroidstimulating hormoneP and the TE was 06 at month and 06 at months P Table Figure shows the shrinkage of the nodules at and months after the procedure comparedwith baseline no hypoechoic blood supplywas observed within the area ofthenodulesBleeding complicationsTwelve patients developed bleeding complicationsincluding a perithyroidal haematoma minor complication in patientsand an intranodular haematoma majorcomplication in patient as shown inTable The haematomas were detectedby US scans which revealed gradualenlargement of a hyperechoic mass in oraround the nodules Figure For thepatient with intranodular haemorrhage 0cHu et alTable Changes in volume before RFA and at each followup visitParameterInitial month laterLargest diameter mm 06 06 Volume mLTechnique efficacy Data are presented as mean 06 standard deviation range 06 06 06 months laterP value 06 06 06 Figure a c e Ultrasound examination and b d f contrastenhanced ultrasound examination of a39yearold woman treated with radiofrequency ablation a b Ultrasound and contrastenhanced ultrasound revealed a cysticsolid nodule before ablation c d One month after ablation ultrasound showed ahypoechoic nodule with a decreased volume d e Six months after ablation the volume of the nodule haddecreased further and no blood supply was observed within the area of the nodule 0cJournal of International Medical ResearchTable Complications and adverse effects in patients who underwent RFA of thyroid nodulesComplication or adverse effectAdverse effectsFeverPainDizzinessSensation of heatMinorPerithyroidal haematomaVomitingnauseaOedemaswellingVoice change for monthMajorVoice change for monthIntranodular haemorrhageData are presented as n the haematoma was controlled throughtimely use of the ablation needle to coagulate the injured blood vessel and by injecting lyophilising thrombin powder into thehaematoma Figure Most of the perithyroidalseriesrequired only observation with or withoutcompression and disappeared within to weeks after the procedure None of the patientssubscapularhaematomahaematomasdevelopedthisinaOther complications and adverse effectsThe adverse effects of RFA included fevern¼ pain n¼ dizziness n¼ and a sensation of heatn¼ Minor complications includedoedemaswelling n¼ and a voicechange for month n¼ vomitingnausean¼ DiscussionImageguided thermal ablation techniquessuch as laser ablation ethanol ablationmicrowave ablation highintensity focusedUS and particularly RFA have recentlybecome more widely used to treat thyroidthe creation ofnodules Brieï¬y the basic mechanism ofRFA involvesthermaldamage by friction and heat conductionwhich is generated from an oscillatinghighfrequency alternating electric currentproduced by the RFA generator and thentransferred through the electrode tip Theenergy of RFA is powerful and accurate2526 RFA is considered an effectiveand safe treatment for control of BTNsIn most cases the incidence of haemorrhage and other complications is low20However haemorrhage is sometimes lifethreatening because serious haematomasmay compress the upper airways Manyreports have described active bleedingduring FNA of thyroid nodules and RFAof hepatocellular carcinomas14 andsome reports have described fatalities14ThusimportantcomplicationhaemorrhageanisThree types of haemorrhage may occurperithyroidal subcapsular and intranodular121427 The mechanism of haemorrhage is thoughtto be related to themechanical or thermal injuries induced bythe RFA electrode tip3031 Thyroid nodulesreportedly have abundant capsular vesselsthat are usually anastomosed with vesselspenetrating into the core32 These numerousvessels are abnormal thinwalled and susceptible to rupture Large thyroid nodulesare another cause of haemorrhage becausemultiple insertions are often required totreat such nodules In additionthepatient cannot coordinate with the physician during the RFA procedure the perithyroidal orintrathyroidal vessels mayeasily be damaged by movement of theneedle tip or production of heat energyifIt is important to manage bleeding associated with RFA of BTNs Based on ourexperience we suggest several steps to preventshouldobtain a thorough medical history of eachpatient before the procedure All risk factorsdrugssuch bleeding Physiciansincludingbleedingfor 0cHu et alFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage and perithyroidal haemorrhage a Ultrasound and contrastenhanced ultrasoundrevealed a hyperechoic mass lesion in the nodule b Ultrasound and contrastenhanced ultrasound revealedperithyroidal haemorrhagedrugsnonsteroidalantiplateletantiinï¬ammatory drugs and anticoagulantsand diseases affecting coagulation shouldbe recorded33 In addition the patientscoagulation function should be thoroughlyevaluated All patients with clinical coagulation disorders should be excluded Evenwhen coagulation indices are normalinpatients with high risk factors for bleedingsuch as liver cirrhosis endstage renal disease anticoagulant use or hypertension34sufï¬cient preoperative preparation shouldbe emphasised A patient with active bleedingthesein the presentstudy metconditions Although his coagulation indices were normal he had a subclinical coagulation disorder due to endstage liverdisease Fresh frozen plasma or human prothrombin complex should be used inpatients with liver cirrhosis and anticoagulants should be withdrawn in these patientswhich will help to improve coagulation function before the procedure If a possibility ofbleeding exists Reptilase haemocoagulaseatrox forinjection Pentapharm BaselSwitzerland can be used preoperativelyDuring the RFA procedure an effectiveclinical strategy and adequate technical 0cJournal of International Medical ResearchFigure Ultrasound examination and contrastenhanced ultrasound examination of patients with intranodular haemorrhage or perithyroidal haemorrhage during ablation a Ultrasound revealed a hyperechoicmass lesion in the nodule b Ultrasound showed an ablation needle inserted into the nodule to coagulatebleeding vessels c After lyophilising thrombin powder was injected into the hematoma ultrasound andcontrastenhanced ultrasound showed disappearance of the hyperechoic mass lesion and microbubbleextravasation d Ultrasound showed a hyperechoic mass lesion around the thyroid and contrastenhancedultrasound showed no microbubble extravasation around the thyroid e After lyophilising thrombinpowder was injected into the haematoma no microbubble extravasation was observedskills are both essential Patient cooperationis the ï¬rst requirement When the needle tipis in the patients body any uncooperativemotion of the patient may lead to injury ofvessels or other structures Most patientscan endure the procedure under local anaesthesia however anxious patients mayrequire general anaesthesia to achieve cooperation If possible smallbore electrodesshould be chosen to decrease the risk ofbleeding35 It is necessary to cauterise thesupplying vasculature of nodules to avoidrecurrence and residue Howeverthepuncture route should be carefully designedto avoid pericapsular vessels and the electrode tip should be closely monitoredActive bleeding during needle puncture isvisible as a rapidly expanding hypoechoicor anechoic signal Locating the haemorrhagic focus is not difï¬cult with CEUSguidance The bleeding pointcan beblocked by RF electrode tip insertion anddirect ablation When the bleeding is toorapid to control with the RF electrode tipby increasing the power drug injection is asuitable alternative Lyophilised thrombin 0cHu et alpowder can be dissolved in normal salineand then injected at the bleeding pointthrough a syringe with US guidance Onereport also described haemorrhage treatedby local injection of hypertonic saline andepinephrine solution in a patient with hepatocarcinoma36 Mildbleeding whichappears as a hypoechoic layer can mostlybe controlled using ice and compression ofthe neck for several minutes after the procedure30 All bleeding can be controlled byconservative methodsthus no surgicalintervention is needed Ecchymosis can befound after the procedure and usually disappears in approximately to weeksPostprocedure CEUS is indispensablefor all patients regardless of whether bleeding occurs CEUS is an objective evaluationtool for active bleeding37 Close clinicalobservation for hours postoperatively isrecommended in our department becausemost bleeding occurs during the ï¬rstlobectomy38Observation of the neck can help to detecta haematoma early and may aid in preventing serious adverse effectsthyroidhoursafterConclusionAcute thyroid bleeding is one possible complication of RFA although rare it is potentially lifethreatening Proper selection ofpatients and sufï¬cient preparation areessential During the RFA procedureboth an effective clinical strategy and adequate technical skills are indispensable Thephysician should trace the electrode tipusing realtime US and sufï¬ciently managebleeding Mild bleeding has limited morbidity and can be easily controlled by compression Active bleeding tends to be rarehowever it may be disastrous if the operator is unaware or careless Direct ablationwith the RF electrode tip and drug injectioninto the bleeding focus are effective modalities for active bleeding CEUS and closeobservation are also recommended afterthe procedureto detect abnormalitiesearly RFA is an effective and relativelysafe alternative for selected patients withBTNs if performed by skilled physiciansAuthor contributionsI Conception and design Jie Ren and Bo LiuII Administrative support Jie RenIIIstudy materials or patientsProvision ofKunpeng Hu and Yufan Lian IV Collectionand assembly of dataJinfen Wang andWenchao Li V Data analysis and interpretation Wenchao Li and Zhicheng YaoVIManuscript writing All authors VII Finalapproval of manuscript All authorsData availabilityData regarding the patients characteristics usedto support the funding are shown in Table Declaration of conflicting interestThe authors declare that there is no conï¬ict ofinterestFundingthe NaturalThis work was supported by the NationalNatural Science Foundation of China CNNoScienceFoundation of Guangdong ProvinceNo2016A030313200 the Science and TechnologyProject of Guangzhou City No the Hengrui Foundation of Hepatobiliary andPancreaticNoCXPJJH1180000120183331NaturalScience Foundation of Guangdong ProvinceNothe FundamentalResearch Funds for the Central UniversitiesSun Yatsen University No 17ykpy67 andthe Clinical Research Project of Sun Yatsen University No 2017A030313580theCancerResearchORCID iDKunpeng Huorcid00000001 0cReferences Guth S Theune U Aberle J et al Very highprevalence of thyroid nodules detected byhigh frequency MHz ultrasound examination Eur J Clin Invest Tan GH and Gharib H Thyroid incidentalomas management approaches to nonpalpable nodules discovered incidentally onthyroid imaging Ann Intern Med Haugen BR Alexander EK Bible KC et al AmericanThyroid AssociationManagement Guidelines for Adult Patientswith Thyroid Nodules and DifferentiatedThyroid Cancer The American ThyroidAssociation Guidelines Task Force onThyroid Nodulesand DifferentiatedThyroid Cancer Thyroid Ceccarelli C Bencivelli W Vitti P et alOutcome ofradioiodine131 therapy inhyperfunctioning thyroid nodules a years retrospective study Clin EndocrinolOxf Reiners C and Schneider P Radioiodinetherapy of thyroid autonomy Eur J NuclMed Mol Imaging S471S478 Nieuwlaat WA Hermus AR SivroPrndeljF et al Pretreatment with recombinanthuman TSH changes the regional distribution of radioiodine on thyroid scintigramsof nodular goiters J Clin Endocrinol Metab LinosDEconomopoulosKPKiriakopoulos A et al Scar perceptionsafter thyroid and parathyroid surgery comparison of minimaland conventionalapproaches Surgery Jeannon JP Orabi AA Bruch GA et allaryngeal nervethyroidectomy a systematicDiagnosis ofpalsy afterreview Int J Clin Pract recurrent Lang B Woo YC and Chiu KW Identifyingpredictive factors for efï¬cacy in high intensity focused ultrasound HIFU ablationof benign thyroid nodules a retrospectiveInt J Hyperthermia analysis Mauri G Pacella CM Papini E et alImageguided thyroid ablation proposalforterminology andstandardization ofJournal of International Medical Researchreportingcriteria Thyroid Sato M Tateishi R Yasunaga H et alMortality and hemorrhagic complicationsassociated with radiofrequency ablation fortreatment of hepatocellular carcinoma inendstagepatients on hemodialysissurveyrenalJGastroenterol Hepatolnationwidediseasefora Krokidis M Spiliopoulos S Jarzabek Met al Percutaneous radiofrequency ablationof small renal tumours in patients with asingle functioning kidney longterm resultsEur Radiol Lim HK Lee Dupuy DE Monchik JM Decrea C et alRadiofrequency ablation of regional recurrence from welldifferentiated thyroid malignancy Surgery JH Ha EJalRadiofrequency ablation of benign nonfunctioning thyroid nodules 4year followup results for patients Eur Radiol et Braga M Cavalcanti TC Collaco LM et alEfï¬cacy of ultrasoundguided ï¬neneedleaspiration biopsy in the diagnosis of complex thyroid nodules J Clin EndocrinolMetab Kakiuchi Y Idota N Nakamura M et alA fatal case of cervical hemorrhage after ï¬neneedle aspiration and core needle biopsy ofthe thyroid gland Am J Forensic Med Pathol Donatini G Masoni T Ricci V et al Acuterespiratory distress following ï¬ne needleaspiration of thyroid nodule case reportand review of the literature G Chir Roh JL Intrathyroid hemorrhage and acuteupper airway obstruction after ï¬ne needleaspirationthyroidglandLaryngoscope theofofAssociation Gharib H Papini E Garber JR et alAmericanClinicalEndocrinologists American College ofEndocrinology and Associazione MediciEndocrinologi medical guidelines for clinicalpractice for the diagnosis and managementthyroid nodules update EndocrofPract 0cHu et al Na DG LeeetJHJung SLalRadiofrequency ablation of benign thyroidnodules and recurrent thyroid cancers consensusstatement and recommendationsKorean J Radiol Ha EJ Baek JH and Lee JH Movingshotversus ï¬xed electrode techniques for radiofrequency ablation comparison in an exvivo bovine liver tissue model Korean JRadiol Jeong WK Baek JH Rhim H et alRadiofrequency ablation of benign thyroidnodules safety and imaging followup inpatients Eur Radiol Cardella JF Kundu S Miller DL et alSociety of Interventional Radiology clinicalpractice guidelines J Vasc Interv Radiol S189S191 Sacks D McClenny TE Cardella JF et alSociety of Interventional Radiology clinicalpractice guidelines J Vasc Interv Radiol S199S202 Goldberg SN Radiofrequency tumor ablation principles and techniques Eur JUltrasound Rhim H Goldberg SN Dodd GR et alEssential techniques for successful radiofrequency thermal ablation of malignanthepatic tumors Radiographics Spec No S17S35 S36S39 Korkusuz Y Erbelding C Kohlhase Ket al Bipolar Radiofrequency Ablation ofBenign Symptomatic Thyroid Nodules initial Experience Rofo Garberoglio R Aliberti C Appetecchia Met al Radiofrequency ablation for thyroidnodules which indications The ï¬rst Italianopinion statement J Ultrasound Baek JH LeeJH Sung JYet alComplications encountered in the treatmentof benign thyroid nodules with USguidedradiofrequencya multicenterstudy Radiology ablation Chen MH Dai Y Yan KalIntraperitoneal hemorrhage duringandafter percutaneous radiofrequency ablationof hepatic tumors reasons and managementChin Med J Engl et Rhim H Dodd GR Chintapalli KN et alRadiofrequency thermal ablation of abdominal tumors lessons learned from complications Radiographics Terry WI Radium emanations in exophthalmic goiterblood vessels of adenomas ofthyroid J Am Med Assoc Hor T and Lahiri SW Bilateral thyroidhematomas after ï¬neneedle aspiration causing acute airway obstruction Thyroid Minami Y Hayaishi S and Kudo MRadiofrequency ablation for hepatic malignancies is needle tract cauterization necessary for preventing iatrogenic bleeding DigDis Baek JH Kim YS Lee D et al Benign predominantly solid thyroid nodules prospective study of efï¬cacy of sonographicallyguided radiofrequency ablation versus control condition AJR Am J Roentgenol Koda M Murawaki Y Hirooka Y et alComplications of radiofrequency ablationfor hepatocellular carcinoma in a multicenter study an analysis of treated nodules in patients Hepatol Res Wiggermann P Wohlgemuth WA Heibl Met al Dynamic evaluation and quantiï¬cationof microvascularization during degradablestarch microspheres transarterial chemoembolisation DSMTACE of HCC lesionsusingultrasoundCEUS a feasibility study Clin HemorheolMicrocirc enhancedcontrast Rosenbaum MA Haridas M and McHenryCR Lifethreatening neck hematoma complicating thyroid and parathyroid surgeryAm J Surg 0c' | Thyroid_Cancer |
evaluation of biochemical and hematological parameters in adults with Down syndromeDavid de Gonzalocalvo123 Isabel Barroeta45 Madalina Nicoleta Nan6 Jos Rives6 Diana Garzn45 Mara CarmonaIragui457 Bessy Benejam457 Laura Videla457 Susana fern¡ndez7 Miren Altuna45 Slvia Valldeneu45 Rafael Blesa45 Alberto Lle45 Francisco BlancoVaca689 Juan Fortea457 Mireia Tondo6Down syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting liveborn infants In addition to intellectual disability individuals with DS have other comorbidities and complex medical conditions The increase in the life expectancy of patients with DS requires expanding the knowledge about their clinical characteristics and related laboratory parameters Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism Therefore our main goal was to describe the biochemical and hematological findings in a DS cohort and to compare the values to those of a control population A total of DS individuals and control subjects were enrolled DS individuals had a higher frequency of several clinical conditions compared to control individuals and presented with significant differences with respect to the controls in both biochemical and hematological parameters We found age and sexrelated differences in several of the parameters A good understanding of the differences in our cohort might be of aid in the clinical followup of adults with DS especially considering that the lifespan of DS individuals may reach years of age in developed countriesAbbreviationsAD AF ALT AST B12 CKDEPI DS ESR FT4 eGFR GGT HbA1c Alzheimers disease Alkaline phosphatase Alanine aminotransferase Aspartate aminotransferase Vitamin B12 Chronic kidney disease epidemiology collaboration Down syndrome Erythrocyte sedimentation rate Free thyroxine Estimated glomerular filtration rate Gammaglutamyl transferase Glycated hemoglobin1Biomedical Research Institute Sant Pau IIB Sant Pau Barcelona Spain 2Institute of Biomedical Research of Barcelona IIBB Spanish National Research Council CSIC Barcelona Spain 3Translational Research in Respiratory Medicine University Hospital Arnau de Vilanova and Santa Maria IRBLleida Lleida Spain 4Sant Pau Memory Unit Department of Neurology Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau Universitat Aut²noma de Barcelona Barcelona Spain 5Center of Biomedical Investigation Network for Neurodegenerative Diseases CIBERNED Madrid Spain 6Department of Biochemistry Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau CSant Quint Barcelona Spain 7Barcelona Down Medical Center Fundaci Catalana de Sndrome de Down Barcelona Spain 8Center of Biomedical Investigation Network for Diabetes and Metabolic Diseases CIBERDEM Madrid Spain 9Department of Biochemistry and Molecular Biology Universitat Aut²noma de Barcelona Barcelona Spain email mtondosantpaucatScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cHDLc LDLc MCH MCHC MCV MDRD4 MPV K RDW Na TG TSH Highdensity lipoprotein cholesterol Lowdensity lipoprotein cholesterol Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume Modification of diet in renal disease Mean platelet volume Potassium Red blood cell distribution width Sodium Triglycerides Thyroid stimulating hormoneDown syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting liveborn infants with an estimated birth prevalence of per live births1 Despite the shorter life expectancy when compared to healthy subjects and adults with other causes of intellectual disability4 there has been a progressive increase in the life expectancy of patients with DS in recent decades currently reaching nearly years5 This fact has increased the need to expand the knowledge about the clinical characteristics of DS individuals and the health problems differentiating them from both pediatric and adult populations6 DS is associated with a distinct phenotype involving many body systems In addition to intellectual disability individuals with DS present with a high number of comorbidities and complex medical conditions whose frequencies are modified throughout the lifespan of the individuals7 The increase in life expectancy has led to a higher prevalence of agerelated pathologies including premature Alzheimers disease AD8Since optimal medical management is associated with improved quality of life and functioning among persons with DS910 medical professionals including pediatricians and other physicians should closely supervise this population throughout their lifespan and evaluate their laboratory results Previous investigations in DS cohorts have focused on select biochemical parameters such as uric acid and thyroid function biomarkers bone mineral density nutritional zinc status gonadal and endocrine function and glucose and lipid metabolism parameters11 However no previous work has described a comprehensive panel of biochemical and hematological parameters in a large cohort of DS patientsOur hypothesis is that a thorough analysis of the biochemical and hematological parameters will provide a basis to establish whether commonly observed alterations in DS individuals are intrinsic of the disease or have clinical implications similarly as for the general population Therefore our goals were to describe the biochemical and hematological findings in our DS cohort and to compare the values to those of a control populationMaterial and methodsStudy participants This was a singlecenter descriptive study of adults with DS recruited at Barcelona Down Medical Center Fundaci Catalana Sndrome de Down and Hospital de la Santa Creu i Sant Pau Barcelona in Catalonia Spain according to a populationbased health plan to screen for neurological comorbidities1718 The Down Medical Center provides medical care specifically for individuals with DS and possesses over medical records more than of the estimated Down syndrome population in Catalonia therefore it reflects the population with DS in our geographic area The period of patient recruitment for this study was February to June In adults with DS ¥ a0years a biochemical and hematological analysis was performed as part of their annual health plan visit A total of patients were enrolled in the study Six further patients were ultimately excluded for presenting with conditions unrelated to DS according to their medical records patients with hepatitis C patient with hepatitis B and patient with breast cancer resulting in a final total number of DS individuals included age range a0years A total of healthy control participants in the same age range a0years were enrolled in the study Volunteers were recruited from the SPIN Sant Pau Initiative on Neurodegeneration cohort santp aumem oryun itcomourresea rchspincohor t or social media SantPauMemory Further details on the clinical protocol of the SPIN cohort can be found elsewhere19Based on current guidelines1720 associated clinical conditions were obtained through a systematic review of the medical records including the following history of arterial hypertension dyslipidemia diabetes mellitus congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology psychiatric pathology epilepsy and Alzheimers disease Treatment data with a special focus on the treatment of hypothyroidism were also collectedBiochemical and hematological data Analyzed biochemical and hematological parameters were selected according to a defined laboratory blood profile as recommended in the guidelines for management of patients with DS1720Blood collection and processing were performed in accordance with the Standard Operating Procedures for Serum and Plasma Collection from the Early Detection Research Network EDRN Consensus Statement and Standard Operating Procedure Integration Working Group21 Blood samples were collected by venipuncture after an overnight fastWhole blood samples were collected in VACUTAINER tubes and fractionated by centrifugation at a0g for a0min at room temperature to obtain serum Serum was aliquoted into a0mL tubes and the following parameters were measured according to standard commercially available assays adapted to an Architect C4000 Abbott Diagnostics USA using automated procedures thyroid stimulating hormone TSH free thyroxine FT4 Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cAge yearsMalefemaleArterial hypertensionDyslipidemiaDiabetes mellitusCongenital heart diseaseGastrointestinal pathologyDermatological pathologyBone pathologyHypothyroidismTreatment for hypothyroidismHearing problemsOtolaryngology pathologyOphtalmological pathologyPsychiatric pathologyEpilepsyAlzheimers disease Controln Median P25P75n Down syndromenMedian P25P754n pvalue Table Characteristics of the Study Population Data are presented as frequencies percentages for categorical variables Continuous variables are presented as median interquartile range Differences between groups were analyzed using Wilcoxon ranksum test or Fishers exact testsodium Na potassium K glucose urea creatinine total bilirubin triglycerides TG total cholesterol aspartate aminotransferase AST alanine aminotransferase ALT alkaline phosphatase AF gammaglutamyl transferase GGT total proteins vitamin B12 and folate The estimated glomerular filtration rate eGFR was calculated according to the MDRD4 Modification of Diet in Renal Disease and CKDEPI Chronic Kidney Disease Epidemiology Collaboration formulasWhole blood samples in EDTAK3 were also obtained for determining blood cell count and indices The tubes were immediately inverted times to mix the anticoagulant additive with blood The blood was processed within a0h of extraction Using the impedance channel of the automated hematology analyzer Sysmex XE2100 Roche Diagnostics Kobe Japan the following parameters were determined red blood cell count RBC white blood cell count WBC platelet count hemoglobin hematocrit mean volume MCV mean corpuscular hemoglobin concentration MCHC mean corpuscular hemoglobin MCH red blood cell distribution width RDW and mean platelet volume MPV The erythrocyte sedimentation rate ESR was calculated with a VES cube Sysmex Analyzer Roche Diagnostics Kobe JapanValues were compared to normal reference ranges used in our laboratory established in a healthy population from our geographical area according to standardized guides22Statistical analysis Descriptive statistics were used to summarize the characteristics of the study population Data are presented as medians [25th percentile P2575th percentile P75] for continuous variables and as frequencies percentages for categorical variables Data normality was analyzed using the KolmogorovSmirnov test Continuous variables were compared between groups using the Wilcoxon ranksum test ANCOVA models adjusted for age and sex were used to compare continuous variables across the study groups Variables were logtransformed to achieve a normal distribution For clarity the original values are shown Categorical variables were compared between groups using Fishers exact test Spearmans rho coefficient was used to assess the correlation between continuous variables The statistical software package R wwwrproje ct was used for statistical analyses A Pvalue was considered statistically significantEthical aspects The study was approved by the Sant Pau Ethics Committee following the standards for medical research in humans recommended by the Declaration of Helsinki and in accordance with Spanish legislation for research in people with intellectual disabilities All participants or their legally authorized representatives gave written informed consent before enrolment in accordance with the guidelines of the local ethics committeeResultsStudy cohort characteristics We enrolled a total of individuals with DS males and females with a median age of years and control subjects males and females with a median age of years The clinical features of the DS and control populations are listed in Table a0 The frequency of the following clinical conditions was significantly higher in the DS group than in the control group history of congenital heart disease gastrointestinal pathology dermatologiScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0ccal pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology epilepsy and AD No differences were observed in the frequency of diabetes mellitus or psychiatric pathology for either group DS individuals presented with a lower frequency of arterial hypertension and dyslipidemia compared to the control group See Table a0 for further details on the cohort characteristicsBiochemical and hematological parameters in patients with Down syndrome We performed a detailed biochemical and hematological analysis of the DS cohort and compared the profiles obtained with our control population The reference values of the studied parameters the number and percentage of patients out of range and the median P25P75 of the whole study population are shown in Table a0 Seventythree percent of the studied hematological parameters and of the studied biochemical parameters were significantly different between the DS individuals and the control population The DS individuals presented with higher TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW values and lower TG total cholesterol folate eGFR MPV and WBC values These differences remained significant or close to signification after adjusting for confounding factors such as age and sex Statistical differences for RBC and MCHC were observed after adjustment An additional analysis to evaluate the impact of hypothyroidism treatment on TSH was performedNo differences were observed for TSH between both studied groups treated DS individuals vs untreated DS individuals Pvalue For categorical variables the percentage of DS individuals out of range for some parameters was also statistically significant compared to the control population Parameters with a higher percentage of values out of range in the DS group were TSH urea creatinine total proteins RBC MCV ESR MCH and WBC whereas those with a lower percentage of values out of range were K TG total cholesterol and ASTThe differences in the biochemical and hematological parameters and the number and percentage of patients out of range between DS individuals and the control population according to sex are displayed in Supplemental Tables a0 and For the female DS cohort parameters with significantly higher values were TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol GGT eGFR RBC MPV and WBC For categorical variables parameters with significantly higher percentages of values out of range were TSH creatinine total proteins MCV ESR MCH and WBC whereas those with a significantly lower percentage of values out of range were total cholesterol and B12 Supplemental Table a0 For the male DS cohort parameters with significantly higher values were TSH hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol eGFR MPV and WBC Regarding categorical variables parameters with significantly higher percentages of values out of range were TSH ESR and MCH whereas those with significantly lower percentages of values out of range were K TG total cholesterol and GGT Supplemental Table a0The differences in the biochemical and hematological parameters between males and females as well as the frequency and percentage of patients out of range in the control and DS groups are displayed in Supplemental Table a0 and Table a0 respectively For the control group parameters with significantly higher values in the male subgroup were K creatinine TG ALT hemoglobin hematocrit RBC and MCHC whereas those with significantly lower values were AF eGFR and ESR Among the categorical variables K had a significantly higher percentage of values out of range in the male subgroup and ESR had a significantly lower percentage of values out of range Supplemental Table a0 For the DS cohort parameters with significantly higher values in the male subgroup were creatinine total bilirubin TG ALT GGT hemoglobin hematocrit RBC MCHC and WBC whereas those with significantly lower values were folate MCV ESR RDW platelet count and MPV Regarding categorical variables parameters with significantly higher percentages of values out of range in the male subgroup were total bilirubin B12 RBC and MPV whereas those with significantly lower percentages of values out of range were MCV ESR and MCHC Table a0The correlation between the biochemical and hematological data with age was also explored in both study groups As shown in Table a0 for the control population urea creatinine total cholesterol and AST showed a significant positive correlation with age while eGFR showed a significant negative correlation For the DS population Na urea creatinine TG total cholesterol AST AF MCV ESR MCH and RDW showed a significant positive correlation with age while eGFR ALT B12 hemoglobin hematocrit RBC MCHC and platelet count showed a significant negative correlationDiscussionThe present study evaluated several biochemical and hematological parameters in a large sample of adults with DS Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism11 DS is among the most complex genetic conditions compatible with life characterized by accelerated aging and affecting gene expression beyond chromosome The sheer number of affected genes and epigenetic changes suggests that numerous pathways of human metabolism are altered and subsequently might be reflected in laboratory test parameters Here we performed a comprehensive approach by analyzing parameters related to different physiological mechanisms We found significant differences with respect to nontrisomic controls in both biochemical and hematological parameters even after adjusting for potential confounding factors Furthermore we found age and sexrelated differences in several of the parameters The fact that women with DS experience menopause earlier than healthy women24 may explain some of these sexrelated differencesClinically and as previously described48925 our DS cohort presented with a higher incidence of congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism otolaryngology pathology ophthalmological pathology epilepsy and AD than the control population Arterial Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT UL ¤ a0years a0years Females Males Females Males Females Males Females Males Females Males Total proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count 109LMPV fLWBC 109LFemales Males Females Males Females Males nn OOR n OOR ControlDown syndromeReference valuesMedian P25P75nMedian P25P75pvalue categoricalpvalue continuouspvalue continuous adjusted Table Biochemical and hematological parameters in the control group and the cohort of patients with Down Syndrome Differences between groups were analyzed using Wilcoxon Ranksum test ANCOVA models adjusted for age and sex or the Fishers exact test OOR out of range NA not applicablehypertension and dyslipidemia were less prevalent whereas no difference was observed regarding the diabetes mellitus incidence as discussed belowWith respect to laboratory studies the hematological profile was largely altered in DS individuals when compared to the control population Of note significant differences were found for almost all the hematological parameters when comparing males and females suggesting the need to consider sex when evaluating the hematological profile in a DS individual It is well known that trisomy impacts hematopoietic cell biology through multiple and complex pathways In adults the metabolic and redox derangements observed in the RBCs from individuals with DS have been previously linked to alterations in cell survival and size in particular macrocytosis26 Different studies have also proposed that the additional copy of chromosome has a profound impact on fetal hematopoiesis which ultimately impacts the function and number of hematopoietic lineages27 Additionally between and of newborn infants with DS develop transient myeloproliferative disorder32 Although the disease usually resolves without treatment in the first few months of life it is estimated that of individuals with transient myeloproliferative disorder will go on to develop subsequent leukemia3536 Finally the fact that folate concentrations are significantly lower in DS individuals matches the observed hematological alterations Taken together these impaired hematological parameters suggest the existence of abnormalities Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cFemalenVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count 109LMPV fLWBC 109L n OOR Median P25P75n OOR Median P25P75pvalue categoricalpvalue continuousMalen Table Differences between sex in the Down syndrome group Differences between groups were analyzed using Wilcoxon Ranksum test or the Fishers exact test OOR out of range NA not applicablein hematopoiesis and provide information on how an extra copy of chromosome may lead to phenotypic consequencesConcerning the biochemical profile our results support the findings from previous independent studies We showed that of our DS individuals presented with values out of range for TSH level Of those out of were treated for hypothyroidism Impaired TSH and FT4 levels have been largely described in DS populations37 Moreover subclinical hypothyroidism in children with DS is an abundantly common occurrence with a prevalence of approximately and has been attributed to the dysregulation of the hypothalamicpituitarythyroid axis37 Regarding urea metabolism of our DS individuals presented with a high urea concentration which may be due to impaired renal function among other causes Indeed and as previously reported39 almost of our DS individuals also presented with impaired creatinine values Serum creatinine is the most reliable parameter for detecting kidney damage due to its high diagnostic specificity From its concentration and based on formulas in which age sex and weight are taken into account it is possible to estimate the glomerular filtration rate eGFR Our DS cohort also presented with a lower eGFR which is in agreement with a previous study exploring renal disease in DS individuals40 Despite the significantly altered parameters related to renal function our DS individuals presented with a very low frequency of arterial hypertensionConcerning the lipid profile we found significantly lower total cholesterol and TG concentrations in DS individuals compared to the control population It would have been interesting to study the fractioned forms of cholesterol together with their apolipoprotein concentrations however because the current study was not designed to answer questions regarding lipid metabolism lowdensity lipoprotein cholesterol LDLc and highdensity lipoprotein cholesterol HDLc were not measured Several works measuring circulating total cholesterol Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cControlnSpearmans rhoDown syndromenSpearmans rhopvaluepvalueBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal Proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count 109LMPV fLWBC 109L Table Correlations between biochemical and hematological parameters and age NA not applicableLDLc HDLc and TG concentrations in the DS population exist However they report contradictory results and prevent firm conclusions from being drawn Some studies have reported an unfavorable41 or favorable lipid profile46 However most of the studies reported no change in serum TC LDLc or HDLc in individuals with DS compared to a control group or to population norms414547 In our study these lower total cholesterol and TG concentrations may have translated into a significantly lower prevalence of hyperlipidemia in DS individuals It has been described that DS individuals may be protected against atherosclerosis4752 leading to a low incidence of cardiovascular events53 However a work carried out with individuals with DS found that they were at high risk of cerebrovascular events but a lower risk of coronary events in males55 Therefore risk of major cerebrovascular events in people with DS should not be ruled out Concerning diabetes mellitus a similar incidence of type diabetes mellitus50 and a higher incidence of type diabetes mellitus has been described for individuals with DS56 We found no difference in type diabetes mellitus frequency among our DS and control populations as previously described in a different study16 In regard to arterial hypertension prevalence our results are in line with numerous studies that have described a lower incidence of this condition in DS individuals50515758 Despite these observations cholesterol fractioned forms and glycated hemoglobin HbA1c concentrations were not measured making it difficult to draw conclusions regarding dyslipidemia and diabetes mellitus in our cohort Yet an increased degree of hypolipidemia should not be ruled out Overall future studies elucidating the mechanisms behind the low cholesterol and TG concentrations and lower prevalence of arterial hypertension observed in our DS cohort should be performedIt is important to emphasize that our main goal was to help determining if the observed biochemical and hematological alterations have direct clinical implications for DS individuals While the altered biochemical and hematological profiles may be developmental features ie a consequence of the specific genetic characteristics of individuals with DS or the result of accelerated aging it should be recalled that they may also be reflecting comorbidities or the use of medication From a clinical standpoint to elucidate if the observed differences are consequence of concomitant conditions or features of the syndrome itself could be of help in the management of DS individuals Unfortunately due to the design of our study these questions remain unanswered Future Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cstudies focusing on specific areas of metabolism of DS individuals with different comorbidities could shed some light on this matterOur study has several strengths We collected relevant clinical biochemical and hematological data in a large DS cohort and performed a systematic analysis The fact that our controls were chosen from a healthy background broadens the actual differences and strengthens the present results Ultimately according to the wide inclusion criteria and the broad range of represented ages we believe that the results from our study may help clinicians when interpreting laboratory analyses in DS individuals Some limitations should also be taken into account The control and DS populations were not strictly age and sex matched and the control group had a reduced number of males when compared to females Nonetheless both populations were within the same age range and additional analysis including adjustment for age and sex were performed Furthermore despite our large cohort of DS individuals the number was still not sufficient to perform statistical analysis stratification according to the observed clinical conditions Moreover and as stated previously some of the observed biochemical andor hematological alterations may have been a consequence of the use of drugs for the treatment of other comorbidities Finally our defined clinical biochemical and hematological profiles were somehow general and unable to cover all the possible comorbidities present in DS individualsIn conclusion adults with DS show a specific profile of biochemical and hematological parameters A good understanding of the differences in our cohort with those in the general population might aid in the clinical followup of adults with DS especially considering that the life span of DS individuals can now reach a0years of age in developed countriesReceived March Accepted July References Parker S E et al Updated national birth prevalence estimates for selected birth defects in the United States Birth Canfield M A et al National estimates and raceethnicspecific variation of selected birth defects in the United States Defects Res A Clin Mol Teratol Birth Defects Res A Clin Mol Teratol Besser L M Shin M Kucik J E Correa A Prevalence of down syndrome among children and adolescents in metropolitan Atlanta Birth Defects Res A Clin Mol Teratol Yang Q Rasmussen S A Friedman J M Mortality associated with Downs syndrome in the USA from to A populationbased study Lancet Bittles A H Glasson E J Clinical social and ethical implications of changing life expectancy in Down syndrome Dev Med Child Neurol Morris J K Alberman E Trends in Downs syndrome live births and antenatal diagnoses in England and Wales from to Analysis of data from the National Down Syndrome Cytogenetic Register BMJ b3794 Startin C M et al Health comorbidities and cognitive abilities across the lifespan in Down syndrome J Neurodev Disord Hithersay R et al Association of dementia with mortality among adults with Down syndrome older than years JAMA Neurol Bull M J Health supervision for children with Down syndrome Pediatrics Roizen N J Patterson D Downs syndrome Lancet Hawli Y Nasrallah M ElHajj Fuleihan G Endocrine and musculoskeletal abnormalities in patients with Down syndrome Nat Rev Endocrinol Sakadamis A Angelopoulou N Matziari C Papameletiou V Souftas V Bone mass gonadal function and biochemical assessment in young men with trisomy Eur J Obstet Gynecol Reprod Biol Niegawa T et al Evaluation of uric acid levels thyroid function and anthropometric parameters in Japanese children with Down syndrome J Clin Biochem Nutr Costa R et al Bone mineral density distribution curves in Spanish adults with Down syndrome J Clin | Thyroid_Cancer |
"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagans nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming Peoples Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expb·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expbD f · expbD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Beggs test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deeks plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Beggs testor Deeks test indicated the potential of publication bias[] Inconsistency index I2 and Cochrans Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagans nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The Ï2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ± NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA ± NA ± Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Beggs Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of BeggsTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Beggs Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Beggs Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deeks funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagans nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagans nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming PeoplesHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L | Thyroid_Cancer |
"elucidate the molecular mechanism underlying the involvement of abnormal DNA methylation in the development of glioma and identify potential newtargets for glioma therapyMethods The GSE79122 chip achieved from the Gene Expression Omnibus GEOdatabase containing glioma samples and normal samples was analyzed Methylationspeciï¬c polymerase chain reaction MSPCR or MSP reverse transcriptionPCR andWestern blot analysis were used to conï¬rm the methylation level and expression level ofthe interleukin receptorassociated kinase IRAK3 gene in glioma cells glioma samplesand the corresponding normal samples In vitro the proliferation apoptosis rate migrationand invasion abilities of glioma cells were detected by Cell Counting Kit8 assay Transwellassay enzymelinked immunosorbent assay and ï¬ow cytometry respectively Besides thexenograft assay of nude mice was used to conï¬rm the effect of the IRAK3 on glioma in vivoResults Microarray analysis showed that the IRAK3 was one of the most hypermethylated genesin glioma and the related mitogenactivated protein kinase MAPK signaling pathway wasactivated More experiments supported the higher methylation level and lower expression levelof the IRAK3 in glioma tissues and cell lines The viability migration and invasion ability ofglioma cells significantly reduced and the apoptosis rate increased with the overexpression anddemethylation of the IRAK3 in vitro Besides treatment with the MAPK signaling pathwayinhibitor PD325901 alone or the overexpression or demethylation of the IRAK3 had a similareffect as the overexpression or demethylation of the IRAK3 alone in glioma cells In vivoxenotransplantation experiments in nude mice conï¬rmed that the overexpression and demethylation of the IRAK3 and suppression of the MAPK signaling pathway inhibited the development ofgliomaConclusion IRAK3 inhibited the development of glioma progression through the MAPKsignaling pathwayKeywords glioma IRAK3 MAPK signaling pathway methylationIntroductionGlioma also known as glioblastoma is one of the most common primary malignant braintumors The average incidence rate of glioma is in individuals1 Despiteimprovements in neurosurgery and radiotherapychemotherapy most patients die within months of diagnosis1 and less than patients survive for years or even more2Recently the molecular mechanisms of glioma have gained increasing attention so as toï¬nd some better methods to defeat this disease Previous studies evaluated that longtermsurvivors of glioma often displayed some favorable molecular characteristics such as thesubmit your manuscript wwwdovepresscomDovePresshttp102147CMARS252772Cancer Management and Research Wu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphpand incorporate the Creative Commons Attribution Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the workyou hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWu et alDovepresshypermethylation of O6methylguanine DNA methyltransferase MGMT promoter3 which is known as a meaningfulpredictive biomarker for the favorable prognosis of the chemotherapeutic drugs4 Therefore this study proposed that epigenetic regulation might play a key role in the development ofglioma which deserves further research for understanding thiscancerDNA methylation isan epigenetic modiï¬cationinvolved in many biological processes especially geneexpression regulation5 The DNA methylation patterns ofnormal cells are controlled well by a balance betweenmethylation and demethylation However this balance isalways disturbed in cancer cells through ectopic deï¬cientor excessive methylation leading to abnormal biologicalactivities6 Hypermethylation of CpG islands on speciï¬cpromoters inhibiting the transcription of downstreamtumor suppressor genes has been discovered in manycancers which may provide clinicians a new strategy forpatients with cancer7 For instance the promoter region ofSEPT9 is hypermethylated in colorectal cancer Hence theSEPT9 gene methylation assay might become a potentialoption for the early detection and screening of colorectalcancer8 CpG islands also display aberrant hypermethylation at a large number of loci and deï¬ne the subgroup ofglioma910 However the underlying molecular events ofDNA methylation and glioma development remain poorlyunderstoodRecently technical advances in genomewide expression analysis have enabled an improved understanding ofthe diagnosis and prognosis of many types of tumors11Genomewide DNA methylation analysis allows comprehensive DNA methylation proï¬ling of the whole genomehelping in the effective identiï¬cation of novel genes thathave a potential value in clinical practice12 Previous studies have reported many speciï¬c methylation signaturegenes in differenttypes of cancers such as thyroidcancer13 lung squamous cell carcinoma14 hepatocellularcarcinoma15 prostate cancer16 and so on using DNAmethylation analysisThis study aimed to examine the genomewide DNAmethylation proï¬ling of glioma tissuesrevealing thehypermethylation of several genes in glioma Then oneof these hypermethylated genes IRAK3 was selected toconductSubsequentlya relationship between IRAK3 and MAPK signaling pathway was demonstrated by using DNA methyltransferaseinhibitor overexpression of IRAK3 and MAPK signalingpathway inhibitor which can disrupt the development ofcomprehensiveaanalysisgliomas in vitro and in vivo The ï¬ndings might providesome clues for the regulatory role of DNA methylationand the underlying application of targeted treatment ingliomaMethodsTissue SamplesGlioma tissues and adjacent normal tissues were collectedfrom patients with primary glioma n admitted to theZhangye Peoples Hospital Afï¬liated to Hexi UniversitySample collection and use was performed according to theapproval of the ethics committee of the Afï¬liated Hospitalof Shandong University Written informed consent wasprovided by every patient with glioma All samples werefrozen in liquid nitrogen and stored at °C All humanspecimens were obtained with the approval by theInstitutional Ethics Committee of Zhangye PeoplesHospital Afï¬liated to Hexi UniversityCell CultureHuman gliocyte cell line HEB and glioma cell linesSHG44 U251 GOS3 HS683 and SF539 wereobtained from Bena Culture Collection Beijing ChinaHEB U251 HS683 and SF539 cells were grown in highsugar Dulbeccos modiï¬ed Eagles medium DMEMwith fetal bovine serum FBS SHG44 cells weregrown in RPMI1640 medium containing NaHCO3 gL glucose gL and sodium pyruvate gLwith FBS The GOS3 cells were grown with minimum essential medium with Earles Balanced Saltswith FBS and mML glucose All cells were cultured at °C under a humidiï¬ed atmosphere with CO2Cell TransfectionpcDNA31interleukin receptorassociated kinase pcDNA31IRAK3 was obtained from GenePharma ShanghaiChina and 5azadC 5aza2ʹdeoxycytidine 5azadCand signaling pathway inhibitor PD325901 were obtainedfrom SigmaAldrich MO USA U251 cells were seeded insixwell plates at cellswell and cultured at °C andthe cell conï¬uence reached CO2 untilTransfections were executed using Lipofectamine Invitrogen CA USA following the manufacturers protocols The medium was changed with the complete mediumafter h of transfectionsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressTable qRTPCR PrimersWu et alAccession NumberSequence 5ʹ3ʹForwardReverseForwardReverseForwardReverseForwardReverseACCCAAACTAACTGATTTTGCCAAGAGAAATTCCGAGGGCAGGCGACCTCCCATGGCAATTTTAACAGAGACAGGCATGGGAAGCCATCTCGACCAGTCCGTCTAGTTGGTCTGTCTCCGCTAAATACGGACTGCAGCCCTGAGGTCAATGAAGGGGTCGTGeneIRAK3MEK1CfosNM_007199NM_002755NM_005252GAPDHNM_008084GenomeWide DNA MethylationAnalysisFor DNA methylation proï¬ling Inï¬nium HumanMethylation450K BeadChip illuminaga_rnaseqv2100 was obtainedfrom the Gene Expression Omnibus GEO database TheDNA methylation data of chip number GSE79122 whichcontained glioma tissues glioblastomas diffuseastrocytomas and anaplastic astrocytomas and controlbrain tissues were analyzed The Inï¬nium MethylationAbbiotec CA USA and Illumina BeadStudio softwareGenetech Biotech Taipei Taiwan were used to measurethe methylation proï¬les of modiï¬ed DNA and loci CpGThe methylated signal intensity at particular CpG loci and450K BeadChip assay were presented as β values and percentage respectivelyQuantitative RTPCRTotal cellular RNA was extracted using PureLink Invitrogenfollowing the manufacturers protocol RT was performed on µg total cellular RNA using a HighCapacity cDNA ReverseTranscription Kit with RNase Inhibitor Applied Biosystemspurchased from Thermo Fisher Scientiï¬c MA USASubsequently quantitative reverse transcriptionpolymerasechain reaction RTPCR was performed using ArcturusParadise Plus qRTPCR Kit Applied Biosystems ThermoFisher Scientiï¬c Comparative expression values were calculated by the ÎÎCt method GAPDH was used for internalreference All primer sequences involved are listed in Table converted into uracil without changing the state of methylated cytosine The IRAK3 methylation level in glioma wasidentiï¬ed using methylationspeciï¬c PCR MSP The MSPprimers are listed in Table EnzymeLinked Immunosorbent AssayThe human interleukin IL6 enzymelinked immunosorbent assay kit Sangon Biotech Shanghai China was usedto test the IL6 level in the glioma cell culture medium Theglioma cell culture medium was centrifuged at rpm for min to remove cells and polymers The supernatant ï¬uidstored at °C was preserved in the supernatant ï¬uid at °C A normal glioma cell culture medium was used as thecontrolWestern Blot AnalysisLysis buffer RIPA Thermo Fisher Scientiï¬c and NPERThermo Fisher Scientiï¬c were used to extract proteinsfrom glioma cells and tissues respectively Then μg totalprotein per sample was separated using SDSPAGE andtransferred to polyvinylidene ï¬uoride membranes ThermoFisher Scientiï¬c The membranes were probed with primaryantiIRAK3 antibody ab8116 Abcam MA USA antiMEK1 phospho S298 antibody [EPR3338] ab96379 antiERK1 ERK2 antibody [ERK7D8] ab54230 anticFosphospho T232 antibody ab17933 and antiGAPDH antibody [6C5] ab8245 Abcam as control The number ofTable MethylationSpeciï¬c PrimersDNA Methylation AssayGenomic DNA in glioma tissues and cells was extracted andtreated with bisulï¬te using CpGenome DNA Modiï¬cationKit Chemicon CA USA following the manufacturersprotocol All unmethylated cytosine residues in DNA wereGeneIRAK3 forwardIRAK3 reverseIRAK3 forwardIRAK3 reverseSequence 5ʹ3ʹ5ʹTCGGGATAGTGGTTAATATTTC3ʹ5ʹTTTTTTTCGTTTTTCGTAAAA3ʹ5ʹ AGTTTGGGATAGTGGTTAATATTTT 3ʹ5ʹ TTTTTTTCATTTTTCATAAAAAAA 3ʹCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Genomewide methylation data were obtained from the GEO database for available glioma9 adjacent mucosa A Density of methylated DNA intensity byeach sample B Type I and Type II assays showed variant β value distributions The differences between probe types were regulated by normalization procedures whichshowed that represented unmethylated sites while represented fully methylated sites C Multidimensional scaling plot showing differential clustering of control versustumor tissues D Dendrogram produced for probes in normal and tumor tissues E Heatmap of top differentially methylated imprinted CpG sitesAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Distribution of top differentially methylated imprinted CpG sites A Distribution of top differentially methylated imprinted CpG sites according toCpG islands island sea shelf and shore B Distribution of top differentially methylated imprinted CpG sites according to the position relative to genes 1stexon ² UTRs or ² UTRs body IGR TSS1500 and TSS200 C Combined genetic and epigenetic annotation information revealed the distribution of the top differentially methylated imprinted CpG sitesbinding proteins was measured using AlphaEaseFC softwareGenetic Technologies FL USACell Counting Kit8 AssayU251 cells were seeded in 96well plates and allowed toadhere for h at °C Then these cells were transfectedwith pcDNA31IRAK3 and treated with 5azadC orPD325901 After h Cell Counting Kit8 DojindoKumamoto Japan was used to test the cell viability at respective time points To summarize mL of the triazoliumsubstrate was added to each well and coincubated with cellsat °C for h The absorbance was measured at nm andCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure DNA methylation analysis of tumor tissues and normal tissues and analysis of methylation degree for seven CpG sites of IRAK3 A The expression of the top candidate genes was analyzed IRAK3 was hypermethylated significantly in tumor tissues compared with normal tissues B The number of IRAK3methylated CpG islands ineach isosite CI Seven CpG sites for IRAK3 which are presented in the boxplot displayed a decreased methylation in the tumor group The boxplot for cg Ccg D cg E cg F cg G cg H and cg IAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related signaling pathway A The top signaling pathways with the highest and lowest correlation with glioma B The top signalingpathwayrelated genes enriched in the glioma the MAPK signaling pathway was highly expressed C The MAPK signaling pathway was activated in glioma D The status ofthe top enriched signaling pathways in gliomaAbbreviation MG malignant gliomaCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressthe results were normalized to untreated cells at respectivetime pointsCell Migration and Invasion AssayBoth cell migration and invasion were performed using theTranswell assay For cell migration assay U251cells were seeded in a serumfree medium in the upperchamber which contained a polyethylene terephthalatemembrane with mm in diameter and μm in poresize The bottom chamber was prepared with FBS asa chemoattractant After incubating at °C for hnonmigrated cells were scraped from the upper surfaceof the membrane with a cotton swab and the cells migrating to the bottom chamber were ï¬xed with paraformaldehyde and stained with crystal violet Finally the stainedcells were counted under a microscope at magniï¬cation in ï¬ve randomly selected ï¬elds for quantiï¬cationinvasion assay U251 cells weresuspended in mL of serumfree DMEM and thentreated using the same procedure as for the migrationassay following the manufacturers protocol but the chambers mm BD Biosciences San Jose CA USA wereplated with BD BioCoat MatrigelFor cellFlow CytometryEach treatment of U251 cells was washed with phosphatebuffered saline and resuspended in μL of AnnexinV binding buffer After staining with Alexa Fluor Annexin V and 7AAD Viability Staining Solution for min in the dark at room temperature these cells were analyzed using multicolor ï¬ow cytometer Data were analyzedusing Kaluza softwarethe mice were keptXenograft StudiesMale BALBc nude mice weeks were maintained underpathogenfree conditions Allina temperaturecontrolled airconditioned conventional animal house with a h lightdark cycle and given free accessto food and water Besides animal health and behaviour weremonitored every two days All experiments were approvedby the Ethics Committee of Zhangye Peoples Hospital toguarantee that all studies involving experimental animalswere performed in full compliance with National Institutesof Health Guidelines for the Care and Use of LaboratoryAnimals The U251 cells100 μL cells were transfected with pcDNA31IRAK3 and then transferred to micen12 Normal U251 cells were transferred to mice in the5azadC and PD325901 groups and then 5azadC andPD325901 were subcutaneously injected respectively intothe posterior ï¬ank of nude mice After and daysculture the mice were sacriï¬ced and the tumor size wasmeasured The tumor volume was measured using a caliperfollowing the formula length width22Statistical AnalysisAll data were collected from three independent experiments and presented as mean ± standard deviationStatistical analyses were performed using GraphPad software Differences between groups were analyzed usingStudent' ttest or chisquare test Statistical signiï¬cancewas set at P ResultsGenome Methylation Proï¬le in GliomaA total of glioma tissues and adjacent normal sampledata publicly available at GEO were analyzed to revealthe global DNA methylation patterns of glioma Firstdensity plots of β values generated from each samplewere used identifying a poor performance of methylatedsignals for raw data Figure 1A Inï¬nium Type I and TypeII probe assays also showed somewhat different distribution of β value ranging from unmethylated sites to fully methylated sites Figure 1B Therefore these datawere regulated by normalization procedures to reduce thepotential impact later Multidimensional scaling MDSplot and dendrogram exhibited a differential clusteringbetween tumor and normal groups which distinguishedglioma from adjacent normal tissues Figure 1C and DFurther heatmap of the top differentially methylatedCpG sites indicated a visible difference in DNA methylation proï¬les between the tumor and normaltissueand general hypermethylation occurred insamplesglioma Figure 1EDistribution of Genomic Regions forDifferentially Methylated CpG SitesBased on the position relative to gene [1st exon ² untranslated region UTR ² UTRs body transcription start sites bp TSS1500 and TSS200] as well as CpG islands andneighborhood content shores shelves islands and sea the distribution of genomic regions for the differentiallymethylated CpG sites was exhibited More than and methylation differences occurred in bodyIGR and CpGisland sea respectively which was obviously higherthan that in other regions Figure 2A and B From thesubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related gene expression level A Based on the differential genes in the disease data the distribution of upregulated genes anddownregulated genes in the GOrelated pathway was enriched B The expression level of top MAPK signaling pathwayrelated genesAbbreviation MG malignant gliomaperspective of both genetics and epigenetics gene and CpGcontent regions were combined for more analyses whichshowed that CpG sites in genetic bodyisland sea andIGRisland sea were differentially methylated betweenglioma and adjacent normal samples Figure 2CIRAK3 Was Hypermethylated in GliomaThe heatmap was used to present the top hypermethylationgenes in glioma compared with normal tissues so as to ï¬gureout the most characteristic gene with methylation change intumors indicating that the IRAK3 was hypermethylated theCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressmost in glioma Figure 3A Then the methylation differentialdistribution of the IRAK3 was upregulated at each siteFigure 3B Furthermore the β value of CpG sitesIRAK3such as cg cg cg cg cg cg cg andso on on the IRAK3 was significantly higher in the tumorindicating that IRAK3group than in the normal groupwas hypermethylated in the tumor group due to CpGsFigure 3CI The aforementioned results showed that theIRAK3 CpG sites were highly methylated in glioma tissuesthan in normal tissuestissues and the top signaling pathwayrelated genesenriched in the glioma were listed Figure 4A and B Theresults suggested that the MAPK signaling pathway washighly expressed and activated Figure 4C and D Based onthe differential genes in the disease data the distribution ofupregulated genes and downregulated genes in the GOrelatedpathway was enriched Figure 5A The expression level ofMAPK signaling pathwayrelated genes in glioma is shown inFigure 5B The results of Figures and manifested thatIRAK3related MAPK signaling pathway was highly activatedin glioma However whether any correction existed with thedevelopment of glioma remained to be veriï¬edMAPK Signaling Pathway Expression Leveland State in GliomaFor the identiï¬cation of gliomarelated signaling pathwaysthat might be inï¬uenced by aberrant DNA methylation thetop IRAK3related signaling pathways in glioma and normalMethylation and Expression Level of theIRAK3 in Glioma Tissues and CellsThe impact of hypermethylation on the expression of IRAK3in glioma tissues and cells was elucidated The IRAK3 wasFigure Methylation level and expression level of the IRAK3 in glioma tissues and cells A The IRAK3 was highly methylated in glioma tissues compared with adjacent tissues BThe IRAK3 was less expressed in glioma tissues than in adjacent tissues C The IRAK3 in the ï¬ve glioma cells SHG44 U251 HS683 SF539 and GOS3 was hypermethylatedcompared with U251 glioma cells D The IRAK3 was less expressed in the ï¬ve glioma cells than in normal glioma cells The difference was significant P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Effect on glioma cells after the overexpression and demethylation of the IRAK3 A The expression level of the IRAK3 was significantly higher in the glioma U251 cells of thepcDNA31IRAK3 and 5azadC groups than in the control group B and C The protein expression level of the IRAK3 significantly increased in the pcDNA31IRAK3 and 5azadC groupscompared with the control group D The expression level of the inï¬ammatory factor IL6 significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with thecontrol group E The activity significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantlyincreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group H and I The migration capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups compared with the control group J and K The invasiveness capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups than in the the control group Allthe mentioned differences were significant P The IRAK3 had a suppressive effect on glioma cells in vitroCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepresshighly methylated and less expressed in glioma tissues than inadjacent normal tissues Figure 6A and B Next the IRAK3 inï¬ve glioma cell lines SHG44 U251 HS683 SF539 andGOS3 also showed hypermethylation and less expressioncompared with normal gliocytes Figure 6C and D U251cells were selected for subsequent experiments because themethylation level of the IRAK3 in U251 cells was the highestFigure 6COverexpression or Demethylation of theIRAK3 Inhibited the Development of GliomaCellsA systematic test with the overexpression or demethylation ofthe IRAK3 was conducted to understand whether the correctionof abnormal IRAK3 methylation and expression affected thedevelopment of glioma After using pcDNA31IRAK3 tooverexpress or 5azadC to demethylate the IRAK3 the proteinexpression level of the IRAK3 in glioma U251 cells increasedin the tumor group compared with the normal group Figure7AC IL6 an inï¬ammatory factor secreted by the MAPKsignaling pathway [PMID ] so that its level in theculture medium could be used to represent the status ofMAPK signaling pathway activation decreased in thepcDNA31IRAK3 and 5azadC groups compared with thecontrol groups Figure 7D Surprisingly the viability ofglioma cells significantly decreased while the apoptosis rateincreased Figure 7EG and the migration and invasivenesscapability decreased in the pcDNA31IRAK3 and 5azadCFigure Effect on glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The expression level of MAPKsignaling pathwayrelated genes expression level was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group B and C Theexpression level of MAPK signaling pathwayrelated proteins significantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the controlgroup D The level of inï¬ammatory factor IL6 was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group E The activitysignificantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantly increased inthe PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group All the mentioned differences were significant P P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et algroups Figure 7HK which elucidated the associationbetween IRAK3 and the development of glioma The aforementioned results veriï¬ed that the overexpression or demethylation of the IRAK3 inhibited the development of glioma cellsin vitroMAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited the Development ofGlioma CellsThe MAPK signaling pathway inhibitors PD325901pcDNA31IRAK3 and 5azadC were used alone or incombination in vitro to conï¬rm the inï¬uence of the IRAK3and MAPK signaling pathways on glioma In PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901groups the mRNA and protein expression levels of theMAPK signaling pathwayrelated genes such as MEK1ERK and cFos as well as the level of IL6 decreased inglioma Figure 8AD The cell viability significantlythe apoptosis rate increased Figure 8EGdecreasedand the migration and invasiveness capability significantlydecreased in PD325901 pIRAK3PD325901 and 5azadCPD325901 groups Figure 9AC The aforementioned results veriï¬ed that the MAPK signaling pathwaysuppression alone or combined with the overexpression ordemethylation of IRAK3MAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited Glioma in vivoFinally whether MAPK signaling pathway suppression aloneor combined with the overexpression or demethylation of theFigure Effect on the migration and invasiveness capability of glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and5azadC A and B The migration capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group A and C Theinvasiveness capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group All the mentioned differences weresignificant P Cancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Effect on mice after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The tumor in the transplantationPD325901 p IRAK3 PD325901 and 5azadC PD325901 treatment groups after and days B The tumor volume was smaller in the PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901 groups compared with the control group C The tumor weight followed the same trend as the volume D Theexpression level of the IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups compared with thetransplantation control group E and F The protein expression level of IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups than in the transplantation control group All the mentioned differences were significant P P IRAK3 had a suppressive effect on glioma in vivo was studiedAfter transplanting glioma U251 cells treated with PD325901pcDNA31IRAK3 PD325901 or 5azadC PD325901the tumorigenesis significantly decreased compared with thatin the control group mice Figure 10AC Finally mRNAand protein expression levels ofthe MAPK signalingsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alpathwayrelated genes in xenograft glioma were detectedthey were found to be significantly decreased in thePD325901 pcDNA31IRAK3 PD325901 or 5azadC PD325901 group Figure 10DFDiscussionAccording to the World Health anization the overallsurvival of patients with cancer has increased significantlyover the years with the improvement in diagnosis and treatment However glioma is still a highly fatal disease1Therefore this study aimed to elucidate the detail of itsprogression mechanisms and search for new therapeutic strategies One recent study reported that the use of MGMTpromoter methylation test in hospitals had a strong inï¬uenceon the prognosis of glioma17 suggesting a significant clinicalapplication prospect of DNA epigenetic regulation Hencethe main focus of the present study was on the relationshipbetween DNA methylation and glioma IRAK3IRAK3 belongs to the IL receptorassociated kinaseIRAK family involved in inhibiting Tolllike receptorsignaling by changing the activity of other members ofthe IRAK family to decrease inï¬ammatory response1819Increasing evidence supported thatthe expression ofIRAK3 in tumorassociated macrophages led to compromised immune surveillance of cancer cells when it proï¬tably prevented excessive inï¬ammation contributing tocancer development Therefore the growth of transplantable cancer cells could be inhibited by enhancing hostimmune responses in IRAK3deï¬cient mice20 A smallnumber oftumor cellintrinsicIRAK3 could also support the progression of tumor cellsin colorectal and lung cancers depending on the dysfunctional innate immune system indirectly Interestingly theexpression of IRAKM in colorectal and lung cancers correlated with poor prognosisin patients with thesecancers2122 However little is known about the molecularmechanism of the IRAK3 in glioma This study revealedthe downregulation of IRAK3 glioma tissues and cellsthrough DNA methylation analysis which seemed to becontrary to the previous ï¬ndings on other cancers Thisstudy investigated whether a special signaling pathwayexisted that connected IRAK3 and glioma progressionshowed thatstudiesAn ongoing study has validated that all members of theIRAK family mediate the activation of MAPK and nuclearfactorκB NFκB signaling pathways23 MeanwhileIRAK3 a general negative regulator was conï¬rmed toinhibit MAPK and NFκB activation2425 Likewisethese results identiï¬ed that the MAPK signaling pathwaywas highly activated in glioma and its related geneexpression level was downregulated after overexpressionof the IRAK3 In fact upstream genomic events andordifferent extracellular stimuli could activate the MAPKsignaling pathway mediating a wide range of cellularprocesses26 The activation of the MAPK signaling pathway often led to abnormal cell growth and tumorigenesisThe predominant effect relied on the signal intensity andthe context or tissue in which the signal occurred2728Zhang revealed that aberrant DNA methylation inthe promoters of MMPTIMP axis genes upregulated theMAPK signaling pathway promoting the progression oftumor cells Correction of the abnormal DNA epigenotypes attenuated the migration and invasion of tumorcells in vitro and reduced tumorigenicity in vivo29Intriguingly these results were consistent with those ofthe present studyConsidering the reverse expression pattern of IRAK3between glioma and other cancers2122 different epigeneticmodiï¬cation was regarded as the major reason causing thisdistinction In many cancers the DNA methylation patternsbecame aberrant with tissue speciï¬city serving as diagnosticmarkers and therapeutic targets30 Hence based on thelower expression and the tumorigenic function of the IRAK3in glioma its epigen | Thyroid_Cancer |
Treatment Strategy for Metastatic Spinal Tumors A Narrative ReviewSam Yeol Chang Sujung Mok Sung Cheol Park Hyoungmin Kim BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Seoul Korea Metastatic spinal tumors are common and their rising incidence can be attributed to the expanding aging population and increased survival rates among cancer patients The decisionmaking process in the treatment of spinal metastasis requires a multidisciplinary approach that includes medical and radiation oncology surgery and rehabilitation Various decisionmaking systems have been proposed in the literature in order to estimate survival and suggest appropriate treatment options for patients experiencing spinal metastasis However recent advances in treatment modalities for spinal metastasis such as stereotactic radiosurgery and minimally invasive surgical techniques have reshaped clinical practices concerning patients with spinal metastasis making a demand for further improvements on current decisionmaking systems In this review recent improvements in treatment modalities and the evolution of decisionmaking systems for metastatic spinal tumors are discussedKeywords Spinal metastasis Decisionmaking system Radiosurgery Minimally invasive surgical procedures Separation surgeryIntroductionThe spine has been identified as the most common site for malignant metastasis in the musculoskeletal system and vice versa spinal metastasis is considered the most common malignant lesion in the spine [] The incidence of metastatic spinal tumors has seen an increasing trend due to the growing aging population worldwide and continued improvements in survival rates among cancer patients [] Symptomatic spinal metastasis is often the first clinical manifestation for of cancer patients [] whereas up to of cancer patients may experience spinal metastasis at some point during the course of their disease [] The objectives of surgical treatment in patients with metastatic spine tumors are mostly palliative Spine surgeons make an effort to maintain or improve the patients quality of life during the remainder of their survival by reducing pain and preserving ambulatory function via surgical treatment []Because clinical manifestations and treatment responses vary widely among cancer patients a multidisciplinary decisionmaking process that integrates medical and radiation oncology together with surgery along with assistance from pathology and diagnostic radiology deemed is essential when deciding to conduct surgical treatment for spinal metastasis [] In the literature various decisionmaking systems have been developed and introduced to date in an effort to aid in this decisionmaking process Received Jul Revised Jul Accepted Jul Corresponding author BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Daehakro Jongnogu Seoul KoreaTel Fax Email bschangsnuackrCopyright ¸ by Korean Society of Spine SurgeryThis is an Access distributed under the terms of the Creative Commons Attribution NonCommercial License httpcreativecommonslicensesbync40which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly citedAsian Spine Journal ¢ pISSN eISSN ¢ wwwasianspinejournalAsian Spine JournalASJAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J [] However recent advancements in the treatment of metastatic spine tumors have injected more complexity into this decisionmaking process and demanded the evolution of the decisionmaking system itself these recent advances include the development of stereotactic spine radiosurgery SRS the introduction of minimally invasive surgical techniques and the evolution of various target therapies for individual primary cancers [] In this review we discussed the development and evolution of various decisionmaking systems in spinal metastasis treatment Current concepts and recent trends in radiotherapy and surgery for spinal metastasis are also includedDecisionMaking Systems for Managing Metastatic Spinal TumorsPrognostic factors for metastatic spinal tumorsWhen attempting to choose an appropriate treatment for a patient with spinal metastasis establishing an accurate estimation of the individuals life expectancy is the most crucial To do this one must first identify prognostic factors associated with the survival of patients with spinal metastasis As such many authors have conducted studies to try and identify prognostic factors associated with survival among spinal metastasis patients and have developed various decisionmaking systems in order to estimate survival based on these prognostic factors []In a recently published metaanalysis Luksanapruksa [] have identified independent prognostic factors associated with the survival of patients with spinal metastasis Among these factors nine factors can be classified as relating to the preoperative performance or neurological status of a patientfor example the Karnofsky Performance Score or Eastern Cooperative Oncology Group grade [] Meanwhile four factors involve the presence or the number of metastases spine bone or visceral and two factors were found to be related to primary tumors in the Tomita classification scheme [] Finally male sex and the time interval from cancer diagnosis to the start of radiotherapy are the two remaining prognostic factors independently associated with survival in spinal metastasis patients Among these primary tumor histology the presence and number of metastases and performance status are proposed as the three most important prognostic factors associated with survival in spinal metastasis patients not only in this study but also in most previous investigations []In other studies the patients age and comorbidities assessed using the American Society of Anesthesiologists physical status [] and Charlson Comorbidity Index [] have also been identified to be prognostic factors in spinal metastasis [] Other authors have identified laboratory abnormalities such as leukocytosis and low hemoglobulin and albumin levels as prognostic factors and included these into their decisionmaking systems [] In addition previous systemic treatment or chemotherapy has also been suggested as an independent prognostic factor by multiple authors [] Further not only preoperative chemotherapy but also the presence of available systemic treatments in the postoperative period has been hypothetically regarded as a potential prognostic factor in the literature [] In a recently published study by Chang [] the authors verified the presence of the remaining systemic treatment options to be independently associated with improved postoperative performance status and survivalClassificationbased decisionmaking systemsBased on these prognostic factors numerous decisionmaking systems or scoring systems have been developed and introduced to estimate the life expectancy among spinal metastasis patients [] In these classificationbased decisionmaking systems scores for each prognostic factor identified by multivariate logistic or proportional hazards regression analyses are integrated in order to obtain a total prognostic score that reflects the estimated survival of the patient Surgeons can adopt these prognostic scores to identify patients with an estimated survival profile that is sufficient to warrant surgical treatment Although the prognostic factors included in each system vary primary tumor histology and visceral metastases are included in most systems Table In the New England Spinal Metastasis Score NESMS was introduced by Ghori [] The NESMS was developed using multicenter data and retrospectively validated in the following investigations [] The NESMS consists of modified Bauer score components and score serum albumin level and ambulatory status of the patient More recently the NESMS was validated prospectively in the Prospective Observational Study of Spinal Metastasis Treatment trial which aimed to verify the 0cTreatment of Spinal Metastasis Table Prognostic factors in decisionmaking systemsReferencesBauer [] modifiedTomita [] To kuhashi [] revisedKa tagiri [] revisedGhori [] Paulino [] Primary tumorPerformance statusNo of vertebral metastasesBone metastasisVisceral metastasisPrevious systemic treatmentOther factorsOOOOOOOOOOOOOOOOOOOOOOOOOOBr ain metastasis WBC Hb platelet albumin bilirubin Creactive protein lactate dehydrogenaseSerum albuminAge WBC Hb brain metastasisWBC white blood cell Hb hemoglobin NESMS as a reliable predictive tool in spinal metastasis patients []There have been efforts to develop novel decisionmaking systems using evolving methodologies In the Skeletal Oncology Research Group S compared multiple prognostic survival algorithms including classic nomogram and boosting algorithms using the same retrospective dataset obtained from patients [] In their study the nomogram was found intuitive and demonstrated a comparable level of performance Then in the S used machinelearning algorithms to develop a novel prognostic model for metastatic spinal disease [] which was externally validated in subsequent studies []Although these various classificationbased decisionmaking systems are helpful and widely used for predicting the survival of spinal metastasis patients recent studies have reported that the degree of accuracy of these classic systems eg Tomita Tokuhashi decreases over time especially in cancers with poor prognoses such as lung cancer [] This pitfall of classificationbased decisionmaking systems reportedly stems from the inability of these systems to reflect survival improvement due to recent evolutions in systemic treatment for primary cancers [] Another existing limitation is that these systems cannot directly guide the selection of specific treatments appropriate for patients with spinal metastasisPrinciplebased decisionmaking systemsAs an alternative to these classificationbased decisionmaking systems that are incapable of reflecting recent advances in oncology and guiding specific treatments several authors have proposed principlebased decisionmaking systems These principlebased systems do not score the patient and estimate survival but instead provide advice regarding which treatment is more appropriate in individual cases based on the integration of rapidly evolving treatment modalities including target therapies radiosurgery and minimally invasive surgical techniquesThe neurologic oncologic mechanical and systemic NOMS decision framework was first introduced in [] The NOMS decision framework consists of neurologic N oncologic O mechanical M and systemic S considerations integrating novel multimodal therapies including SRS and minimally invasive surgical techniques [] As a neurological N assessment approach the grading system developed by Bilsky and Smith [] was used while surgical decompression is recommended for highgrade spinal cord compressions During oncological O assessment the responsiveness of spinal metastasis to currently available treatments especially the level of tumor sensitivity to radiotherapy is evaluated Mechanically M instability of the spinal column as determined by the Spinal Instability Neoplastic Score SINS indicates the need for surgical stabilization regardless of the neurologic or oncologic status [] Finally systemic S assessment focuses on the patients ability in tolerating the suggested treatment Meanwhile if the general condition performance status and medical comorbidities of a patient do not allow surgery to be performed radiotherapy is instead recommendedA modification of the NOMS decision framework has also been presented in the literature Paton introAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J duced the LMNOP system as an improvement to the NOMS approach [] With this development these authors added two additional key considerations to the NOMS as follows the location and levels of metastasis L and the patients response to previous therapy P The P in LMNOP stands for not only the response to prior therapy but also includes patient fitness and prognosis which was considered previously as part of the systemic S assessment in the NOMS decision framework The authors emphasized that the response of primary cancer to previous treatments including chemotherapy and radiotherapy is considered a significant factor when determining the appropriate treatment for spinal metastasis patients For instance it is anticipated that a patient diagnosed with symptomatic spinal metastasis at the initial presentation of primary cancer synchronous metastasis who would have multiple potential treatment options is more likely to experience a better prognosis than a patient diagnosed with spinal metastasis despite previous treatment metachronous metastasis Differences in the survival rates between the patients with synchronous and metachronous spinal metastasis have been confirmed in a previous research [] Therefore a more aggressive approach including surgical treatment can be considered for patients with a synchronous spinal metastatic lesion In summary principlebased decisionmaking systems relative to classificationbased systems are able to better incorporate evolving treatment modalities and guide the selection of appropriate treatments for patients in a timely fashion Table Current trends and future directions in the development of decisionmaking systemsAdvances in cancer biology and treatment modalities are necessitating the evolution of decisionmaking systems for spinal metastasis Possible future directions to take to improve decisionmaking systems include the following the use of multicenter or multinational databases the integration of histologyspecific data the application of computational methodologies such as machinelearning algorithms and the combination of classificationbased and principlebased systems Some recent studies are already covering these trendsThe size of the study sample under assessment determines the performance and accuracy of prognostic models Although spinal metastasis is found to be relatively common data from a larger sample population beyond that of just a single institution is usually required to develop a powerful enough prognostic model For this reason recently introduced prognostic models or decisionmaking systems are generated from multicenter databases [] Future prognostic models should also have the freedom to rely on even larger databases such as multinational tumor registriesBiologic therapy including molecular target therapy and immunotherapy is believed to be an emerging gamechanger in modern cancer treatment Genetic subtype analysis of the primary cancer histology which guides selection among t hese therapies has become more essential [] In a revised prognostic system proposed by Katagiri Table The NOMS decision frameworkNeurologic NOncologic OMechanical MSystemic SDecisionLowgrade ESCCno myelopathyHighgrade ESCC±myelopathyRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadioresistantRadioresistantStableUnstableStableUnstableStableUnstableStableStableUnstableUnstablecEBRTStabilization followed by cEBRTSRSStabilization followed by SRScEBRTStabilization followed by cEBRTAble to tolerate surgeryD ecompressionstabilization followed by SRSUnable to tolerate surgerycEBRTAble to tolerate surgeryDecompressionstabilization followed by SRSUnable to tolerate surgeryStabilization followed by cEBRTNOMS neurologic oncologic mechanical and systemic ESCC epidural spinal cord compression cEBRT conventional external beam radiation SRS stereotactic radiosurgery 0c [] the authors considered the availability of molecular target therapy when classifying the primary tumor For example lung cancer treated with targeted drugs was designated as an example of a moderategrowth tumor while lung cancer without targeted drugs is regarded as an example of a rapidgrowth tumor [] This application of genetic profiles to decisionmaking systems is likely to grow more specific and tailored corresponding to the evolution of molecular genetics in the futureAs previously described machinelearning algorithms have been applied in the development of prognostic models Classically prognostic models for spinal metastasis have been developed using logistic or proportional hazards regression analyses As part of its research efforts the S was able to develop prognostic models using machinelearning algorithms such as gradient boosting decision trees random forests and neural networks [] and these algorithms were externally validated elsewhere [] Like in other fields of medicine evolving computational methodologies including machinelearning algorithms should be assessed extensively in terms of their potential in the management of spinal metastasisFinally the combination of classificationbased and principlebased decisionmaking systems should be considered Classificationbased systems or prognostic models seek to estimate the patients remaining survival Based on this survival estimation principlebased systems then may suggest the most appropriate treatment option Until now surgeons and physicians have been employing these two separate systems in the same decisionmaking process A novel decisionmaking system could integrate these two systems together and provide survival estimations and appropriate treatment options simultaneouslyRadiotherapy for Metastatic Spinal TumorsTreatment of Spinal Metastasis single session of SRS achieved durable longterm control of spinal metastasis regardless of histology and tumor size Of note the only significant factor associated with tumor control was the radiation dose These results suggest that SRS can be effective even in cases of metastasis previously considered to be radioresistantSRS can also be a definitive treatment for the management of solitary metastasis without spinal cord compression [] Excellent local control rates of have allowed SRS to replace curative surgeries with high morbidities such as total en bloc spondylectomy for addressing these solitary metastases [] In patients with highgrade spinal cord compression SRS can be applied after separation surgery which will be discussed further in the following section Overall the effectiveness of SRS has been changing the role and extent of surgical treatment and in turn shifting the focus of the treatment of spinal metastasisVertebral compression fracture following stereotactic spine radiosurgeryA pitfall of SRS is an increase in vertebral compression fracture VCF following radiotherapy Risk estimates for VCF after SRS are reported to be up to as compared with just in relation to conventional radiotherapy [] The occurrence of VCF is dosedependent and caution is required if the radiation dosage exceeds Gy per fraction in highrisk patients [] Highrisk patients of older ages with lytic lesions andor with spinal malalignment can reportedly benefit from undergoing preventive stabilization surgery before SRS When determining the necessity of stabilization surgery before SRS SINS can be used to identify potentially unstable lesions [] SINS will be further covered in the following sectionStereotactic spine radiosurgery is triggering a paradigm shiftTiming of radiotherapy after surgeryEvidence in the literature supports that radiosurgery is a safe and effective modality for local tumor control with low associated complication rates in patients with spinal metastasis [] Technical improvements including intensitymodulated and imageguided radiation delivery and processing software have allowed SRS to be a gamechanger in the treatment of spinal metastasis [] A recent study by Yamada [] reported that a highdose Adequate timing of radiotherapy following surgery whose determination is related to the risk of wound complications continues to be debated among spine surgeons and radiation oncologists It is also controversial whether the interval can be shortened in patients receiving SRS Lee [] sent questionnaires to radiation oncologists and spine surgeons to gather opinions on the optimal timing of surgery and radiotherapy in spinal metastasis Based on the procured comments the auAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J thors recommended that the interval be at minimum weeks regardless of radiation modalities Interestingly as compared with radiation oncologists surgeons tended to favor a shorter interval of time between surgery and radiotherapy when SRS is performed although there was no statistically significant difference in this regard []A recently published systemic review also advocated for weeks with a minimum of days between surgery and radiotherapy [] When the rates of wound complications were compared between SRS and conventional radiotherapy many studies reported reduced wound complications in SRS patients [] However due to limited highlevel evidence no definite conclusion was made regarding whether the interval could be reduced in patients undergoing SRSSurgery for Metastatic Spinal TumorsSurgical indicationsThe objective of surgical treatment in spinal metastasis was to provide pain relief support neurological improvement and in turn enhance the quality of life during the remaining survival period Clinical benefits of direct surgical decompression in patients with metastatic spinal cord compression MSCC have been well described in the literature [] The most important prerequisite for surgical treatment in spinal metastasis has been identified as the sufficient enough estimated survival time to make surgery a reasonable approach Researchers have largely recommended that a minimum of to months of remaining survival should exist when considering whether to perform surgery [] At this point a number of prognostic scoring systems previously described are being used to estimate a patients survival The patient should also have a good enough general condition or performance status to in order to endure surgery If these conditions are satisfied then surgery can be performed in patients with symptomatic MSCC or mechanical instabilityIn spinal metastasis the instability is assessed by the SINS [] Table The SINS is an independent and unique tool that integrates clinical and radiological components to help surgeons decide whether to conduct surgery for stabilization A score of to points suggests impending instability while that of points or more indicates existing spinal instability which requires stabilization As previously mentioned the SINS has also been incorporated into principlebased decisionmaking systems such as the NOMS and LMNOP systems [] Recent studies have reported the reliability and accuracy of the SINS system in predicting spinal adverse events including VCF especially in those patients who received radiotherapy [] although some components may still require revision []Separation surgery and minimally invasive surgeryWhen considering the surgical techniques used for spinal metastasis the literature shows a trend toward the adoption of less invasive techniques which is thought to have Table Spinal Instability Neoplastic Score systemComponentLocationJunctional occiputC2 C7T2 T11L1 L5S1Mobile spine C3C6 L2L4Semi rigid T3T10Rigid S2S5PainaYesOccasional pain but not mechanicalPainfree lesionBone lesionLyticMixed lyticblasticBlasticRadiographic spinal alignmentSubluxationtranslation presentDe novo deformity kyphosisscoliosisNormal alignmentVertebral body collapse collapse collapseNo collapse with body involvedNone of the abovePosterolateral involvement of spinal elementsbBilateralUnilateralNone of the aboveScoreaPain improvement with recumbency andor pain with movementloading of spine bFacet pedicle or costovertebral joint fracture or replacement with tumor 0cTreatment of Spinal Metastasis primarily resulted from recent advances in radiotherapy as previously described [] With the use of advanced radiation techniques surgeons can minimize surgical morbidities by avoiding extensive debulking surgery [] Fig During the separation surgery circumferential spinal cord decompression is performed only to the extent required to facilitate safe radiosurgery In a study by Laufer [] separation surgery followed by postoperative SRS resulted in a low local progression rate Other studies have also reported that this hybrid surgeryradiosurgery approach is a safe and effective treatment option for MSCC []BCADFig A 68yearold male with spinal metastasis of renal cell carcinoma at T9 The patient also had lung metastasis A Preoperative MRI shows spinal cord compression and involvement of posterior elements and left rib head at the T9 level B Following a separation surgery without spinal instrumentation MRI at postoperative 2weeks shows a decompressed spinal canal but residual metastatic tumor around the left 9th rib head C In the postoperative 1year MRI the patient showed a nearcomplete response following a single session stereotactic radiosurgery 18Gy1 fraction which was performed weeks after the separation surgery MRI magnetic resonance imaging D Planning images for the postoperative stereotatic radiosurgery following a separation surgery Asian Spine Journal 0cSam Yeol Chang et alAsian Spine J Minimalaccess surgery is also used in treating spinal metastasis while reducing surgical morbidities In the anterior approach retractor systems and thoracoscopic assistance have been described [] Minimally invasive posterior approaches for decompression and corpectomy have also been introduced and reviewed in the literature [] Other minimally invasive techniques for spine surgery such as the intraoperative stereotactic navigation system and percutaneous pedicle screw instrumentation are being incorporated into spinal metastasis surgery as well []Studies comparing minimally invasive surgery and surgery showed that minimally invasive surgery provided equivalent or superior outcomes with reduced surgical morbidity and complications in spinal metastasis patients [] However because the quality of evidence is deemed low in the current literature no definite conclusion regarding the superiority of minimally invasive surgery over surgery can be derived and no strong recommendations have been made at this point []Role of curative surgery en bloc resectionMost surgeries for spinal metastasis are found palliative and the role of en bloc resection in spinal metastasis is decreasing even further due to improvements in radiotherapy Generally curative surgical resection of spinal metastasis has been considered in the context of a single metastasis of a slowgrowing tumor such as in renal cell thyroid and breast cancers Fig Favorable outcomes in this regard have been reported in the literature [] However some authors recently reported that curative ABECDFig A 63yearold male with spinal metastasis of thyroid carcinoma at T8 A Preoperative MRI shows pathologic fracture and spinal cord compression at the T8 level B Postoperative Xray at month shows removal T8 vertebra and reconstruction with an expandable cage following total en bloc spondylectomy C MRI at postoperative 3years shows a widely decompressed spinal canal with no tumor recurrence D Postoperative Xray at postoperative 5years shows wellmaintained instrumentation E Bone scan at postoperative 5years shows no evidence of bone metastasis MRI magnetic resonance imaging 0csurgical resection en bloc spondylectomy did not impact the oncologic outcomes of spinal metastasis patients [] Therefore a more careful and thorough decisionmaking process is required before performing curative resection surgery for spinal metastasis especially when the extended role of radiosurgery is consideredPostoperative complications and preventive measuresThe overall complication rate following surgery for spinal metastasis ranges from to in the literature [] Because surgery for spinal metastasis is performed to improve the quality of life of a patient surgeons should try to minimize all possible surgical and medical complications by implementing multidisciplinary interventions Among diverse complications those that require additional attention when found in spinal metastasis patients eg wound infection instrumentation failure and intraoperative bleeding are briefly discussed hereThe incidence of surgical site infection SSI is determined to be higher in spinal metastasis surgery reaching up to as compared with during other spine surgeries [] SSI has also been identified as the most common cause for reoperation in of reoperations following surgery for spinal metastasis [] Poor nutrition and exposure to adjuvant therapies chemotherapy and radiotherapy put spinal metastasis patients at risk for SSI [] Surgeons should provide adequate nutritional support perioperatively and secure a sufficient time interval between radiation and surgery as previously discussed to minimize SSIs Additional risk factors such as smoking obesity and medical comorbidities should also be considered []The second cause for reoperation in spinal metastasis surgery is the failure of instrumentation [] Risk factors associated with instrumentation failure include the number of operated levels prior chest wall resection and history of radiotherapy [] The necessity of additional fusion procedures while performing surgery for spinal metastasis is debated but highlevel evidence remains to be lacking at this time [] In future studies we suggest that instances of early and late failure be distinguished when assessing instrumentation failure because the mechanisms of failure between the two types seem to differ ie insufficient stability of the construct in the context of early failure versus the progression of deformity or lack of fusion in the context of late failureTreatment of Spinal Metastasis Intraoperative bleeding during spinal metastasis surgeries can be massive and can further lead to serious complications such as cardiovascular or cerebral events [] For the spinal metastasis of hypervascular tumors such as renal cell hepatocellular and thyroid cancers preoperative embolization is recommended Previous studies have verified the effectiveness of preoperative embolization in reducing intraoperative bleeding in spinal metastasis surgeries [] Surgery should be performed within hours following embolization to avoid the diminished effect of preoperative embolization []ConclusionsThe determination of appropriate treatment for a patient with spinal metastasis is a challenging task that requires multidisciplinary considerations Recent advances in radiotherapy and surgery for spinal metastasis have brought about improvements in the management of these patients Evolving decisionmaking systems are also crucial contributors to stateoftheart care of patients with metastatic spinal tumorsConflict of InterestNo potential conflict of interest relevant to this was reportedReferences White AP Kwon BK Lindskog DM Friedlaender GE Grauer JN Metastatic disease of the spine J Am Acad Orthop Surg Laufer I Sciubba DM Madera M Surgical management of metastatic spinal tumors Cancer Control Schiff D ONeill BP Suman VJ Spinal epidural metastasis as the initial manifestation of malignancy clinical features and diagnostic approach Neurology Klimo P Jr Schmidt MH Surgical management of spinal metastases Oncologist Nathan SS Healey JH Mellano D Survival in patients operated on for pathologic fracture implications for endoflife orthopedic care J Clin Oncol Curtin M Piggott RP Murphy EP Spinal metaAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J static disease a review of the role of the multidisciplinary team Orthop Surg Ahmed AK Goodwin CR Heravi A Predicting survival for metastatic spine disease a comparison of nine scoring systems Spine J Barzilai O Fisher CG Bilsky MH State of the art treatment of spinal metastatic disease Neurosurgery Tomita K Kawahara N Kobayashi T Yoshida A Murakami H Akamaru T Surgical strategy for spinal metastases Spine Phila Pa Tokuhashi Y Matsuzaki H Oda H Oshima M Ryu J A revised scoring system for preoperative evaluation of metastatic spine tumor prognosis Spine Phila Pa Bauer HC Wedin R Survival after surgery for spinal and extremity metastases prognostication in patients Acta Orthop Scand Van der Linden YM Dijkstra SP Vonk EJ Marijnen CA Leer JW Dutch Bone Metastasis Study Group Prediction of survival in patients with metastases in the spinal column results based | Thyroid_Cancer |
"Many cancerassociated single nucleotide polymorphisms SNPs are located in the genomic regionsof long noncoding RNAs lncRNAs Mechanisms of these SNPs in connection to cancer risk are not fullyunderstoodMethods Association of SNP rs140618127 in lncRNA LOC146880 with nonsmall cell lung cancer NSCLC wasevaluated in a casecontrol study of individuals The mechanism of the SNPs biologic influence was exploredwith in vitro and in vivo experiments including plasmid transfection siRNA knockdown flow cytometry assessmentand assays of cell proliferation migration invasion and colony formationResults Association analysis showed that A allele of SNP rs140618127 was associated with low risk of NSCLC in theChinese population Lab experiments indicated that SNP rs140618127 contained a binding site for miR5395p andthe binding between miR5395p and LOC146880 resulted in declined phosphorylation of an oncogene ENO1 Thereduced phosphorylation of ENO1 led to decreased phosphorylation of PI3K and Akt which is further linked to thedecline in cell proliferation and tumor progressionConclusion The study demonstrates that SNP rs140618127 in lncRNA loc146880 provides an alternate binding sitefor microRNA miR5395p which affects the phosphorylation of ENO1 and activation of the PI3K and Akt pathwayKeywords NSCLC lncRNA SNP miRNA ENO1BackgroundLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in the world []The majority oflungcancer NSCLC which accounts for around of alllung cancer is nonsmall cell Correspondence qianbiyunsjtueducn Tienan Feng and Nannan Feng contributed equally to this work1Hongqiao International Institute of Medicine Shanghai Tongren HospitalClinical Research Institute Shanghai Jiao Tong University School of MedicineShanghai China7Second Affiliated hospital of Chengdu Medical College China NationalNuclear Corporation Hospital Chengdu Sichuan ChinaFull list of author information is available at the end of the lung cancer cases Genetic factors may play an importantrole in an individuals susceptibility to NSCLC Longnoncoding RNAs lncRNAs are a class of noncodingtranscripts with nucleotides or more Increasingevidence suggests thatlncRNAs are involved in theoccurrence oflung cancer due to their functions asoncogenes or tumor suppressors [] Our previous studies indicated that a lncRNA on chromosome 17q243named LOC146880 was expressed higher in tumortissues than in adjacent normal tissues and high expression was associated with poor prognosis of NSCLC []Singlenucleotide polymorphisms SNPs in the noncoding regions of the genome have been shown to affect The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cFeng Journal of Experimental Clinical Cancer Research Page of rs140618127 the A allelecancer risk via regulating the transcription andor changing the structure of lncRNA [] A previous studyidentified SNPs in more than humanlncRNAs and a large number of SNPs were predicted tohave a potentialimpact on the microRNA miRNAlncRNA interaction [] Here we report the identificationof SNP rs140618127 in LOC146880 as a new susceptiblelocus to NSCLC Bioinformatics analysis predicts thatin LOC146880variantprovides an altered secondary structure which maycreate a binding site for microRNA miR5395p []sequestering its action on other molecules Shiraishi conducted a GWAS on lung adenocarcinoma andidentified SNP rs7216064 in BPTF 17q243 in association with the cancer risk OR p 7e11 []Seow confirmed that SNP rs7216064 was associated with the risk of lung cancer based on a GWASstudy of Asian female nonsmokers [] We found thatSNP rs140618127 was in strong linkage disequilibriumwith SNP rs7216064 LD r2 and this lncRNASNP was associated with lung cancer risk OR p in our casecontrol study of individualsToSNPrs140618127 in NSCLC development and progressionweconsequence ofLOC146880 and miR5395p interaction and found thatthe microRNA behaved like a tumor suppressor []which prevented LOC146880 from interacting withprotein ENO1 an oncogene product [] reducing itsphosphorylation As a resultthe phosphorylation ofPI3KAKT was also reduced after the suppression ofENO1 phosphorylation [] which further inhibitedtumor growth and metastasisleading to a betterprognosis of NSCLCthe molecular mechanism ofexploreevaluated thatthe biologicalMaterials and methodsStudy populationsSuspected NSCLC individuals had histopathological orcytologically confirmed diagnosis according to theWorld Health anization classification These studysubjects including suspected individuals diagnosed withlung cancer or normal were recruited from the ChinaMedical University CMU Distributions of the basiccharacteristics ofthe study subjects are provided inTable At recruitment an informed consent wasIlluminated Only ifthe subject agreed heshe wasincluded This study was approved by the InstitutionalReview Board at CMUSNP selection and genotypingSNPs with r2 were considered to be in the same LDblock With this criterion one SNP was selected in eachLD block and genotyped using the TaqMan genotypingmethod in the ABI RealTime PCR systemApplied Biosystems For quality control we implemented several measures in our genotyping assays including each plate contained both case and controlsamples technicians were blinded to the casecontrolstatus of the samples both positive and negativecontrol no DNA template samples were included ineach 384well plate and nearly of the sampleswere assayed in duplicate and the concordances werebetween and Cell linesHuman NSCLC cell lines A549 and PC9 and humanlung epithelial BEAS2B cells were purchased from theCell Bank of Type Culture Collection at the ChineseAcademy of Sciences Shanghai Institute of Biochemistryand Cell Biology These celllines were passaged forfewer than months All the cells were tested for mycoplasma and were found to be free from infection Thecells were maintained in DMEM supplemented with FBS and grown without antibiotics in an atmosphere of CO2 and relative humidity at °C² and ² RACE and coding prediction of LOC146880We used ² and ² RACE to determine the transcripinitiation and termination sites of LOC146880tionalwith a SMARTe RACE cDNA Amplification kit Clontech The Alignment File of a fulllength sequence ofLOC146880 obtained from ² and ² RACE is availableupon requestConstruction of reporter plasmids transient transfectionsand luciferase assaysA reporter plasmid in the psiCHECK2 vector Promegawas created which contains a 1000bp LOC146880 exonregion flanking rs140618127 [G] or rs140618127 [A]with the restriction enzymes XhoI and NotI FermentasA549 and PC9 cells were seeded at cells per wellin 24well plates and ng of the reporter plasmid and pmol of miR5395p mimic Ambion were cotransfected into the cells h later using Lipofectamine Invitrogen These cells were collected h aftertransfection Renilla luciferase activity was measured andused to normalize the efficiency of transfectionRNA extraction and qRTPCR analysisTotal RNA from the NSCLC tissue specimens and celllines used in this study was extracted using the TRIzolreagent Firststrand cDNA was synthesized using theSuperScriptInvitrogenRelative RNA levels determined by qPCR were measuredon an ABI sequence detection system AppliedBiosystems using the SYBR Green method Îetaactinwas employed as an internal control for the quantification of LOC146880 and the mRNA levels of other genesreverse transcriptase kitII 0cFeng Journal of Experimental Clinical Cancer Research Page of For miRNA quantification small nuclear RNA U6 wasused as an endogenous control The relative expressionof RNA was calculated using the comparative CtmethodSubcellular fractionationCytosolic and nuclear fractions of A549 and BEAS2Bcells were prepared and collected according to theinstructions ofthe NuclearCytoplasmic Isolation kitBiovision LOC146880 was mainly detected in thenuclear fraction although it was also present in the cytoplasm Fig S1belowBrieflydescribedRNA pulldown and mass spectrometry analysisRNA pulldown assays were performed following theprotocolbiotinylatedLOC146880 or antisense LOC146880 was incubated withcellular protein extracts from A549 cells and streptavidin beads were then added Recovered proteins associated with LOC146880 or antisense LOC146880 wereexcised and proteomics screening was accomplished bymass spectrometry analysis on a MALDITOF instrumentIn vitro transcription ofLOC146880 and its deletion fragments were analyzedwith primers containing the T7 promoter sequenceBruker DaltonicsPlasmid construction and transfectionTo construct a lentiviral vector expressing humanLOC146880 NR_026899 a fulllength of LOC146880cDNA containing rs140618127 [G] or rs140618127 [A]was commercially synthesized GeneChem and subcloned into the AgeI and NheI sites of the GV367IRESPuromycin lentiviral expression vector GeneChem Toproduce lentivirus containing LOC146880 T cellswere cotransfected with the vector described above andlentiviral vector packaging system GeneChem using Lipofectamine Infectious lentiviruses were collectedat h after transfection and filtered through 045μmPVDF filters for analysis of genotype After conformation these lentiviruses were designated to LOC146880[G] or LOC146880 [A] We used the GV367IRESPuromycin empty vector as a negative control Theviruscontaining pellet was dissolved in DMEM and aliquots of the solution were stored at °C A549 andPC9 cells were infected with concentrated virus in thepresence of polybrene SigmaAldrich The supernatantwas replaced with complete culture medium after hfollowed by selection with puromycin and the expression of LOC146880 in infected cells was verified byqPCRRNA immunoprecipitation assaysRIP experiments were performed using the Magna RIPProteinRNABindingkitMillipore Antibodiesagainst ENO1 Abcam orcontrol proteins were diluted at Total RNA inputcontrol and precipitation with the isotype control IgGfor each antibody were assayed simultaneously The coprecipitated RNAs were detected by RTqPCRImmunoprecipitationCell lysis and immunoprecipitationCells were homogenized in RIPA buffer supplemented with ProteasePhosphatase Inhibitor CocktailPierce Cell lysates were centrifuged and the supernatants were prepared for immunoblotting or immunoprecipitation withbelowImmunoblot signal was detected using Clarity WesternECL Substrate Thermo FisherantibodiesdescribedtheTable Characteristics of lung cancer patients and healthy controlsVariablesP valueControlN NSCLCN Age at dx year ¥ GendermalefemaleSmoking statusnoyesSNP rs140618127G alleleA allele adjusted by smokinggenderage OR valueP valueOR value 0cFeng Journal of Experimental Clinical Cancer Research Page of from cells orImmunoblot assaysProtein extractsimmunoprecipitationsamples were prepared using detergentcontaining lysisbuffer Total protein μg was subjected to SDSPAGE and transferred to PVDF membrane MilliporeAntibodies against ENO1 Abcam ab155102 ENO1phosphorylated at Cterminalinhibitory site Tyr44StressMarq Bioscience spc965D PI3K CST 13666SPI3K phosphorylated at Tyr458 CST 4228S AKTCST 2938S AKT phosphorylated at Ser473 CST9018S PCNA CST 2586S NFkB CST 8242SVimentin Abcam ab92547 βCatenin CST 8480S Ecadherin CST 14472S NCadherin Abcam ab18203and βActin SigmaAldrich A1978200UL were usedMembranes were incubated overnight at °C withprimary antibody diluted and proteins were detected with the Odyssey near infrared dualcolor laserimaging system LICORAnalysis of cell proliferation migration invasion cellcycle and colony formationCells were seeded in 96well flatbottom plates with cells in μl cell suspension in each well Afterculture cell viability was measured with the CCK8assay Each experiment with six replicates was repeatedthree times For cell cycle analysis cells were collectedand fixed in ethanol overnight at °C Singlecellsuspensions were labeled with μgml Propidium Iodide Sigma and analyzed by flow cytometry BeckmanCoulter For colony formation cells were seededin 65mm culture dishes and allowed to grow until visible colonies formed in complete growth medium weeks Cell colonies were fixed with methanol stainedwith crystal violet and counted Invasion assays wereperformed in Millicell chambers in triplicate The 8μmpore inserts were coated with μg of Matrigel BD Biosciences Cells were added to the coated filtersin serumfree medium PMI1640 medium containing FBS was filled in the lower chambers as a chemo attractant After h at °C in an incubator suppliedwith CO2 cells that migrated through the filters werefixed with methanol and stained with crystal violet Cellnumbers in three random fields were counted The migration assay was conducted in a similar fashion withoutcoating the filters with MatrigelExperiments on xenograft animalsTen male BALBc mice weeks old were kept in aspecific pathogenfree grade environment All animalexperiments were approved by the Animal Care and UseCommittee of Shanghai Jiao Tong University School ofMedicine Shanghai China All applicable guidelines ofthe Animal Care and Use Committee of Shanghai JiaoTong University School of Medicine for the care and usethe hind flank regions ofof animals were followed PC9 cells of rs140618127 [A]and rs140618127 [G] type were collected and resuspended in PBS at a concentration of cellsmLand mixed with Matrigel® at a ratio of respectivelyThe mixture mL was subcutaneously injected intotwo sides ofthe micers140618127 [A] and rs140618127 [G] cells in the samemouse Tumor size was measured once every daysusing a Vernier caliper across its two perpendicular diameters and tumor volume was calculated using the following formula V 12ab2 where V is the tumorvolume a is the largest diameter and b is the smallestdiameter After weeks oftreatment all mice weresacrificed and their tumors were collected and weighedHistological evaluation ofsamples wasperformedthe tumorHistopathological analysesTumor tissues from the animals were fixed in paraformaldehyde BOSTER Wuhan Chinafor h atroom temperature The fixed tissues were then dehydrated in a graded series of alcohol cleaned in xyleneand embedded in paraffin A rotary microtome was usedto section paraffin the blocks into 4μm thick sectionsThe sections were deparaffinized and stained withhematoxylin and eosin HE A light microscopeOlympus was used to examine the stained tissuesectionsStatistical analysisThe association between SNP rs140618127 and NSCLCrisk was analyzed under an additive model using the unconditionallogistic regression model adjusted for agesex and smoking status Results of laboratory experiments were presented as Means ± SD Students t testwas used to compare means between two groups andANOVA was employed for comparison of more thantwo groups Repeated ANOVA was employed for comparison of more than two groups which contained repeated measure data All the statistical analyses wereperformed using Statistical Product and Service Solutions SPSS software version and GraphPad PrismVersion GraphPad Software San Diego CA USAResultsSNP rs140618127 G A in LOC146880 and NSCLC riskSNP in LOC146880 rs140618127 is in strong linkagedisequilibrium with SNP rs7216064 r2 which is aGWASdiscovered risk allele for NSCLC We found thatSNP rs140618127 G A in the exon of LOC146880chr17 was associated with the risk of NSCLC the A allele compared to G had an adjusted oddsratio OR in a casecontrol study of subjects Table Stratified analyses suggested 0cFeng Journal of Experimental Clinical Cancer Research Page of that this effect was more evidence in those who were ¥female and nonsmokers Supplemental years oldTable S1 The minorfrequency of SNPrs140618127 is low globally but can be high as in some American populations see Suppl alleleEffects of LOC146880 with rs140618127 [a] on cellproliferation and behaviorsWe examined the effects of LOC146880 on cell proliferation by its allele at rs140618127 and found that overexpression of rs140618127 [A] in the NSCLC celllinesA549 and PC9 both with the G allele at rs140618127substantially reduced the rate of cell proliferation whencompared with rs140618127 [G]Fig 1A Colonyformation ability in both A549 and PC9 cells wasmarkedly suppressed by rs140618127 [A] when compared with rs140618127 [G] Fig 1B Overexpression ofrs140618127 [A] significantly suppressed the invasionand migration of NSCLC cells Fig 1C 1D Tumorsize in a xenograft animal model of PC9 was decreasedin both genotype groups but the decline in tumor sizewas greater for rs140618127 [A] than for rs140618127[G] P There was no significant difference intumor size between the vector control group and wildtype rs140618127 [G] Fig S2 HE staining showedthat tumors of rs140618127 [A] possessed less malignantmorphology Fig 1E Together these results indicatethat rs140618127 [A] can inhibit the growth of lung cancer more in vitro and in vivo compared to rs140618127[G]such a possibilityInteraction between LOC146880 and miR5395pEvidence suggests that SNPs in lncRNAs may generatenew interacting sites between lncRNAs and other transcripts such as miRNAs [] Using an online softwarelncRNASNP httpbioinfolifehusteducnlncRNASNP[] we found that several SNPs in LOC146880 werepredicted to haveand SNPrs140618127 was indicated to lie within a putative binding site for miR5395p The G A mutation atrs140618127 was predicted to change the local foldingstructures and free energy of LOC146880 which mightcreate a binding site for miR5395p Following this prediction we investigated whether miR5395p interactswith LOC146880 based on its genotype at rs140618127Luciferase reporter assays showed that in comparison tothe construct containing rs140618127 [G] the constructwith the A allele had significantly reduced luciferase activity in the presence of miR5395p suggesting moreinteraction of miR5395p with LOC146880 [A] thanwith LOC146880 [G] Fig 2A The interaction betweenmiR5395p and LOC146880 [A] could be blocked bythe miR5395p inhibitor miR5395p is constitutivelyexpressed in both A549 and PC9 cells In cells stablyoverexpressing LOC146880 miR5395p only decreasedthe levels of LOC146880 with rs140618127 [A] not alleleG indicating that allele A is a target of miR5395p Fig2Bthe RNA pulldown assay weInteraction between LOC146880 and ENO1isolated aUsingLOC146880 withrs140618127[G]protein complexMass spectrometry analysis showed that there were threeproteins in this complex and the most abundant onecompared to antisense one was ENO1 Fig 3A Wethen selected ENO1 for validation detecting ENO1 inthree independent RNA pulldown assays RNA immunoprecipitation RIP assays also showed enrichment ofLOC146880 in the complexes precipitated with ENO1antibody as compared with IgG or another irrelevantantibody indicating that ENO1 may be a key target protein of LOC146880 Fig 3b Next we evaluated the consequences of the interaction between LOC146880 andENO1 We found that ENO1 mRNA expression andprotein level were not significantly different P see Fig S3 in the cells overexpressing LOC146880 withrs140618127[A] or rs140618127[G] in the presence ofmiR5395p Fig 3C 3D However HE staining ofxenograft tumors in mice showed that phosphorylatedENO1 was higher in rs140618127 [G] than in [A] Fig3E The expression of CMYC a downstream target ofENO1 was decreased remarkably when the cells weretransfected with a siRNA against ENO1 Fig 3FRegulation of PI3KAKT signal by LOC146880 via ENO1phosphorylationWe found that ENO1 phosphorylation was markedly decreased in cells overexpressing rs140618127 [A] as compared with those overexpressing rs140618127 [G] in thepresence of miR5395p mimics Fig 4A Fig S4 andFig S5 Using insilico prediction tools [ ] weidentified a phosphorylation site at Tyr44 in the proteinbased on the PDB database Fig S5 [] We next investigated the impact of altered LOC146880 levels on thedownstream signal of ENO1 Since our results describedaboveoverexpressionincreased cell proliferation migration and invasion wefocused our investigation on the PI3KAKTNFkB signaling The total amount of PI3K and AKT proteins wasnot significantly different between cells overexpressingrs140618127 [A] and [G] However we observed thatprotein phosphorylation levels affected the expression ofdownstream molecules in the PI3KAKT signaling inA549 Fig S4 Cells overexpressing LOC146880 withrs140618127 [A] showed substantial decreases in NFkB PCNA Vemintin and Ncadherin levels while theirβcatenin and Ecadherin levels weresignificantlyincreased when compared withthesamecellsLOC146880indicatedthat 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs140618127[A] inhibits NSCLC cell proliferation and EMT process A proliferation assay of different samples which were compared byrepeated ANOVA B clone formation ability of different samples which were compared by ANOVA C wound healing assay of different sampleswhich were compared by ANOVA D cell invasion assay of different samples which were compared by ANOVA e cancer protective effect ofrs140618127[A] in xenograft animals which were illuminated by repeated ANOVA and ttest 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig rs14061812[G][A] expression of different condition in A549 and PC cell lines a luciferase activity of different conditions which wereilluminated by ANOVA b LOC146880 expression level of different conditions which were compared by ttestoverexpressing LOC146880 with rs140618127 [G] Fig4B Fig 4C Fig S7 and Fig S8 Immunohistochemical staining of xenograft tumors showed that pPI3KpAKT TWIST NCadh and SNAIL were all significantly higher in rs140618127 [G] than in [A] Fig 4Dand Fig S9DiscussionIn this study we found that SNP rs140618127 inLOC144680 contained a binding site for miR5395pand the binding between miR5395p and LOC146880resulted in declined phosphorylation of an oncogeneENO1 which was found to be a downstream target ofLOC146880 Furthermore the reduced phosphorylationof ENO1 led to decreased phosphorylation of PI3K andAkt which was linked to the decline in tumor cell proliferation and progress The entire process of how SNPrs140618127 influences NSCLC is depicted in Fig Our casecontrol study supports the notion that SNPrs140618127 genotype [A] may have a protective effecton NSCLC compared to genotype [G] In a previousstudy we found that LOC146880 expression was significantly higher in NSCLC tumors than adjacent normaltissuesforLOC146880 []a possible oncogenicsuggestingroleThere has been an increasing interest in understandingthe mechanisms of rare genetic variants in lncRNAs inrelation to the complex traits and diseases [ ] Ingle suggested that genetic polymorphisms in lncRNAMIR2052HG offer a pharmacogenomic basis for the response of breast cancer patients to aromatase inhibitortherapy [] Tang indicated that SNP rs9839776in SOX2OT was significantly associated with breast cancer possibly via influencing the expression of SOX2OT[] Redis demonstrated that the GWASidentifiedSNP rs6983267 on 8q24 is in a lncRNA gene calledCCAT2 which regulates cancer cell metabolism in anallelespecific manner through binding to the cleavagefactor I complex This complex is implicated in anallelespecific regulatory mechanism of cancer metabolism orchestrated by alleles ofthe lncRNA [ ]Russell et alidentified a neuroblastoma susceptibilitylocus rs9295534 located in the upstream enhancer of atumor suppressor CASC15S The SNP could decreasethe transcriptional activity of CASC15S and be associated with the disease outcome [] Wang foundthat SNP rs965513 a locus on 9q22 in the FOXE1 geneand lncPTCSC2 was associated with the risk of papillary thyroid carcinoma []In this study we found that a lncRNA could regulatethe function of a protein via its phosphorylation with 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 promotes ENO1 activation in a variantspecific manner A pulldown assaymass spectrum of identificationsilver staining BRIP assay with A549 and PC9 cell lines which were compared by ANOVA C D ENO1 mRNA expression and protein level of rs140618127[G][A]by overexpression plasmid transfection which were compared by ttest E HE staining of ENO1 phosphorylation in vivo which were comparedby ttest F ENO1 LOC146880 and cMYC expression level after ENO1siRNA transfection which were compared by ttestlittle influence on gene expression or protein concentration Similar findings have been reported before in whichthe phosphorylation site of a protein can be blocked by alncRNA leading to decreased phosphorylation For example NFkB can be inhibited by a long noncodingRNA which directly blocks IKB phosphorylation inbreast cancer [] LncRNA can also bind to proteinsincreasing or decreasing their phosphorylation viaanother protein LncRNA DANCR and PANDAR influence the phosphorylation of serine in RXRA and SFRS2via GSK3β and P53 in breast and ovarian respectively[ ] GSK3βs phosphorylation in breast cancer wasreported to be reduced by lncRNA NLIPMT [] Thephosphorylation of ULK1 can be suppressed by lncRNAHOTAIR in NSCLC [] LINC00675 enhances the phosphorylation of vimentin on Ser83 to suppress gastriccancer progression [] Our finding of a lncRNAs impact on the phosphorylation of a protein was quiteunique and interesting because it is achieved by a microRNA through a polymorphic site in LOC146880In our study we found that a G to A transition atrs140618127 in LOC146880 could turn into a bindingsite for a microRNA and miR5395p was indeed the target Interestingly the wildtype of LOC146880 had no 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig LOC146880 regulates PI3KAKT signaling via ENO1 A549 PC9 A ENO1pENO1 PI3KpPI3K AKTpAKT protein level usingrs140618127[G][A] overexpression plasmid transfection B PCNA NFkB protein level using rs140618127[G][A] overexpression plasmidtransfection C βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection D HEstaining of pPI3K pAkt TWIST NCadhersin and SNAIL 0cFeng Journal of Experimental Clinical Cancer Research Page of Fig Diagrammatic sketch of rs14061812mediated NSCLC tumorigenesis LOC146880 rs14061812[G] increases ENO1s phosphorylationresulting in activating PI3KAKT signaling pathway and NSCLC tumorigenesis while LOC146880rs14061812[A] binds to miR5395p decreasesENO1s phosphorylation resulting in deactivating PI3KAKT signaling pathway and NSCLC tumorigenesisinteraction with the microRNA at all This SNP has notbeen reported before in any studies [] However miR is known to be a tumor suppressor [ ] Ourfinding of miR539s binding to loc146880 provided newinsights into a possible mechanism that explains the biologic function of miR539 as a tumor suppressor Thiseffect takes place when a microRNA and lncRNA interact through a polymorphic site which results in changesin phosphorylation in a protein ENO1 that the lncRNAmay target on Low levels of LOC146880 did not influence the mRNA expression or protein levels of ENO1but suppressed the phosphorylation of ENO1 ENO1 is ametabolic enzyme involved in the synthesis of pyruvateIt also acts as a plasminogen receptor and mediates theactivation of plasmin and extracellular matrix degradation In tumor cells ENO1 is upregulated and supportsthe Warburg effect The protein is located on the cellsurface where it promotes cancerinvasion and issubjected to substantial posttranslational modificationsnamely acetylation methylation and phosphorylation[] Reduced phosphorylation of ENO1 lowers thePI3KAkt signal which results in slower cell migrationor invasion of NSCLCThe SNPbased interaction between miRNA andlncRNA in regulation of protein function has beenhypothesized and predicted by YaRu but fewstudied have provided evidence [] Our study was thefirst to show the interaction between LOC146880 andmir5395p in the NSCLC and to elucidate the downstream mechanism involving tumor growth and metastasis The modulation model of the lncRNA and miRNA isnot the classical competing endogenous RNAs ceRNAHow LOC146880 interacts with ENO1 to regulate itsphosphorylation and downstream signals remains to beelucidated Although the A allele of rs140618127 is lowin general some racial groups still have a relatively highfrequency In some Caucasian populations the A allelefrequency is close to ConclusionsWe found in a casecontrol study of Chinese thatSNP rs140618127 in LOC146880 was associated with therisk of NSCLC People with the G allele of rs140618127had higher risk than those with the A allele Our in vitroand in vivo experiments demonstrated that LOC146880was an oncogene and the G allele of rs140618127 hadstronger oncogenic effects on lung cancer cells than theA allele in LOC146880 This differential effect appearedto come from the binding of a microRNA miR539 tothe A allele but not the G allele at rs140618127 ThemicroRNA binding prevented the lncRNAs interactionwith its downstream target ENO1 which led to the reduction of ENO1 phosphorylation and suppression ofthe PI3KAKT signaling resulting in lower tumor cellproliferation and less aggressive cell behaviors 0cFeng Journal of Experimental Clinical Cancer Research Page of Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s13046020016525Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestAdditional file Fig S1 Locations of LOC146880 A549 BEAS2BLOC146880 locals mainly in cytoplasm The comparison between twogroups using ttestAdditional file Fig S2 Comparison of tumor size between vectorcontrol group and the wide type There was no significant difference oftumor size between vector control group and the wide typers14061812[G]Additional file Fig S3 Comparison of ENO1 protein level ofrs140618127[G][A] by overexpression plasmid transfection which werecompared by ttestAdditional file Fig S4 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of A549 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S5 Comparison of ENO1pENO1 PI3KpPI3KAKTpAKT protein level of PC9 cell lines using rs140618127[G][A]overexpression plasmid transfection which were compared by ttestAdditional file Fig S6 Predicting site of phosphorylation of ENO1A predicting phosphorylation site of ENO1ENO1 chain A usingNetPhos B predicting phosphorylation site of ENO1ENO1 chain Ausing PhosphoELM BLAST C The predicting phosphorylation site ofENO1 chain A using data of PDB databaseAdditional file Fig S7 Comparison of PCNA and NHkB protein levelof using rs140618127[G][A] overexpression plasmid transfection whichwere compared by ttest A results of A549 cell lines B result of PC9cell linesAdditional file Fig S8 Comparison of βCatenin Vimentin NCadherin ECadherin protein level using rs140618127[G][A] overexpression plasmid transfection which were compared by ttest A results ofA549 cell lines B result of PC9 cell linesAdditional file Fig S9 Comparison of the HE staining of pPI3K pAkt TWIST NCadhersin and SNAIL which were compared by ttestAdditional file Distribution of | Thyroid_Cancer |
Neck Tissues A a0Systematic ReviewJerome a0R a0Lechien1234 Stphane a0Hans13 a0· Maria a0R a0Barillari18 a0· Giovanni a0Cammaroto19 a0· Graldine a0Descamps12 a0· Julien a0Hsieh110 a0· Luigi a0Vaira111 a0· Giacomo a0De a0Riu111 a0· Leigh a0Sowerby112 a0· Isabelle a0Gengler113 a0· Justin a0Michel15 a0· Sven a0Saussez124 a0· Thomas a0Radulesco15 a0· Christian a0CalvoHenriquez16 a0· Carlos a0M a0ChiesaEstomba17 a0· Received July Accepted August Springer ScienceBusiness Media LLC part of Springer Nature AbstractTo review the data regarding the expression of angiotensin converting enzyme2 ACE2 and transmembrane protease serine2 TMPRSS2 in head and neck tissue Scopus Cochrane Library Medrxiv Google Scholar and PubMEDMEDLINE were searched by four independent investigators for studies investigating ACE2 or TMPRSS2 expressions in head and neck tissues The following outcomes were considered sample origin animal versus human detection method anatomical location and cell types PRISMA checklist and modified population intervention comparison outcome timing and setting PICOTS framework were used to perform the review Of the identified studies met our inclusion criteria Thirteen studies were conducted during the severe acute respiratory syndrome a0coronavirus2 SARSCoV2 pandemic ACE2 and TMPRSS2 were expressed in oral pharyngeal sinusonasal human mucosa The following cell types expressed ACE2 basal apical goblet minor salivary and endothelial cells TMPRSS2 was found in goblet and apical respiratory cells ACE2 and TMPRSS2 were found in the olfactory region especially in sustentacular nonneural and neural stem cells Animal studies suggested that ACE2 expression may vary regarding age There was an important heterogeneity between studies in the methods used to detect ACE2 and TMPRSS2 leading to a potential identification bias The SARSCoV2 receptors ACE2 and TMPRSS2 are both expressed in many head and neck tissues enabling the viral entry into the host anismKeywords ACE2 a0· TMPRSS2 a0· SARSCoV2 a0· COVID a0· Coronavirus a0· Head NeckIntroductionThe renin angiotensin aldosterone system is one of the most important systems regulating the homeostasis of cardiovascular and pulmonary function this involves many molecules including angiotensin converting enzyme2 ACE2 [] ACE2 is also known to be the functional receptor of some coronavirus species as initially discovered in during the severe acute respiratory syndrome coronavirus SARSCoV epidemic [] The current pandemic of coronavirus disease Jerome R Lechien and Thomas Radulesco have contributed equally to the paper and are joint as cofirst authorsJustin Michel and Sven Saussez equally contributed to the paper and are cosenior authorsjeromelechienumonsacbe Jerome R Lechien Extended author information available on the last page of the COVID19 has brought to light the importance of ACE2 regarding development of infection viral spread and the development of the clinical COVID19 [] At the same time another SARSCoV2 receptor has been identified the transmembrane protease serine2 TMPRSS2 []ACE2 and TMPRSS2 tissue expressions are particularly important to identify viral entry pathways and to better understand the anrelated clinical presentation of the disease [ ] Further evaluation of ACE2 and TMPRSS2 expression in ear nose and throat mucosa is warranted to shed light on the pathophysiology of disease in the head and neck []The aim of this systematic review is to summarize the current data about the expression of ACE2 and TMPRSS2 in head neck tissueVol01234567891 0c MethodsThe review was conducted regarding the Preferred Reporting Items for a Systematic Review and Metaanalysis PRISMA checklist [] A modified population intervention comparison outcome timing and setting PICOTS framework was used to structure the review process [] For this review the PICOTS structure was kept but adapted to experimentalbasic research studies on human and animal tissuesStudiesAnimal and human experimental published studies in Englishlanguage peerreviewed journals were considered Preprint studies were also considered in light of the current pandemic and the significant wealth of knowledge derived over the last few months All studies where investigators assessed ACE2 or TMPRSS2 expressions in head neck tissues through immunochemistry IHC in a0situ hybridization Western Blot RNA sequencing RNAseq or reverse transcription polymerase chain reaction RTPCR were evaluatedParticipants and a0Inclusion CriteriaThe papers had to include either human or animal subjects The authors extracted substantial information about the sample characteristics including species involved and ACE2 and TMPRSS2 identification methodOutcomesThe primary outcome studied was tissue expression of ACE2 and TMPRSS2 The anatomical location the types of cells that expressed both receptors were recorded Particular attention was paid to the method used to detect ACE2TMPRSS2 in tissues Additional useful information such as viral impact on the functioning of the tissuecell that expressed the receptor or interindividual differences were also collectedIntervention and a0ComparisonBecause the aim of the study was to investigate the tissue ACE2 and TMPRSS2 expression we did not consider potential intervention on patient or animal modelsHead and Neck PathologyTiming and a0SettingWe included the studies where the receptor analysis was made on normal subjects andor infected patientsSearch StrategyThe PubMedMEDLINE Google Scholar Medrxiv Scopus and Cochrane search was conducted by independent authors JRL TR CCH GD CMCE to identify papers published between January and April The authors screened publications with database s and available full texts referring to the condition The following keywords were used for the search strategy ACE2² TMPRSS2² COVID19² COVID SARS coronav coronavirus salivary gland Receptor Head Neck Nasal ear nose throat ENT Tissue and Cell The authors investigated papers for number of samplesindividuals study type design inclusion criteria and ACE2TMPRSS2 detection outcomesResultsThe electronic search identified papers of which met our inclusion criteria Table a0 [] A total of studies investigated the expression of ACE2 and TMPRSS2 in human head and neck tissues while five papers focused on mouse and two on monkey samples respectively Table a0 One study focused on ACE2 genetic analysis without reporting sitespecific anatomical expression [] The flow chart of the study process is available in Fig a0 Five studies were preprint [ ]Tissue Expression in a0HumanACE2 ExpressionACE2 was assessed in studies [] The expression of ACE2 was found in all mucosa of the respiratory upper tract including trachea [ ] sinus and nasal cavities [ ] Among the respiratory mucosa ACE2 was expressed in several types of cells including epithelial goblet and endothelial cells [ ] One study reported that ACE2 was expressed on ciliated epithelial cells and not on nonciliated goblet cells [] Butowt et a0al compared the intensity of expression of ACE2 in the upper and lower respiratory tract [] They found that nasal epithelial cells had lower levels of ACE2 expression compared with epithelial cells of the lower respiratory tract [] Among the nasal region two studies investigated the ACE2 expression in the 0cHead and Neck Pathology Table Studies reporting ACE2 or TMPRSS2 head and neck expressionAuthorsDesignVaarala Mixed [] StudyHamming HumanSamples MethodsHuman TMPRSS2MouseHuman ACE2RNAseqACE2 TMPRSS2 expressionSalivary glandsTMPRSS2 humanOral Nasal Nasopharyngeal Epithelium endotheliumACE2 human all mucosa [] StudyIHCHumanJia [] StudyHuman ACE2Tracheal Epithelium endotheliumIHC biotinylation ACE2 humanLiuExperimenal Monkey ACE2 [] StudyIHCNaso Oro Hypopharyngeal Tracheal EpitheliumACE2 MonkeyVirion in SalivaBilinska Animal [] StudyBrannMixed [] StudyButowtMixed [] StudyMouse ACE2 TMPRSS2 Olfactory EpitheliumRNAseq RTPCRACE2 sustentacular cellsIn situ hybridization TMPRSS2 sustentacular cellsWB IHCMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 TMPRSS2 Mouse nonneuronal cellsACE2 TMPRSS2 Human glial and neuronal stem cellsMouse ACE2 TMPRSS2 Olfactory EpitheliumHuman RNAseqACE2 Mouse Human non neuronal cellsCaoHumanHuman ACE2 gene [] StudyGenetic AnalyzisChenHuman [] StudyHikmetHuman [] StudyHuman ACE2RNAseqHuman ACE2IHCLeeHumanHuman ACE2TMPRSS2 Mouse Human neuronal nonneuronal cellsRespiratory EpitheliumACE2 TMPRSS2 Human Lower Airway NasalNo localization providedSalivary glandsACE2 humanNasopharyngeal EpitheliumACE2 human no expression in nasopharynxTracheal Nasal Sinusal EpitheliumFindings TMPRSS2 is expressed in human salivary gland tissues ACE2 was found in endothelial arteries veins and epithelial cells of nasal rhinopharyngeal and oral mucosa Precisely the epithelium expression concerned the basal layer cells ACE2 was more expressed on the apical than the basolateral surface of polarized airway epithelia ACE2 is expressed in salivary gland ducts of the pharyngeal glands ACE2 was expressed in epithelial cells lamina propria respiratory tract Virus was found in saliva of infected monkeys ACE2 TMPRSS2 are expressed in sustentacular cells of the olfactory epithelium but notmuch less in most olfactory receptor neurons Expression of the entry proteins increases in animals of old age In human ACE2 TMPRSS2 were not identified in purified olfactory neurons ACE2 was identified in glial cells olfactory stem cells Nasal epithelial cells have lower levels of ACE2 TMPRSS2 compared with epithelial cells of the lower respiratory tract ACE2 has nonneuronal expression in olfactory epithelium The expression of ACE2 TMPRSS2 mouse were increased in elderly mouse unique expression quantitative trait loci variants were found for ACE2 The genotypes of ACE2 gene polymorphism may be characterized by higher expression levels of ACE2 in East Asian population There would be different susceptibility or response to SARSCoV2 in different populations ACE2 is expressed in human granular cells of salivary glands There was no ACE2 expression in nasopharyngeal cells ACE2 is expressed in ciliated epithelial cells cilia anelle 0c Table continuedAuthorsDesign [] StudyHumanLiHikmetHuman [] Study [] StudySungnak Human [] StudySamples MethodsIHCACE2 TMPRSS2 expressionACE2 humanHuman ACE2Human ACE2IHCRNAseqThyroidNasopharyngeal EpitheliumACE2 human no expression in nasopharynxACE2 humanHuman ACE2 TMPRSS2 Airway Nasal epitheliumRNAseqACE2 humanTMPRSS2 human subset of ACE2 cellsXuHumanHuman ACE2 TMPRSS2 Oral Epithelium [] StudyRNAseqHumanXu [] StudyWuHuman [] StudyHuman ACE2RNAseqHuman ACE2RNAseqACE2 humanACE2 humanOral T cells B cells fibroblastsACE2 humanMinor salivary glandsACE2 humanNasal Oral EpitheliumHead and Neck PathologyFindings2There was no ACE2 expression in the nonciliated goblet cells ACE2 expression is influenced by patient demographics clinical characteristics comorbidities or medication use The use of ACE inhibitor drugs did not increase ACE2 protein expression ACE2 is expressed by thyroid cells There was no ACE2 expression in nasopharyngeal cells ACE2 was expressed in airway epithelial cells ACE2 is more expressed in nasal epithelial cells compared with other respiratory cells goblet ciliated cells TMPRSS2 is only expressed in a subset of ACE2 cells ACE2 is expressed in the oral cavity epithelial cells ACE2 expression was higher in tongue than buccal and gingival tissues ACE2 is expressed in minor salivary glands ACE is expressed in nasal epithelial cells The was a higher virus concentration in the nasalswab comparing with throatswab which is attributed to ACE2expression in nasal epithelial cells ACE2 TMPRSS2 are coexpressed in nasal goblet secretory cellsMixedZiegler [] StudyACE2 Angiotensin Converting Enzyme2 IHC Immunohistochemistry RTPCR reverse transcription polymerase chain reaction SARSCoV2 severe acute respiratory syndrome a0coronavirus2 TMPRSS2 transmembrane protease serine2 WB Western BlottingHuman ACE2 TMPRSS2 Sinusal Nasal goblet epithelial cellsMonkey RNAseqACE2 TMPRSS2 Humanmucosa of the olfactory region including olfactory bulb [ ] The ACE2 receptor was identified in sustentacularnonneuronal cells of the olfactory tissues Moreover ACE2 was found in a low proportion of neuronal stem cells in the olfactory bulb [ ] The expression of ACE2 in olfactory neurons nonstem cells remains uncertain because Butowt et a0al and Brann et a0al observed that ACE2 has only nonneuronal expression pattern in olfactory epithelium [ ]Five studies investigated ACE2 expression in oral and pharyngeal regions including oral and hypo oro and nasopharyngeal spaces [ ] The study that explored ACE2 expression in human nasopharynx [] did not exhibit significant ACE2 immunostaining in nasopharyngeal cells [] ACE2 receptor was identified in oral endothelial [] epithelial [ ] and salivary [] cells Xu et a0al found that ACE2 was also expressed in T and B cells as well as fibroblasts of the oral cavity [] Moreover ACE2 was expressed in major salivary gland tissues [] and thyroid tissue [] In many publications authors reported the type of cells goblet versus epithelial versus stem cells that expressed ACE2 or TMPRSS2 Table a0 Interestingly Xu et a0al almost as much ACE2 expression in the thyroid as in the lungs []The genetic analysis of Cao et a0al reported that there are unique expression quantitative trait loci variants in the East Asian population supporting a gene polymorphism and tissuerelated differences between individuals [] 0cHead and Neck Pathology Fig Flow chart ACE2 Angiotensin Converting Enzyme2 TMPRSS2 transmembrane protease serine2TMPRSS2 ExpressionTMPRSS2 expression was investigated in studies [ ] Similarly to ACE2 TMPRSS2 was identified in nasal [ ] and respiratory mucosa cells [] including both epithelial and goblet cells with higher expression in lower airway compared with upper airway [] Moreover TMPRSS2 receptor was identified in sustentacular and neuronal olfactory cells [ ] but not in olfactory neurons [] TMPRSS2 was also identified in salivary major gland tissue []ACE2 TMPRSS2 Tissue Expression in a0Mouse and a0MonkeySix studies used animal models to assess ACE2 or TMPRSS2 expressions in head and neck tissues [ ] The mouse studies of Butowt et a0al and Bilinska et a0al revealed that elderly mice had higher expression of both ACE2 and TMPRSS2 in nasal mucosa compared with younger mice [ ] In olfactory tissue ACE2 was identified in sustentacularnonneuronal and neural stem cells of mice [] Liu et a0al analyzed ACE2 expression in monkeys [] 0c Head and Neck PathologyTable Summary of Cell Expression of ACE2 and TMPRSS2AuthorsBilinska []Brann []SamplesMouseHuman MouseTissueOlfactoryOlfactoryButowt []Human MouseNasalOlfactoryChen []Hamming []Hikmet []Jia []Lee []Li []Liu []Sungnak []Vaarala []Xu []Xu []Wu []Ziegler []HumanHumanHumanHumanHumanHumanMonkeyHumanMajor Salivary GlandOral Nasal NasopharyngealNasopharyngealTracheal Nasal SinusalThyroidPharyngealTrachealTracheal NasalHuman MouseHumanMajor Salivary GlandOralHumanHumanHuman Mouse MonkeyMinor Salivary GlandNasalOralNasal SinusalCell typesSustentatorialSustentatorialNeuronalStem NeuronalEpithelialSustentatorialNeuronalGranularBasal layerEndothelialEpithelialApical EpithelialEndothelialApical EpithelialGobletUnspecifiedMinor salivary ductalBasal layerGobletApical EpithelialUnspecifiedApical EpithelialFibroblastT and BcellsUnspecifiedUnspecifiedBasal layerGobletACE2NATMPRSS2NANANANANANANANANANANANANANANANANAACE2 Angiotensin Converting Enzyme2 NA not available TMPRSS2 transmembrane protease serine2reporting a higher ACE2 expression in tracheal naso oro and hypopharyngeal tissues as well as in the salivary ducts of the pharyngeal gland and consequently in saliva In this study the cell expression was mainly localized in the lamina propria In the same vein Vaarala et a0al reported TMPRSS2 expression in mouse salivary tissues []Cell Detection MethodsThe following methods have been used for detecting ACE2 and TMPRSS2 in cells of human and animal tissue RNAseq N IHC N RTPCR N in a0situ hybridization ISH N and WB N Different detection approaches were used in studies [ ] One study reported specific genetic analysis [] There were significant differences between studies regarding methods used While Ziegler et a0al and Sungnak et a0al detected ACE2 by RNAseq in goblet cells Lee et a0al did not find any immunohistochemical labeling [ ] However the results reported in sustentacular cells agree in the same direction whatever the technique used whether by RNAseq or by ISH and immunocytochemistry [] The discrepancies are rather observed between studies having performed immunohistochemistry Indeed using two different antibodies Hikmet did not find ACE2 expression in nasopharynx epithelium whereas others demonstrated the staining of the apical surface of epithelia and ciliated epithelial cells [ ] Interestingly all the studies which carried out RNAseq found an expression of ACE2 or TMPRSS2 at the epithelial level which implies that the technique used could generate biases between the studies [ ] 0cHead and Neck Pathology DiscussionThe presentation of COVID19 infection may be in several clinical forms ranging from anosmia in isolation to severe multiple an failure and death The mechanisms underlying the COVID19 polymorphism are still unknown To infect tissues SARSCoV2 needs to entry into the cells which is allowed through ACE2 and TMPRSS2 receptors [] The identification of virus receptor expression in the tissues makes particularly sense to better understand the clinical expression of the disease This systematic review sheds light on many pointsFirst ACE2 and TMPRSS2 receptors are expressed in epithelial and nonepithelial cells throughout the head and neck The head and neck expression may support the otolaryngological clinical picture of the disease which was recently found in European and North American COVID patients [ ] By entering the body via the epithelial cells of the upper aerodigestive tract mucosa the SARSCoV2 virus leads to an inflammatory reaction and the development of otolaryngological symptoms Nasal entrance of the virus through high ACE2 expression was supported in the study of Wu et a0al who found a higher virus concentration in nasal swabs compared with throat swabs []The olfactory cleft is a nasal region that has drawn the attention of many researchers over the past few weeks Indeed recent data supported that more than of COVID19 patients developed subjective olfactory dysfunction especially when patients suffered from mildtomoderate forms of the disease [ ] Because ACE2 and TMPRSS2 are both expressed in the nasal mucosa of the olfactory cleft entrance into the olfactory bulb seems plausible Once in the bulb according to some human studies [ ] the virus could infect cells that express ACE2 or TMPRSS2 namely glial and neuronal stem cells Fig a0Integrating the molecular clinical and radiological characteristics of SARSCOV2 olfactory loss may shed light about its pathophysiological process Taking into account that the loss is often temporary SARSCOV2 may primarily infect the sustentacular cells supporting the olfactory sensory neurons This infection may cause rapid disruption of the olfactory epithelium structure and function with a possible inflammatory response inducing sudden onset smell loss This inflammation is observed in a minority of patient with congested olfactory cleft who underwent CT scan [ ]ACE2 and TMPRSS2 are also found in horizontal basal olfactory stem cells located in the basal layer They are less exposed to the external environment thus less likely to be infected in first line the loss would have been more Fig Epithelium of Olfactory Cleft The figure summarizes the olfactory cleft epithelium 0c Head and Neck Pathologyprogressive However once infected they might slow down recovery time because horizontal basal cells give rise to many cell type in the olfactory epithelium They may also contribute to virus spread to the olfactory bulb vascular pericytes Magnetic resonance imaging MRI studies of the olfactory bulb [ ] in which ACE2 are only expressed in vascular pericytes but not in neurons may show inflammatory signs suggesting that the infection process can extend more centrally and promote inflammatory response [] Inflammatory causes are often quickly reversible for example after a oneweek trial of high dose of corticosteroids or simply days after the resolution of the viral infection suggesting that the olfactory neurons and bulbs are still somewhat intact This seems to be the case for a majority of patients In contrast with more sustained destruction of neuronal olfactory structures the recovery time is much longer and may take a0years given the slow neuroregeneration process [ ]In this systematic review we found that three studies reported high ACE2 expression in major or minor salivary gland human tissues [ ] These data corroborate the literature findings that reported a salivary pattern of SARSCoV2 and related parotitis [ ] Moreover the virus spread into the salivary gland tissues allows us a better understanding the mechanisms underlying salivary transmission Interestingly in Liu et a0al observed that monkeys infected by SARSCoV had a salivary viral spread which was associated with a salivary virion excretion [] These data support the need to conduct future studies investigating the presence of SARSCoV2 in the saliva of infected human and to corroborate the saliva findings with the ACE2 salivary gland expressionThe head and neck expression of ACE2 and TMPRSS2 and the related otolaryngological symptom pattern seems obvious but could vary across individuals and populations In support of this Lee et a0al observed that ACE2 expression is influenced by patient demographics clinical characteristics comorbidities and medication [] As reported in the genetic analysis of Cao et a0al there would be different susceptibilities or responses to SARSCoV2 in different populations [] The polymorphism of ACE2 and TMPRSS2 expression could explain the clinical differences between individuals Indeed many physicians reported in clinical and epidemiological studies different clinical presentation of the disease [ ] which could be associated with virus mutations [ ] The virus mutations and the related impact on receptor binding and infectivity is another point that has to be considered in future studies Otherwise according to the Bgee database https bgee expression of ACE2 and TMPRSS2 evaluated in murine models may increase with age These findings have to be confirmed in humans but could explain more severe clinical pictures in the elderlyThe present study has several limitations First the heterogeneity between studies about the detection method may lead to detection bias as some approaches are more sensitive than others Some studies interrogate gene expression at mRNA level and others at protein levels both types of analysis having their advantages and limitations Compared to transcriptomic analyzes immunohistochemistry brings additional important spatial information in tissue samples but recently Sorokin et a0al demonstrated high and statistically significant correlations between the RNA sequencing and immunohistochemical measurements [] Interestingly they highlighted the complementarity of both techniques for measuring cancer biomarkers in FFPE samples However differences observed across IHC studies suggest the involvement of many parameters The antibody specificity is a big challenge to ensure reproducibility of antibodybased studies and given the high homology between ACE1 and ACE2 cautions must be taken regarding antibody selection Besides a report from the International Working Group for Antibody Validation IWGAV proposed five scientific approaches to validate antibody specificity [] then such strategies must be considered in future investigations to confirm the published observations In addition it seems essential to enlarge and diversify patient cohorts and to combine transcriptomic and proteomic strategies as well as colocalize different markers of SARSCoV2 such as ACE2 and TMPRSS2 to provide an accurate representation of ACE2 expression through all head and neck areas of the whole populationSecond the majority of studies that were conducted during the SARSCoV2 pandemic did not consider many demographic and clinical factors such as the age of patients from who the tissues were extracted or the use of ACE inhibitor medications among others Third some otolaryngological regions remain uninvestigated such as the vocal folds The investigation of these remaining regions may shed further light on some recently reported unusual clinical phenomena such as severe dysphonia []ConclusionACE2 and TMPRSS2 are both expressed in head and neck tissues which may explain the otolaryngological clinical pattern of the disease and the entry of SARSCoV2 into the host anism Future studies considering demographical and clinical characteristics of patients from who the tissues are extracted are needed to better understand the cell entry mechanisms of SARSCoV2Author contributions JR TR SS JM design acquisition of data data analysis interpretation drafting final approval and accountability 0cHead and Neck Pathology for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved CCH CMCE MRB IG design data analysis interpretation revising the manuscript for important intellectual content final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved LS SH GC GD JH LV GR design acquisition of data data analysis interpretation drafting some parts of the manuscript final approval of the version to be published final approval and accountability for the work final approval of the version to be published agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolvedCompliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interestReferences Crackower MA Sarao R Oudit GY et a0al Angiotensinconverting enzyme is an essential regulator of heart function Nature Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Somasundaran M Sullivan JL Luzuriaga K Greenough TC Choe H Farzan M Angiotensinconverting enzyme is a functional receptor for the SARS coronavirus Nature Wang Z Xu X scRNAseq profiling of human testes reveals the presence of the 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" COVID19 pathways for brain andheart injury in comorbidity patients A role of medical imaging and artificial intelligencebased COVIDseverity classification A review Computers in Biology and Medicine 101016jcompbiomed2020103960 0cThis is a PDF file of an that has undergone enhancements after acceptance such as the additionof a cover page and metadata and formatting for readability but it is not yet the definitive version ofrecord This version will undergo additional copyediting typesetting and review before it is publishedin its final form but we are providing this version to give early visibility of the Please note thatduring the production process errors may be discovered which could affect the content and all legaldisclaimers that apply to the journal pertain Published by Elsevier Ltd 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS1 Anudeep Puvvula MBBS FCD12 Mainak Biswas PhD3 Misha Majhail14 Luca Saba MD5 Gavino Faa MD6 Inder M Singh MD7 Ronald Oberleitner BS MBA8Monika Turk MD PhD9 Paramjit S Chadha BE1 Amer M Johri MD FASE10 J Miguel Sanches PhD11 Narendra N Khanna MD DM FACC12 Klaudija Viskovic MD PhD13 Sophie Mavrogeni MD PhD FESC14 John R Laird MD FACC FACP15 Gyan Pareek MD16 Martin Miner MD17 David W Sobel MD16 Antonella Balestrieri MD5 Petros P Sfikakis MD18 Gee Tsoulfas MD19 Athanasios Protogerou MD PhD20 Durga Prasanna Misra MD FRCP21 Vikas Agarwal MD FRCP21 Gee D Kitas MD PhD FRCP22 Puneet Ahluwalia MS MCh24 Raghu Kolluri MD25 Jagjit Teji MD26 Mustafa AlMaini MD27 Ann Agbakoba MD28 Surinder K Dhanjil MSc FSVU29 Meyypan Sockalingam MBBS FRCR30 Ajit Saxena MD12 Andrew Nicolaides MS PhD FRCS31 Aditya Sharma MD32 Vijay Rathore MD33 Janet N A Ajuluchukwu MD34 Mostafa Fatemi PhD FAIMBE FIEEE FASA FAIUM35 Azra Alizad MD FAIMBE FAIUM36 Vijay Viswanathan MD PhD37 P K Krishnan MD38 Subbaram Naidu PhD Life FIEEE39 1Stroke Monitoring and Diagnostic Division AtheroPoint¢ Roseville CA USA 2Annus Hospitals for Skin and Diabetes Nellore AP INDIA 3JIS University Kolkata West Bengal INDIA 4Oakmont High School and AtheroPoint¢ Roseville CA USA 5Department of Radiology Azienda Ospedaliero Universitaria Cagliari ITALY 6Department of Pathology AOU of Cagliari Italy 7Stroke Monitoring and Diagnostic Division AtheroPoint¢ Roseville CA USA 8Behavior Imaging USA 9The HanseWissenschaftskolleg Institute for Advanced Study Delmenhorst GERMANY 10Department of Medicine Division of CardiologyQueens University Kingston Ontario CANADA 11Institute of Systems and Robotics Instituto Superior Tecnico Lisboa PORTUGAL 12Department of Cardiology Indraprastha APOLLO Hospitals New Delhi INDIA 13University Hospital for Infectious Diseases Zagreb CROTIA 14Cardiology Clinic Onassis Cardiac Surgery Center Athens GREECE 15Heart and Vascular Institute Adventist Health St Helena St Helena CA USA 16Minimally Invasive Urology Institute Brown University Providence Rhode Island USA 17Mens Health Center Miriam Hospital Providence Rhode Island USA 18Rheumatology Unit National Kapodistrian University of Athens GREECE 19Aristoteleion University of Thessaloniki Thessaloniki GREECE 20National Kapodistrian University of Athens GREECE 21Sanjay Gandhi Postgraduate Institute of Medical Sciences Lucknow UP INDIA 22Academic Affairs Dudley Group NHS Foundation Trust Dudley UK 23Arthritis Research UK Epidemiology Unit Manchester University Manchester UK 24Max Institute of Cancer Care Max Superspeciality Hospital New Delhi INDIA 25OhioHealth Heart and Vascular Ohio USA 26Ann and Robert H Lurie Childrens Hospital of Chicago Chicago USA 27Allergy Clinical Immunology and Rheumatology Institute Toronto CANADA 28University of Lagos NIGERIA 29AtheroPoint LLC CA USA 30MV Center of Diabetes Chennai INDIA 31Vascular Screening and Diagnostic Centre and University of Nicosia Medical School CYPRUS Journal Preproof 0c32Division of Cardiovascular Medicine University of Virginia Charlottesville VA USA 33Nephrology Department Kaiser Permanente Sacramento CA USA 34Department of Medicine Lagos University Teaching Hospital Lagos NIGERIA 35Dept of Physiology Biomedical Engg Mayo Clinic College of Medicine and Science MN USA 36Dept of Radiology Mayo Clinic College of Medicine and Science MN USA 37MV Hospital for Diabetes and Professor M Viswanathan Diabetes Research Centre Chennai INDIA 38Neurology Department Fortis Hospital Bangalore INDIA 39Electrical Engineering Department University of Minnesota Duluth MN USA Corresponding Author Dr Jasjit S Suri PhD MBA FAIMBE FAIUM FAPVS Fellow American Institute of Medical and Biological Engineering Fellow American Institute of Ultrasound in Medicine Fellow Asia Pacific Vascular Society Stroke Monitoring and Diagnosis Division AtheroPoint¢ Roseville Roseville CA USA Phone Email jasjitsuriatheropointcom Journal Preproof 0cCOVID19 Pathways for Brain and Heart Injury in Comorbidity Patients A role of Medical Imaging and Artificial Intelligencebased COVID severity Classification A Review Abstract Artificial intelligence AI has penetrated the field of medicine particularly the field of radiology Since its emergence the highly virulent coronavirus disease COVID19 has infected over million people leading to over deaths as of July 1st Since the outbreak began almost s about COVID19 have been published pubmedncbinlmnihgov however few have explored the role of imaging and artificial intelligence in COVID19 patientsspecifically those with comorbidities This paper begins by presenting the four pathways that can lead to heart and brain injuries following a COVID19 infection Our survey also offers insights into the role that imaging can play in the treatment of comorbid patients based on probabilities derived from COVID19 symptom statistics Such symptoms include myocardial injury hypoxia plaque rupture arrhythmias venous thromboembolism coronary thrombosis encephalitis ischemia inflammation and lung injury At its core this study considers the role of imagebased AI which can be used to characterize the tissues of a COVID19 patient and classify the severity of their infection Imagebased AI is more important than ever as the pandemic surges and countries worldwide grapple with limited medical resources for detection and diagnosis We conclude that imaging and AIbased tissue characterization when considered alongside COVID19 symptoms and their pretest probabilities offer a compelling solution for assessing the risk of comorbid patients These methods show the potential to become an integral part of tracking and improving the healthcare system both during the pandemic and beyond Keywords COVID19 comorbidity pathophysiology heart brain lung imaging artificial intelligence risk assessment Journal Preproof 0c Introduction In December a novel coronavirus referred to as severe acute respiratory distress syndrome coronavirus SARSCoV2 [] appeared in Wuhan the capital of Hubei Province in PR China The disease caused by the virus was initially named novel coronavirus pneumonia NCP by the Chinese government but was subsequently renamed coronavirus disease COVID19 by the World Health anization WHO On January 30th it was declared a public health emergency of international concern PHEIC [] It is believed that SARSCoV2 is primarily transmitted through saliva droplets or nasal discharge [] The first evidence of humantohuman transmission was found by Jasper FukWoo Chan et al in their study at The University of Hong KongShenzhen Hospital [] Due to its contagiousness Ro27 the virus has reached epidemic levels affecting countries and causing over million infections and more than deaths as of July 1st [] shown in Figure Recent literature suggests that patients with preexisting diseases are likely to experience severe complications from COVID19 [] In one study on admitted diabetic and nondiabetic COVID19 patients the mortality rate vs and the rate of admission to the intensive care unit ICU vs were significantly higher for diabetic patients Diabetic patients also experienced severe inflammatory responses and cardiac hepatic and renal coagulopathy [] The prevalence of heart and brain injuries was also higher in COVID19 patients with concomitant chronic conditions like diabetes kidney disease dyslipidemia hypertension [] chronic obstructive pulmonary disease COPD and cardiovascular diseases [] Recent studies have shown that SARSCoV2 invades the thin lining of the epithelial cells of the arteries leading to atherosclerosis [] and arterial inflammatory diseaseone of the major causes of cardiovascular diseases CVDs which also causes heart and brain injuries [ ] This could be due to a reduced expression of angiotensinconverting enzyme ACE2 causing endothelial dysfunction which in turn aggravates existing Journal Preproof 0catherosclerosis [ ] It has also been observed that comorbid patients when subjected to imagescreening show mild to severe pretest probability PTP for COVID19 [] The conventional cardiovascular risk factors CCVRF in these comorbid patients appear strongly correlated either to their heart imaging or to surrogate biomarkers of coronary artery disease such as carotid artery disease Both imaging and biomarkers could be helpful in severity predictions for COVID19 [] Figure illustrates the associations between SARSCoV2 and other comorbidities such as diabetes as well as the comparative survival rates for COVID19 patients with and without diabetes Figure World map showing COVID19 spread over countries courtesy John Hopkins University ACE2 expression causes scars in the vessels and can even rupture the walls of the arteries [] For this reason CCVRF should be considered alongside imaging in patients who present with COVID19 and many comorbidities [] The second stage is the one at which a patient is most severely affected by Journal Preproof 0cCOVID19 and has the highest probability of cardiac injury or release of troponin T TnT Imaging has been shown to offer benefits in monitoring the tissue scars caused by COVID19 [] Figure a Association of SARSCoV2 with other comorbidities and b comparison of the mortality rate of diabetic and nondiabetic COVID19 patients reproduced with permission [] Multiple modalities can be utilized to determine whether a patient has the sequelae of COVID19 including magnetic resonance imaging [] computed tomography [] and ultrasound [] The advantage of these imaging modalities is the visual access they provide to the scar tissue caused by the disease A disadvantage however is their inability to provide a risk assessment The application of artificial intelligence AI can enhance the information provided by these imaging modalities resulting in a more accurate characterization of the tissue and the disease process [] In fact the combination of AI and medical imaging has been shown to improve diagnosis and risk stratification speed up patient evaluation enhance disease monitoring and accelerate early intervention [ ] Thus this review will focus on the use of AIbased tissue characterization of images of comorbid patients affected by COVID19 The layout of this paper is as follows Section presents the pathophysiology of the four pathways leading to brain and heart injury Section summarizes the evidence related to the use of Journal Preproof 0cimaging during the COVID19 pandemic Section elaborates on the use of AIbased tissue characterization for risk assessment Finally the paper concludes with a critical discussion The Pathophysiology of SARCoV2 Leading to Brain and Heart Injury Several studies suggest that SARSCoV2 uses the ACE2 receptor to gain access to cells by binding to the SPIKE protein S protein on their surface [] see Figure ACE2 and angiotensinconverting enzyme ACE1 are homolog carboxypeptidase enzymes that have different vital functions in the reninangiotensinaldosterone system RAAS pathway [] ACE2 is widely expressed in myocardial cells [] type pneumocytes enterocytes and astrocytes in the brain [ ] Thus it is recognized as a cause of extrapulmonary complications Figure shows the overall picture of how SARSCoV2 causes brain and heart injuries via four different pathways These include i the neuronal pathway ii the hypoxia pathway iii the RAAS pathway and iv the immune pathway We will discuss these pathways and the injuries they lead to which may manifest as viral encephalitis infectious toxic encephalopathy or acute cerebrovascular disease i The Neuronal Pathway Figure the pathway I Recent epidemiological studies have demonstrated similarities at the genomic level between SARSCoV1 MERS and SARSCoV2 [ ] Meanwhile previous experimental studies have shown that beta coronaviruses in generalsuch as SARSCoV1 and MERScan spread into and directly infect the brain when inhaled as droplets via the nasal epithelium [ ] Figure depicts the olfactory nerve and the olfactory bulb []labeled as a and b respectivelyon the image of the sagittal brain in the neuronal pathway Based on recent reports we are aware that patients infected by SARSCoV2 show symptoms of dysgeusia loss of taste and anosmia loss of smell [ ] Bohmwald et al further validate that coronaviruses that infect through the olfactory nerve and bulb can reach the brain and cerebrovascular fluid CSF within seven days Additionally these viruses have been observed to cause inflammation and demyelination [] The Journal Preproof 0cauthors demonstrated in an experimental study of mice that removing the olfactory bulb from the pathway can lead to the restriction of CoV in the central nervous system CNS [] Based on this evidence we believe that the neuronal pathway is one possible track for SARSCoV2 ii The Hypoxia Pathway Figure pathway II In this pathway decreased levels of ACE2 proliferate in the lung parenchyma cells after the coronavirus has passed through causing exaggerated neutrophils accumulation enhanced vascular permeability and the formation of diffuse alveolar and interstitial exudates This ultimately leads to pulmonary edema and acute respiratory distress syndrome ARDS [] ARDS is characterized by severe abnormalities in blood gas composition resulting from an oxygen and carbon dioxide mismatch which leads to low blood oxygen levels [ ] This ongoing hypoxia can lead to myocardial ischemia and heart injury [ ] see Figure pathway IIA Hypoxia in the brain increases anaerobic metabolism in the mitochondria of the brain cells [] leading to cerebral vasodilatation edema and impaired flow This can result in cerebral ischemia and acute cerebrovascular diseases such as acute ischemic stroke [ ] see Figure pathway IIB iii The RAAS Pathway after SARSCoV2 Figure pathway III The RAAS pathway is crucial in regulating blood pressure as well as the balance of fluid and electrolytes Any disturbance in this pathway can trigger the pathogenesis of cardiovascular diseases [] Before a SARSCoV2 infection triggers the RAAS Angiotensin I Ang I cleaves to Angiotensin II Ang II via ACE1 Ang II causes vasospasm It is also a proinflammatory agent with prothrombotic and proliferative effects that are detrimental to vascular tone and hemostasis [ ] Thus as a counterregulatory mechanism ACE2 degrades Ang II and generates Ang which counteracts the negative impacts of Ang II [ ] Both ACE2 and Ang have cardiocerebral vascular protective effects [] After the triggering of SARSCoV2 infection it results are in the deregulation of RAAS causing heart and brain injury in two different pathways The main culprit is an increase in Ang II which is caused by the decrease in ACE2 levels Figure pathway IIIA First an increase in Ang II levels stimulates the adrenal cortex of the kidney resulting in an increased production of aldosterone Aldosterone is a steroid hormone that causes sodium and water Journal Preproof 0creabsorption to increase at the distal tubule and collecting duct of the nephron [] This reabsorption increases blood volume and causes an elevation in blood pressure which results in the endothelial dysfunction that causes brain and heart injury [] The second effect of an increase in Ang II levels ie as a consequence of decreased ACE2 levels is endothelial dysfunction leading to intimal damage in the arterial walls [] which can be seen during the imaging of the arterial wall see Figure pathway IIIB This pathway can also trigger a cytokine storm as high levels of Ang II can cause an increase in proinflammatory cytokines see the bridge line between the RAAS and immune pathways iv The Immune Pathway Figure pathway IV Several recent studies have reported SARSCov2 viral pneumonia [ ] having an exaggerated inflammatory response known as a cytokine storm This response appears to present at advanced stages of severe COVID19 with increased levels of inflammatory cytokines leading to multiplean failure [] The rise in inflammatory markersincluding IL6 IL7 IL12 IL15 IL22 TNFα and CXCL10results in the destabilization of plaque This in turn can cause plaque rupture resulting in heart and brain injury [ ] The Role of Imaging in Comorbid Patients with COVID19 As the previous section discussed COVID19 uses four pathways ie neuronal hypoxia RAAS and immune to cause critical heart and brain injuries in patients with comorbidities The prevalence of myocardial injury and brain injury caused by COVID19 [ ] points to a need for increased use of medical imaging to expedite assessments differential diagnoses and patient management [ ] with proper safety measures [] The seriousness of a patients COVID19 symptoms helps to determine which imaging modality is appropriate portable or nonportable and invasive or noninvasive BMode ultrasound imaging is portable and can be used for lowrisk patients Meanwhile Xray magnetic resonance imaging [] and computed tomography [] are nonportable and can be used for mediumrisk patients Intravascular ultrasound IVUS [] and ventriculography are invasive imaging modalities used in highly critical cases [ ] Amongst all the imaging modalities ultrasound is noteworthy because it is radiationfree portable quick repeatable inexpensive Journal Preproof 0cand can be performed in isolation thus lowering the chance of spreading the COVID19 infection [ ] There are several examples of medical imaging that have led to proper treatment and healthcare management during the pandemic ultimately reducing the mortality rate Xray imaging of the chest has demonstrated irregular patchy hazy reticular and widespread groundglass opacities indicating the progression of COVID19 at various stages this information can support the healthcare team in developing the most appropriate treatment plan [] Chest CT scans of COVID19 patients revealed in almost of the patients that the disease was affecting at least one of the five lobes of their lungs [] Chest MRI scans of COVID19 patients showed pulmonary tissue consolidation in six diffusionrestricted regions in six and lung damage in seven [] Meanwhile heart MRI studies of recovered patients showed that of the patients had myocardial edema At the same time late gadolinium enhancement was found in implying that COVID19related cardiac injury is longstanding and requires frequent monitoring even after recovery [] In a different study MR scans of a COVID19 patient revealed myocardial inflammation signifying myocardial injury due to a cytokine storm related to the SARSCoV2 infection as discussed in Section Pathway IV [] Several studies have also evaluated the effects of COVID19 on the brain In one MRI scans revealed hemorrhagic rim enhancing lesions within the bilateral thalami medial temporal lobes and subinsular regions [] shown in Figure In another brain MRI scans were completed for patients of which produced abnormal findings [] Additionally evidence of liver injury and gall bladder abnormality was found in a joint CT and ultrasound study of the abdomen [] Recent MRI scans of COVID19 patients olfactory bulbs have revealed the cause of olfactory function loss to be the interaction between SARSCoV2 and the ACE2 protein expressed by the olfactory epithelium which leads to inflammatory obstruction [] Journal Preproof 0cFigure We have shown in four pathways how COVID19 can cause Brain and heart injury Brain image in pathway I httpdebugliescom20200123olfactorydisturbanceshaveimplicationsinmentalandemotionalwellbeinghealth Courtesy of Debug Lies Invasive imaging is another option for diagnosing COVID19 patients who have critical comorbidities In one such study IVUS along with stenting was performed with precautions on a COVID19 patient with myocardial infarction [] shown in Figure A detailed discussion of precautions is included in section In another study takotsubo syndrome a form of myocardial injury triggered by COVID19 was detected using ventriculography [] In various studies the medical imaging of COVID19 patients had been crucial to ascertaining the extent of tissue damage and critical infection although there were no visible symptoms [ ] Therefore medical imaging is the preferred way to ascertain the extent of cardiac and brain tissue damage throughout the lifetime of COVID19 patients COVID19 Journal Preproof 0cpatients with comorbidities are especially vulnerable and so they need to be screened through medical imaging from the first day of their diagnosis Medical imaging can help patients with deep vein thrombosis DVT as they are highly susceptible to severe tissue damage from COVID19 A study showed that COVID19 patients suffering from DVT had a worse prognosis than patients without DVT Patients with DVT were admitted to the ICU more frequently discharged less frequently and suffered more deaths than those without DVT [] Figure MRI scan of COVID19 patient showing hemorrhage MRI images demonstrate T2 FLAIR hyperintensity within the bilateral medial temporal lobes and thalami A B E F with evidence of hemorrhage indicated by hypointense signal intensity on susceptibilityweighted images C G and rim enhancement on postcontrast images D H reproduced with permission [] Journal Preproof 0c Figure Application of chest CT and IVUS for a COVID19 patient suffering from myocardial infarction a Chest CT scan with viral pneumonia showing fibrinous focal exudative changes b when the patient complained of chest pain the ECG report showed the STsegments elevations in V1V5 lead c d CAG radiology that the proximal segment of LAD was occluded e f The blood flow of LAD restoration after DESs was implanted g the dissection distal shown by IVUS to the stent in LAD from o'clock h the low echogenic shadow with scattered higher echogenic flicker indicating a thrombus i after a DES was implanted and the stent was well expanded the dissection could not be seen j The thrombus disappearance after the intervention reproduced with permission [] Journal Preproof 0c Although medical imaging can be very useful to patients and doctors alike the exponential pandemic curve inadequate medical facilities [] and a limited number of radiologists make the assessment diagnosis and management processes challenging tedious and errorprone Therefore although medical imaging can make diagnoses faster as stated above it will be of limited use Given this fact new age techniques such as artificial intelligence AI [] applications in medical imaging for tissue characterization can make computeraided assessments and diagnoses faster The main reason for this is that the AI can be scaled up to match the pandemic curve thereby meeting the immediate demands of medical image diagnoses during the COVID19 pandemic AIbased tests can categorize the nature of a patients risk in one of the categories namely norisk low lowmedium LM highmedium HM lowhigh LH or highhigh HH risk depending on the patients symptoms and their severity [ ] as shown in Figure The imaging modality also varies with the degree of risk as follows no imaging for norisk portable imaging for low and LM risk nonportable imaging for HM and LH and invasive imaging for HH A probability PTP is performed to accurately interpret diagnostic results to categorize the patient into one of the four groups [] After that for norisk patients nonimaging biomarkers can be collected for risk assessment using AIbased data protocols For lowrisk patients portable 2D3D imaging such as ultrasound is used whereas nonportable and invasive 2D3D imaging such as MRICTXRayechocardiography can be used for LM patients For HH patients invasive imaging techniques such as IVUS and ventriculography can be used Based on the data provided by various 2D3D scans AIbased medical imaging is applied for risk assessment Further treatment is then planned based on this imaging process In the next section deep learning DLbased medical imaging is proposed for medical imaging scans particularly ultrasounds for COVID19 patients Machine Learning and Deep Learning for Tissue Characterization Using AI and associated technologies in healthcare can significantly slow down diagnosis times Journal Preproof 0cespecially during the COVID19 pandemic as patient numbers are continually growing and there are few specialists available [] Figure Role of AIbased risk assessment on COVID19 patients having comorbidity Journal Preproof 0cAlthough some caution must be exercised regarding its fullscale deployment [] its overall usefulness in healthcare management during times of crisis cannot be ignored [ ] In general AI in healthcare refers to all artificial intelligencebased technologies that make educated decisions regarding a patients diagnosis monitoring treatment and management The importance of AI has specifically increased many folds when imaging comes into play mainly because of large volumetric data sizes and the extensive need to characterize and quantify the disease via lesion images [] Tissue imaging and its characterization is of prime importance since it has a direct influence on decisions related to COVID19 severity for a patient [] The main benefit of AI methods is that the machines can be used to train by mimicking the physicians cognitive actions and such trained models can be used to predict the diseases severity in asymptomatic patients Within a short period several machine learning MLbased techniques used the power of AI to manage COVID19 [ ] ML and DL Architectures ML Architecture ML is a twostage process In stage I different features are extracted from the lesion COVID images the extractions are then operated on by an ML statistical model called a training system to generate offline coefficients These coefficients are then transformed by the test lesion images which yield an intelligent classification or inference A typical ML system for predicting risk class is shown in Figure CUSIP is an imagebased phenotype that uses the event equivalent gold standard EEGS [ ] model DL Architecture DL refers to a visual cortex that imitates multiple layers of a neural network applied directly to tissue images to extract features and for characterization purposes [] The convolution neural network CNN [] shown in Figure is one such DL network architecture that is widely used to characterize medical images It performs a series of convolution maxpooling operations to extract features and perform characterizations ML and DL both follow the supervised learning Journal Preproof 0capproach by which models are trained using offline data Figure Typical lowcost machine learning architecture utilizing EEGS model As discussed in previous sections there are four pathways through which a COVID19 infection leads to heart and brain injuries AI can be used via medical imaging to detect the extent of tissue damage in these pathways and help medical professionals to develop an effective treatment plan for patients There are several instances in which the AI paradigm has been used for tissue characterization based on Journal Preproof 0cmedical images during the pandemic as well as during standard times Some uses of AI are described anwise following a proposed model for characterizing DLbased tissues Figure A convolution neural network courtesy of AtheroPoint¢ CA USA ML Architecture used for Tissue Characterization for Stroke Risk Stratification There are two types of tissue characterization that can be carried out using AI i MLbased [ ] and ii DLbased [] Various MLbased technologies have been developed to classify symptomatic and asymptomatic plaque from ultrasound images For example an MLbased technique based on support vector machines SVM was developed to characterize the symptomatic and asymptomatic plaques of carotid scans that indicated the presence of plaque [ ] SVM classifiers work by determining the maximum margin between two data clusters First a texture analysis [] is used to extract the features ie standard deviation entropy symmetry and run percentage in | Thyroid_Cancer |
"The coronavirus disease COVID19 is now a worldwide challenge for public health Among million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan ChinaMethods In this retrospective cohort study we included adult inpatients with confirmed moderate disease of COVID19 from the Affiliated Hospital of Jianghan University during February and early March All of these patients were recovered from COVID19 and discharged from hospital Demographic clinical and laboratory data of admission and discharge were extracted from electronic medical records and analyzed using SPSS as well as among young middle age and elderly peopleResults The median age of this cohort of patients was years And the median hospitalization time was days Common clinical manifestations included fever cough asthenia and shortness of breath On admission laboratory results showed normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level and increased inflammatory indicators erythrocyte sedimentation rate ESR and Creactive protein CRP and some patients were complicated with coagulation disorder and myocardial damage Furthermore patients older than years had statistically higher CRP ESR and fibrinogen level As the health condition was improved at discharge the median level of most laboratory results were in the normal range except hemoglobin and related blood cell count as well as Manuscript submitted July accepted July Published online August aWuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan ChinabThe Affiliated Hospital of Jianghan University Wuhan ChinacThe two authors contributed equallydCorresponding Author Binlian Sun Wuhan Institute of Biomedical Sciences School of Medicine Jianghan University Wuhan China Email binlian17jhuneducn 1014740jocmr4293inflammatory indicator ESR And patients older than years showed slower recovery on coagulation parameters when compared to younger patientsConclusions The severe acute respiratory syndrome coronavirus SARSCoV2 infection induces a controllable inflammatory response in moderate disease of COVID19 in Wuhan China Since patients older than years had higher inflammatory state and more dysregulated coagulation condition it might be essential to closely assess their illnessKeywords COVID19 Moderate disease Clinical feature Laboratory findings InflammatoryIntroductionThe novel severe acute respiratory syndrome coronavirus SARSCoV2 was first identified in Wuhan China in December [ ] and now spread all over the world Its infection in human mainly appears as acute respiratory syndrome sometimes along with digestive and nervous disorders [ ] The outbreak of this coronavirus disease COVID19 was declared a pandemic by the World Health anization WHO on March Globally as of June there have been confirmed cases including deaths []A large cohort study showed the spectrum of COVID19 that among patients had mild symptom had severe disease and were in critical condition [] Naturally most studies about COVID19 focus on the severe and critical cases although more than of COVID19 patients show mild to moderate symptoms It should be equally important to understand the average degree of SARSCoV2 infection especially if this virus would become another endemic virus in communities like the influenza virusOur study describes patients that were admitted in a designated hospital in Wuhan the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city during February and early March These patients showed moderate symptom of COVID19 and were confirmed both by s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmrThis is distributed under the terms of the Creative Commons Attribution NonCommercial International License which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly cited 0cLiu et alJ Clin Med Res SARSCoV2 RNA test and chest radiography And they were all discharged from hospital after recovery The aim of our study is to clarify the characteristics of laboratory test index of COVID19 patient with moderate symptoms during the first wave of the pandemic in Wuhan These findings may help us extend our understanding of the pathogenicity in SARSCoV2 infectionMaterials and MethodsPatientsThis retrospective cohort study included adult patients ¥ years old with confirmed COVID19 admitted to the Affiliated Hospital of Jianghan University the sixth hospital of Wuhan city in Wuhan from February to March According to WHO interim guidance [] patients with moderate disease of COVID19 on admission were enrolled in this study they showed clinical signs of pneumonia fever cough dyspnea fast breathing but no signs of severe pneumonia including oxygen saturation SpO2 ¥ on room air All the patients were confirmed by SARSCoV2 RNA test in respiratory secretions for twice as well as by groundglass opacities or bilateral pulmonary infiltration showed in chest computed tomography CT scan During hospitalization patients were kept in regular wards without intensive cares or invasive mechanical ventilation They received supportive therapy effective oxygen therapy antiviral agent and if necessary antibiotics Patients were discharged from hospital when the following criteria were met body temperature normal for more than days respiratory symptoms significantly improved pulmonary imaging significantly improved on CT scan and SARSCoV2 RNA tests showed negative for twice This study was approved by the Ethics Committee of School of Medicine of Jianghan University Wuhan China This study was conducted in compliance with the ethical standards of the responsible institution on human subjects as well as with the Helsinki DeclarationData collectionsThe laboratory tests including blood routine biochemistry coagulation parameters and cardiac injury biomarkers were performed in patients on admission and during the hospitalization The demographic and clinical information laboratory results and outcome data were finally collected from electronic medical recordsStatistical analysisTable Demographics and Clinical Characteristics of Patients on Admission N Female MaleAge median IQR yearSex Hospitalization days median IQR dayMedical history Hypertension Diabetes Heart disease Cancer Other respiratory disease Kidney disease Liver disease Neurological disease Thyroid disease OthersSymptoms Fever Cough Sputum production Nose obstruction rhinorrhea Shortness of breath Headache Chest pain Myalgia Asthenia Vomiting DiarrheaSigns Respiratory rate median IQR bpm Heart rate median IQR bpm Systolic pressure 140mm Hg SpO2 Data are median IQR n or nN IQR interquartile range SpO2 oxygen saturationsion IBMResultsContinuous variables were expressed as median interquartile range IQR and compared with the oneway analysis of variance ANOVA test between different age groups A twosided α of less than was considered statistically significant Statistical analyses were done using SPSS verPatient demographics and characteristicsA total of patients with moderate disease of COVID19 were recruited The demographic and clinical characteristics s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Summary of Laboratory Findings on Admission and at DischargeResults on admissionResults at dischargeLaboratory indexNormal rangeNeutrophil count 109L Median IQR Patient Median IQR NA Patient NA Female MaleNeutrophil ratio Lymphocyte count 109LLymphocyte ratio Red blood cell count 1012L Hemoglobin gL Hematocrit Platelet count 109L Female Male Female MalehsCRP mgLESR mmhDdimer mgLFibrinogen gLFDP mgLNTproBNPa pgmL years years years hscTnL ngmLCK ULLDH ULData are median IQR or nN aThe normal level of NTproBNP is increased with age Three normal ranges for different age groups are listed and results are presented accordingly For increased blood level For decreased blood level hsCRP high sensitive Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product NTproBNP Nterminal prohormone brain natriuretic peptide hscTnL high sensitive cardiac troponin L CK creatine kinase LDH lactose dehydrogenase IQR interquartile range NA not applicableof these patients are shown in Table Of these patients females and males the median age at disease onset was years range years with patients older than years Most patients had fever and cough as their first symptoms some also had asthenia shortness of breath sputum production and diarrhea As for the underlying diseases hypertension heart disease and diabetes were the most common in medical histories of these patients On admission the vital signs of patients were also recorded Notably although the SpO2 of all the patients were ¥ at room air of patients were Patients received symptomatic and pneumonia treatments in hospital and the median of their hospitalization were days IQR Whole blood cell counting findingsThe whole blood cell counting was monitored for all the patients on admission and during their hospitalization median values showed in Table The white blood cell counts were generally in the normal range whereas of patients of patients showed lymphopenia lymphocyte count 109L Table Among patients showed lymphopenia of had a decrease by less than lymphocyte count s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0cLiu et alJ Clin Med Res Figure Illustration of percentage changes of laboratory index according to number of patients Data are presented as number of patients that had changed level in laboratory assessments a Decreased lymphocyte count on admission and at discharge b Decreased hemoglobin level on admission and at discharge c Increased CRP level on admission and at discharge d Increased ESR level on admission and at discharge e Increased fibrinogen level on admission and at discharge f Increased Ddimer level on admission and at discharge g Increased FDP level on admission and at discharge In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation products The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res Table Statistically Significant Biomarkers on Admission and at Discharge With Different AgesCRP inESR inESR outFibrinogen inFDP outD dimer outCK n n n 944a 1073a 983a 53b 41a 082a 798bData are expressed with the median IQR P values comparing different age groups are from oneway ANOVA aP for group and group vs group has statistical difference bP for group vs group has statistical difference In for the data collected on admission out for data collected at discharge CRP Creactive protein ESR erythrocyte sedimentation rate FDP fibrinogen degradation product CK creatine kinase IQR interquartile range 109L Fig 1a while five of of them showed a decrease by more than lymphocyte count 109L Fig 1a Twentythree percent of of patients Table still had lymphopenia when they were discharged from hospital and the decrease was limited for most Fig 1a The change of neutrophil count affects fewer patients on admission of nine of patients showed neutrophilia neutrophil count 109L Table and of patients had neutropenia neutrophil count 109L Table However neutrophil ratio was augmented in of of patients neutrophil ratio Table and the maximum increase was about This ratio appeared to be in the normal range for most patients at discharge Therefore the main change in white blood cell counts for patients with moderate COVID19 was the decrease of lymphocyte And as the health condition improved at discharge lymphocyte count also gradually increased to normal levelThe data showed that more than of all patients had declined level of red blood cell count of hemoglobin of and hematocrit of Table and four female patients were in severe condition hemoglobin gL Fig 1b When discharged from hospital of of patients still had these decreases Table The four female patients mentioned above had their hemoglobin increased to over gL However one female of years had less than gL hemoglobin and that was less than earlier result Furthermore there were five more male patients had hemoglobin declined at discharge Fig 1b These results suggested that SARSCoV2 infection may be accompanied by oxygen transport defect in red blood cellsAs for the platelet counting on admission of patients seven of had decreased platelet level platelet count 109L Table and of had increased level platelet count 109L Table At discharge most patients showed platelet level in the normal range Table Inflammatory biomarkersInflammatory biomarkers were also examined on admission and during hospitalization such as erythrocytes sedimentation rate ESR Creactive protein CRP and procalcitonin PCT For most patients PCT levels were in the normal range data not shown For CRP the median value was mgL IQR among patients on admission Table and patients older than years had significantly higher level Table To be specific CRP levels in serum were increased CRP mgL Table in of patients of and of showed an increase by more than CRP mgL Fig 1c At discharge only of Table had elevated level of CRP and the increased level did not exceed mgL except for one patient Fig 1cLevels of ESR were increased in of patients of ESR mmh Table and of Fig 1d had an increase exceeding mmh Similar to CRP patients older than years had statistically higher ESR level Table The median value of ESR on admission was mmh IQR while the value improved to mmh IQR at discharge Table There were still patients had ESR level more than mmh in serum Fig 1dCoagulation parametersThe coagulation parameters were examined on admission for patients Eighty percent of patients of Table had normal serum levels of Ddimer on admission Among the of patients of who had increased Ddimer level Ddimer mgL Table eight showed an increase by more than Ddimer 2mgL Fig 1f At discharge of patients seven of still had abnormal level of Ddimer though the results were very close to the normal range The fibrinogen level on the other hand had increased in of patients of fibrinogen mgL Table and decreased in of fibrinogen 2mgL Specifically patients of had an increase of fibrinogen level by more than fibrinogen mgL Fig 1e while most decreases were slight Furthermore when compared to patients younger than years fibrinogen level on admission was significantly higher in the patients older than years Table At discharge only seven patients still showed high level of fibrinogen Fig 1e Fibrinogen degradation product FDP was also s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0cLiu et alJ Clin Med Res cardial damage Since the outcome of this cohort is known as recovery and discharge from hospital we also collected the last available laboratory results Although all the patients met the discharge criteria for COVID19 some still had abnormal laboratory findings Especially we found the levels of the inflammatory biomarker ESR were still elevated for half of the patients tested Hence patients were asked to go back to the hospital for followup examination including the virus RNA test chest CT scan blood routine coagulation function and other biochemical indicators on both the 14th and 28th day after dischargePrevious studies have found that white blood cell WBC procalcitonin PCT aspartate aminotransferase AST lactose dehydrogenase LDH Cr hscTnL and Ddimer could indicate the progression of COVID19 on admission especially for the severe and mortal cases [ ] And in the moderate condition we analyzed most patients had these indicators in the normal range Therefore our findings are consistent with other studiesHowever these biomarkers were not all specific to COVID19 Similar to other virus infections including SARS [] and H1N1 influenza [] the neutrophil count was mostly normal in mild to moderate patients while lymphocyte count was significantly decreased As the major antiviral cells lymphocyte count declined down to 109L in this cohort on admission And after receiving antiviral and supportive treatment the lymphocyte counts increased among all the patients at discharge Previous study found that lymphocyte counts would continually decrease in severe and critical COVID19 patients [] These results suggested that SARSCoV2 infection can suppress the level of lymphocyte Therefore closely monitoring lymphocyte counts could be one of the best methods when we evaluate the outcome of moderate patientsThe blood routine results also showed that about of patients had low level of hemoglobin red blood cell and hematocrit and the condition was not improved at discharge Previous studies also reported that of patients had hemoglobin below the normal range [] and the hemoglobin level of severe patients was lower although the difference was not statistically significant [] There might be two explanations First the inflammatory state caused by infection may interfere with erythrocytebone marrow metabolism and iron regulation [] and eventually result in a decline of hemoglobin and red blood cell Second patients could suffer from anemia for some time and appear more vulnerable to SARSCoV2 infection Once infected however correction of anemia could not benefit from all the symptoms of COVID19 and psychological stresses The decrease of functioning hemoglobin may contribute to hypoxia and further aggravate the disease in severe casesexamined and of patients had elevated level of FDP mgL Table Fig 1g on admission Twentyseven percent of patients of still showed increased amount of FDP at discharge Table Therefore fibrinogen and FDP were the most altered parameters in coagulation function from our analysisCardiac injury biomarkersCardiac injury biomarkers were monitored for patients on admission Among the patients assessed all had normal serum levels of high sensitive cardiac troponin L hscTnL ngmL The other cardiac biomarker examined was Nterminal prohormone brain natriuretic peptide NTproBNP and of patients of had elevated level NTproBNP and pgmL according to age Table on admission and five of had an increase by more than At discharge the NTproBNP level had decreased into the normal range except two male patients These two patients still had high level of NTproBNP more than pgmL however other cardiac biomarkers such as hscTnL and creatine kinaseMB CKMB were both in the normal range Considering their history of hypertension they were asked to reevaluate their heart function after the COVID19DiscussionAs an emerging infectious disease COVID19 is now a worldwide challenge for public health Among the million patients about present mild to moderate disease but studies dedicate to these patients are actually scarce Therefore we enrolled adult patients with moderate disease of COVID19 to elucidate the clinical manifestation and laboratory findingsOne of the risk factors of critical and mortal COVID19 is male with older age [] Considering the not severe condition of disease it is rational that female patients present more than male in this cohort Previous studies have found comorbidity such as diabetes and hypertension heart disease as other risk factors [] Similar to other study of patients had diabetes and had hypertension Similar to other studies in Asia [ ] patients mainly present fever cough asthenia and shortness of breath In Europe however a recent study showed that patients with mild to moderate disease mainly had headache loss of smell nasal obstruction and asthenia [] Considering the big differences it is possible that olfactory dysfunction might be neglected during the first wave of SARSCoV2 infection in Wuhan Also the virus is phylogenetically distinct from Asia type B to Europe type C [] which could contribute to different clinical outcomesThrough analysis of laboratory findings we found that patients with moderate disease of COVID19 had common characteristics on admission normal or increased neutrophil ratio low lymphocyte count decreased hemoglobin level increased inflammatory indicators ESR and CRP and some patients were complicated with coagulation disorder and myoExamination of inflammatory biomarkers found that PCT was in the normal range for moderate patients while CRP and ESR were increased in and of patients on admission respectively Other studies including mild to moderate disease showed heterogeneous results [] and found three of or of of patients had increased level of CRP [ ] Thus SARSCoV2 infection is involved with the disorder of inflammatory response Furthermore we find that the increase of both CRP and ESR were correlated with patients age which may indicate that high inflammatory state s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0cClinical Features of Moderate COVID19J Clin Med Res and more severe pneumonia present more often in elderly people In addition the increase of CRP level was more transient as it returned to normal range in most people when they were discharged from hospital Nevertheless it took more time for ESR to return to normal level especially for patients older than years Certainly elder people would need more time to recover and to metabolize the extra inflammatory biomarkersAbnormal coagulation condition is known as associated with poor prognosis of COVID19 In particular serum level of Ddimer and FDP were significantly higher in lethal cases [] Other coronavirus infection such as SARS and Middle East respiratory syndrome MERS showed similar increased coagulation activities in severe cases [ ] The possible mechanism is that local inflammatory response induced by cytokine responses stimulates coagulation cascade and hemodynamic changes [ ] In patients with moderate COVID19 we found about and presented increased level in fibrinogen FDP and Ddimer on admission respectively And when compared to young people patients older than years tend to have higher levels of these coagulation parameters Certainly the hemodynamics and endothelial conditions are more complicate in elder people Furthermore SARSCoV2 infection as well as the inflammatory response it induced could deteriorate the existed dysregulation As the health condition improved at discharge these parameters decreased to normal ranges for most patients Yet again patients over years had significantly higher level of fibrin degradation products In the meantime we found that the platelet level had no obvious decrease in most patients with moderate disease Therefore as reported previously thrombocytopenia happens more often in severe and critical patients [] while abnormal coagulation condition is slight and temporary in moderate patientsThe receptor of SARSCoV2 angiotensinconverting enzyme ACE2 is expressed in myocardial cells and vascular endothelial cells [] It is possible that heart dysfunction is directly targeted by virus infection hence early evaluation and continued monitoring of cardiac damage are important [] We systematically examined the cardiac function biomarkers on admission CKMB and cTnL had no obvious change in these moderate patients while about of patients showed increased NTproBNP This percentage is much lower than previous reported of the severe cases that had elevated NTproBNP Furthermore we only found two patients still had NTproBNP higher than normal range at discharge indicating that moderate patients had little cardiac complicationThis study provides us more information about moderate COVID19 but still has some limitations First this is a relatively small singlecenter study Second due to the retrospective study design a few laboratory tests were not done in all patients In addition several patients were hospitalized during a short period that some tests were not reexamined before they were discharged Third we do not possess any viral kinetic data in these patients further studies are necessary to elucidate the correlation between viral load and laboratory changesIn this ongoing pandemic of infected people showed moderate disease [] it is important to understand more about this moderate spectrum of disease to settle into a longterm problem We conducted a cohort study of adult patients with moderate disease of COVID19 in Wuhan China Based on the clinical characteristics we conclude that SARSCoV2 infection inhibits the immune system of patients and induces a controllable inflammatory response in moderate COVID19 Elderly patients have higher inflammatory state and more dysregulated coagulation condition It is essential to closely assess their condition for a better clinical managementAcknowledgmentsThis study was supported by the Natural Science Foundation of China Hubei Province Department of Education Science and Technology Project B2019232 and the Wuhan Municipal Education Bureau Project Financial DisclosureThere are no financial conflicts of interest to discloseConflict of InterestAll authors declare no competing interestsInformed ConsentAll subjects provided written informed consentAuthor ContributionsYL XZ and BS designed and performed the study YL and BS drafted the manuscript and did critical editing XZ and RG were involved in data collection YL JQ QY JS and WL analyzed the data BS carefully supervised this manuscript preparation and writingData AvailabilityAll data used in the study are available from the corresponding author by request Although some data confidential in nature may only be provided with restrictionsReferences Zhu N Zhang D Wang W Li X Yang B Song J Zhao X et al A novel coronavirus from patients with pneumonia in China N Engl J Med Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species Severe acute respiratory syndromerelated coronavirus classifying 2019nCoV and naming it SARSCoV2 Nat Microbiol Huang C Wang Y Li X Ren L Zhao J Hu Y Zhang s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0cLiu et alJ Clin Med Res L et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Wang D Hu B Hu C Zhu F Liu X Zhang J Wang B et al Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA WHO Coronavirus disease COVID19 Situation Report Available from wwwwhointdocsdefaultsourcecoronavirusesituationreports20200628covid19sitrep160pdfsfvrsn2fe1c658_2 Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA WHO Clinical management of COVID19 Available from wwwwhointpublicationsdetailclinicalmanagementofcovid19 Liu K Chen Y Lin R Han K Clinical features of COVID19 in elderly patients A comparison with young and middleaged patients J Infect 2020806e14e18 Zheng Z Peng F Xu B Zhao J Liu H Peng J Li Q et al Risk factors of critical mortal COVID19 cases A systematic literature review and metaanalysis J Infect 2020812e16e25 Li LQ Huang T Wang YQ Wang ZP Liang Y Huang TB Zhang HY et al COVID19 patients' clinical characteristics discharge rate and fatality rate of metaanalysis J Med Virol Cao Y Liu X Xiong L Cai K Imaging and clinical features of patients with novel coronavirus SARSCoV2 A systematic review and metaanalysis J Med Virol Lechien JR ChiesaEstomba CM Place S Van Laethem Y Cabaraux P Mat Q Huet K et al Clinical and epidemiological characteristics of European patients with mildtomoderate coronavirus disease J Intern Med Forster P Forster L Renfrew C Forster M Phylogenetic network analysis of SARSCoV2 genomes Proc Natl Acad Sci U S A Zhou F Yu T Du R Fan G Liu Y Liu Z Xiang J et al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Velavan TP Meyer CG Mild versus severe COVID19 Laboratory markers Int J Infect Dis Lee N Hui D Wu A Chan P Cameron P Joynt GM Ahuja A et al A major outbreak of severe acute respiratory syndrome in Hong Kong N Engl J Med Cheng Y Zhao H Song P Zhang Z Chen J Zhou YH Dynamic changes of lymphocyte counts in adult patients with severe pandemic H1N1 influenza A J Infect Public Health Chen N Zhou M Dong X Qu J Gong F Han Y Qiu Y et al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Cavezzi A Troiani E Corrao S COVID19 hemoglobin iron and hypoxia beyond inflammation A narrative review Clin Pract Zeng F Huang Y Guo Y Yin M Chen X Xiao L Deng G Association of inflammatory markers with the severity of COVID19 A metaanalysis Int J Infect Dis Chen G Wu D Guo W Cao Y Huang D Wang H Wang T et al Clinical and immunological features of severe and moderate coronavirus disease J Clin Invest Wang F Hou H Luo Y Tang G Wu S Huang M Liu W et al The laboratory tests and host immunity of COVID19 patients with different severity of illness JCI Insight Tang N Li D Wang X Sun Z Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia J Thromb Haemost Wong RS Wu A To KF Lee N Lam CW Wong CK Chan PK et al Haematological manifestations in patients with severe acute respiratory syndrome retrospective analysis BMJ AlAbdallat MM Payne DC Alqasrawi S Rha B Tohme RA Abedi GR Al Nsour M et al Hospitalassociated outbreak of Middle East respiratory syndrome coronavirus a serologic epidemiologic and clinical description Clin Infect Dis Moore JB June CH Cytokine release syndrome in severe COVID19 Science Giannis D Ziogas IA Gianni P Coagulation disorders in coronavirus infected patients COVID19 SARSCoV1 MERSCoV and lessons from the past J Clin Virol Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Liu L et al Clinical Characteristics of Coronavirus Disease in China N Engl J Med MendozaTorres E Oyarzun A MondacaRuff D Azocar A Castro PF Jalil JE Chiong M et al ACE2 and vasoactive peptides novel players in cardiovascularrenal remodeling and hypertension Ther Adv Cardiovasc Dis Guzik TJ Mohiddin SA Dimarco A Patel V Savvatis K MarelliBerg FM Madhur MS et al COVID19 and the cardiovascular system implications for risk assessment diagnosis and treatment options Cardiovasc Res s The authors Journal compilation J Clin Med Res and Elmer Press Inc¢ wwwjocmr 0c" | Thyroid_Cancer |
"rebound effect after stopping treatment with denosumab may be associated with rapid loss ofthe gains in bone mineral density achieved with treatment high levels of bone remodeling markers the occurrenceof vertebral fractures and even hypercalcemiaCase presentation A 64yearold osteoporotic Caucasian woman suffered from a fracture of her second lumbarvertebra in From January she was treated with denosumab for years with good densitometry resultsfor her hip and lumbar areas and no fractures over the last years of treatment Ten months after the treatmentwith denosumab was stopped a cascade of vertebral fractures including some in unusual locations third thoracicvertebra and multiple rib fractures in a context of hypercalcemia suggested possible malignancy A completeevaluation including systemic biological and biopsy analyses ruled out this hypothesis The hypercalcemia wasassociated with normal plasma phosphate and vitamin D concentrations and a high parathyroid hormone levelwith an abnormal fixation of the lower lobe of the thyroid on sestamethoxyisobutylisonitrile scintigraphyHistological analysis of the excised parathyroid tissue revealed hyperplasia The associated thyroidectomy goiterled to the discovery of a thyroid papillary microcarcinomaConclusions We consider the consequences of this rebound effect not only in terms of the major loss of bonedensity return to basal values within years and the multiple disabling fracture episodes but also in terms of thehypercalcemia observed in association with apparently autonomous tertiary hyperparathyroidism Several cases ofspontaneous reversion have been reported in children but the intervention in our patient precluded anyassessment of the possible natural course The discovery of an associated thyroid neoplasm appears to befortuitous Better understanding of the various presentations of the rebound effect after stopping treatment withdenosumab would improve diagnostic management of misleading forms as in this case Bisphosphonates couldpartially prevent this rebound effectKeywords Osteoporosis Denosumab rebound Fracture Hypercalcemia Hyperparathyroidism Correspondence yvesmaugarschunantesfrRheumatology Department Nantes University Hospital place AlexisRicordeau Nantes Cedex France The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMaugars Journal of Medical Case Reports Page of IntroductionSome warning signs need to be explored and are wellknown aftertreatment with denosumab A majorrebound effect after stopping denosumab can be responsible for rapid bone loss with vertebral crushes []Some other manifestations have been described such ashypercalcemia in both children [] and adults []hyperparathyroidism [] and vertebral osteonecrosis[] A suspected increase in the number of cases ofprimary neoplasia has been reported in a recent metaanalysis [] We discuss a new observation with allthese manifestations together which poses diagnosisproblems and several explorations to eliminate a neoplasia This could have been avoided with better knowledge of these rebound manifestationsCase presentationA 64yearold Caucasian woman suffered from a firstvertebral fracture in the second lumbar vertebra L2 in following a fall from her bicycle She did not obtainany treatment Dual Xray absorptiometry DXA relumbar Tscore of standardvealed osteoporosisdeviation SD Our patients characteristics during follow up are summarized in Table Her phosphorus andcalcium levels were normal plasma calcium concentration mmoll normal range to mmollparathyroid hormone PTH concentration was normal ngl normal range to ngl and vitamin D levelwas low ngml normal range to ngl Shewas included in the FREEDOM protocol comparingdenosumab mg subcutaneously every monthsplus mg of calcium and IU of vitamin D dailywith placebo for the treatment of postmenopausal osteoporosis in January The unblinding of the trial years later showed that she had been randomized to thedenosumab group Several vertebral fractures occurredTable Patients characteristics during follow upduring this 3year period fifth thoracic vertebra T5eighth thoracic vertebra T8 and an aggravation of theL2 fracture She continued to participate in the extension protocol in mode for years and then withdrew of her own volition with a finalinjection ofdenosumab in July there were no new vertebralfractures during this entire period DXA in September demonstrated increased bone mineral densityBMD of in her lumbar region Tscore SD and of in her total left hip Tscore SD She was a former tobacco smoker and her medicalhistory included osteoarthritis of the knee a hiatus hernia hypertensionand colonicpolyps Calcaemia monitoring revealed a return to normal values until January nmoll normal range to mmoll Checkups while our patient wasstill on denosumab yielded values of nmoll in January and in September Fig amlodipineallergyIn May our patient complained of acute intensespinal pain that resisted standard painkillers and required treatment with opiates An evaluation was carriedout in hospital in June Spinal Xrays revealed fractures of the fourth thoracic vertebra T4 wedge grade T5 biconcave grade T8 wedge grade ninththoracic vertebra T9 crush grade tenth thoracic vertebra T10 wedge grade 11th thoracic vertebra T11crush grade first lumbar vertebra L1 biconcave grade L2 wedge grade and third lumbar vertebra L3 biconcave grade Fig Bone scintigraphy revealedhypersignals in all these vertebrae except L2 and T5 andin several ribs Magnetic resonance imaging MRI identified vertebra T4 in hypersignal on a T2weighted sequence and hyposignal on a T1weighted sequence withno signs of infiltration or suspected lysis Fig T9 T10T11 L1 and L3 also showed hypersignal and T5 T8 andL2 were older vertebral fractures with no bone marrowFracturesL2Tscore lumbartotalhip BMD SDCalcaemiammollClinicalpresentationBicycle fallNo painFollow up of thepatient January FREEDOM 3yearevaluationJanuary FREEDOM extensionSeptember T5 T8 and aggravationof L2No painNo new fractureTreatmentsInitiation denosumab mg semiannuallycalcium g daily vitamin D IU dailyPursuit denosumab mg semiannuallycalcium g daily vitamin D IU dailyAll treatments stoppedNoneZoledronate two infusions of mg annuallyvitamin D IU quarterlyHospitalizationJune Acute intensedorsal painNew evaluationSeptember Chronic dorsaland lumbar painT4 T9 T10 T11 L1 L3No new fractureNew evaluationAugust BMD bone mineral density L1 first lumbar vertebra L2 second lumbar vertebra L3 third lumbar vertebra SD standard deviation T4 fourth thoracic vertebra T5fifth thoracic vertebra T8 eighth thoracic vertebra T9 ninth thoracic vertebra T10 tenth thoracic vertebra T11 11th thoracic vertebraChronic lumbarpainTreatments stoppedNo new fracture 0cMaugars Journal of Medical Case Reports Page of Fig Calcemia bone mineral density and fracture events for a patient treated with denosumab for years who experienced a rebound effectwhen treatment was stopped with a combination of hypercalcemia related to hyperparathyroidism multiple fractures and rapid bone lossedema Lumbar and dorsal pain remained severe throughout this period of exploration justifying bed rest Biologicaltests revealed hypercalcemia with plasma concentrations of mmoll for calcium normal range to mmoll Fig and mmoll for phosphate normal range to mmoll hypercalciuria mmol24 hoursnormal range to mmol24 hours a 25OH vitaminD3 concentration of ngml normal range to ngla PTH concentration of pgml normal range to ngl a Creactive protein CRP concentration of mglnormal values mgl normal protein electrophoresiswith no Bence Jones proteinuria and a plasma creatinineconcentration of μmollrange to μmoll Blood formula and plasma concentrationsthyroidstimulating hormone TSH parathyroidofhormonerelated peptideand 25OH2D were normal Carboxyterminal collagen crosslinklevels were very high μgl normal valuesCTX μgl but were difficult to interpret in the context ofvertebral fracture DXA performed year after the last injection of denosumab revealed BMD losses of in our patients lumbar region and in her total hipPTHrpnormalcortisolThe association of fractures that are unusual for osteoporosis T4 acute and persistent back pain other rib fractures and hypercalcemia were suggestive of a potentialneoplasia which led to systemic explorations vertebralbiopsy and hyperparathyroidectomy and thyroidectomyknown goiter at the ultrasound exploration A thoracicabdominalpelvic computed tomography CT scan showedonly a heterogeneous multinodular goiter Sestamethoxyisobutylisonitrile MIBI scintigraphy revealed a small areaof fixation of the posterior lower right thyroid lobe and alower lobe nodule displaying clear uptake Fineneedleaspiration results were negative A biopsy of the T4 wascarried out under CT control and produced normal resultsWith hindsight a gassy image of the upper facet of the T4was suggestive of necrosis A parathyroid neoplasia couldhave been evoked too Surgery was performed at the end ofJuly to remove the right upper parathyroid gland à à mm weight g and histological analysissuggested nodular hyperplasia Associated total thyroidectomy led to the detection of a dystrophic goiter withmacrovesicular nodules and a mm isthmic papillarymicrocarcinoma with no associated adenopathy 0cMaugars Journal of Medical Case Reports Page of Fig Lumbar and thoracic Xray in January months before denosumab was stopped and in June months after denosumabwas stopped three old vertebral fractures L2 in and T5 T8 L2 aggravation during the period 20052008stars and new fractures T4 T9T10 T11 L1 L3arrows with denosumab rebound Stars are the old fractures and arrows the new oneHer pain was initially acute but of the mechanical typewith a generally favorable outcome Her calcaemia normalized the day after surgery mmoll with a plasma PTHconcentration of ngl normal range to ngl Anew bone densitometry evaluation was carried out in October at which time bone losses of for the lumbarregion Tscore SD and for the total left hipTscore SD were recorded Fig She continued tocomplain of disabling spinal pain Her phosphorus andcalcium evaluation results remained normal as did her vitamin D levels with the continuation of substitution treatment Given the considerable decrease in BMD she wasplaced on risedronate in September but this wasbadly tolerated She was then placed on zoledronate mgHer calcaemia remained stable at mmoll normalrange to mmoll After two infusions October and October a new DXA in August showed stabilization of the lumbar BMD and a significant loss in the total hip BMD Her plasma calcium levels remained normal mmoll and she did nothave any new vertebral or peripheral fracturesDiscussion and conclusionsThis patient who was included in the initial FREEDOMprotocol [] had benefited from denosumab treatmentwith no new vertebral fractures in the last years oftreatment and an increase in BMD to values exceeding SD with no secondary effects However monthsafter stopping the treatment a cascade of vertebral fractures some in unusual locations T4 although this canbe possible when there are multiple lower vertebral fractures and multiple rib fracturesin a context of 0cMaugars Journal of Medical Case Reports Page of Fig Bone scintigraphy computed tomography scan and magnetic resonance imaging in June Magnetic resonance imaging and bonescintigraphy confirmed that there were six recent vertebral fractures fourth thoracic vertebra ninth thoracic vertebra tenth thoracic vertebra11th thoracic vertebra first lumbar vertebra and third lumbar vertebra small white arrows There were some costal fractures on bonescintigraphy sixth posterior on the right side and laterally tenth 11th and 12th on the left side black arrows A computed tomography scan alsoshowed old vertebral fractures fifth thoracic vertebra eighth thoracic vertebra and second lumbar vertebra white starssuggestedpossible malignancy Ahypercalcemiacomplete evaluation with systemic biological and biopsy T4 analyses ruled out this hypothesis Histological analysis ofthe tissue removed during theparathyroid intervention revealed hyperplasia but no adenoma The hypothesis of coincidental hyperparathyroidism had to be considered Before the treatment withdenosumab our patients calcaemia and PTH levels werenormal Based on retrospective analyses of calcaemia results we concluded that the increase in calcaemia became abnormal after years of denosumab treatmentHyperparathyroidism could have appeared during thedenosumab treatment phase with no obvious link between the two occurrences however there were no clearincreases in calcaemia during the first years and hypercalcemia was markedly aggravated by stopping denosumab The link between hyperparathyroidism anddenosumab was the subject of a recent publication []However the hyperparathyroidism described occurredrapidly after a single injection of denosumab with a fold increase in PTH levels at week normalization at months and normal calcaemia throughout [] This caseresolved within a year In our case calcaemia was highafter years of treatment No PTH determinations werecarried out in parallel during this period It is therefore difficult to determine the date of onset of the hyperparathyroidism as the denosumab treatment may have masked thehypercalcemia Similarly as our patient underwent surgery it is impossible to know what spontaneous course itwould have takenTwo similar cases of hypercalcemia during the rebound effect after stopping treatment with denosumabhave been reported one with low PTH levels and aspontaneously favorable outcome over several months[] and the other after a high dosage of denosumab mg quarterly [] The excessive bone remodelingobserved in the absence of associated fractures may havebeen due to major bone reabsorption potentially accounting for the hypercalcemia as in immobilizationrelated osteoporosis The high hypercalcemia observed isconsistent with this hypothesis Alendronate was administered and the patients plasma calcium concentrationsreturned to normal values within months In contrastour patient had a PTH concentration that was well 0cMaugars Journal of Medical Case Reports Page of nothighandanddisplayedcontrolledrapidnormalization within hours of calcaemia after theintervention Normal phosphate midupper calcaemiaand normal creatinine were not in favor of tertiaryhyperparathyroidism However the hyperparathyroidismdescribed occurred rapidly after a single injection ofdenosumab with a 22fold increase in intact PTHlevels at weeks and normal calcaemiaiPTHthroughout in a case report [] This dramatic increasein iPTH resolved spontaneously within a year Given thesurgical intervention carried out on our patient we cannot determine what the natural course of this hypercalcemia might have been in the same way that half thecases of hyperparathyroidism in kidney transplant recipients resolve within year for example [] The surgerywas carried out because we were concerned that our patient might be suffering from a parathyroid carcinomaThis rebound effect has been reported after stoppingdenosumab administered at an oncological dosage mg monthly for months [] Seven nonmalignantvertebral crushes were observed months after the lastinjection of denosumab At lower dosages denosumab mg halfyearly for years patients with breastcancer receiving aromatase inhibitors developed vertebral crushes after stopping denosumab [] The riskof vertebral fracture was higher if the treatments werelonger and if the patients had preexisting osteoporosistumorssometimesEight other cases of hypercalcemia have been reportedin children Denosumab was administered in these casesfor giantcellfibrous dysplasia brittle bonedisease and juvenile Pagets disease [] The hypercalcemia occurred early to months after the denosumab injection and wassevere plasmacalcium concentrations of up to mmoll but it occurred in a context of high doses and bone remodelingnot comparable with the contextin adults and therebound effect The hypercalcemia regressed over a fewmonths either spontaneously or on zoledronate Reactional bone hyperreabsorption was again suggestedThis hyperreabsorption seems to be related to therelease of receptor activator of nuclear factor kappaBligand RANKL with high crosslinked carboxyterminaltelopeptide of type collagen CTX1 and low Dickkopf1DKK1 and sclerostin [] Osteocytes are known to be theprincipal source of RANKL [] We have suggested thatosteocytes may undergo apoptosis during this reboundeffect when the treatment with denosumab is stoppedpotentially accounting for the necrosis and strong hyperreabsorption reported in certain patients presenting thisrebound effect []The discovery of a thyroid papillary microcarcinomaappears to have been fortuitous in this case Other studies have shown that denosumab may not only decreasethe frequency of bone tumor events but even have adirect or indirect antitumoral effect [] However ametaanalysis comparing denosumab and zoledronateand including four randomized trials patientsfound a significantly higher risk of primary neoplasiawith a cumulative annual incidence of on denosumab versus on zoledronate [] No particular cancer type profile was identified but the number of caseswas small n The cumulative doses in our patientamounted to approximately g of denosumab corresponding to months at the dose of mgmonth prescribed to prevent secondary bone tumor complicationsIt is not possible to draw any firm conclusions concerningour case as goiter is itself a risk factor for thyroid cancerwith a poorly defined incidence of between and forcancer in patients undergoing surgery for goiter []In conclusionit is important to be aware of thisrebound effect with strong bone hyperreabsorption incertain patients after stopping treatment with denosumab Several explorations to eliminate a neoplasm couldbe avoided with knowledge of these cases It can takeseveral different forms a simple loss of the BMD gainedon the treatment vertebral fractures or transient hypercalcemia which in this context may raise concerns of orsimulate malignant bone diseases or hyperparathyroidism as in our case The rebound after stopping treatment with denosumab makes it necessary to checkcalcaemia and CTX early before the risk of further vertebral fractures DXA will be carried out at the end ofthe denosumab sequence but an early new comparativeDXA would be less sensitive than CTX dosage We canpropose CTX and calcaemia months after the last injection of denosumab as a reference and then or months later A marked increase will require preventionThe most appropriate therapeutic approach remains unclear The effects of gradually decreasing the dose ofdenosumab have not yet been reported Bisphosphonatesseem to be only partially effective according to publishedpreliminary results []AbbreviationsBMD Bone mineral density CRP Creactive protein CT Computedtomography CTX Carboxyterminal collagen crosslink CTX1 Crosslinkedcarboxyterminal telopeptide of type collagen DKK1 Dickkopf1 DXA DualXray absorptiometry iPTH Intact parathyroid hormone L1 First lumbarvertebra L2 Second lumbar vertebra L3 Third lumbar vertebraMRI Magnetic resonance imaging PTH Parathyroid hormonePTHrp Parathyroid hormonerelated peptide RANKL Receptor activator ofnuclear factor kappaB ligand SD Standard deviation T4 Fourth thoracicvertebra T5 Fifth thoracic vertebra T8 Eighth thoracic vertebra T9 Ninththoracic vertebra T10 Tenth thoracic vertebra T11 11th thoracic vertebraTSH Thyroidstimulating hormone MIBI MethoxyisobutylisonitrileAcknowledgementsNAAuthors contributionsAll authors contributed to the work contribution to the observation YMCDL the discussion JMB BLG PG JG and writing the manuscript YMCDL All authors read and approved the final manuscript 0cMaugars Journal of Medical Case Reports Page of function Curr Drug Saf httpsdoi102174 Maugars Y Bart G Guillot P Multiple vertebral osteonecrosesKümmell's Disease after years on denosumab is osteocyte apoptosis toblame Calcif Tissue Int Chen F Pu F Safety of denosumab versus zoledronic acid in patients withbone metastases a metaanalysis of randomized controlled trials Oncol ResTreat Papapoulos S Lippuner K Roux C The effect of or years ofdenosumab treatment in postmenopausal women with osteoporosisresults from the FREEDOM Extension study Osteoporos Int Nakamura Y Kamimura M Ikegami S Changes in serum vitamin D andPTH values using denosumab with or without bisphosphonate pretreatment in osteoporotic patients a shortterm study BMC Endocr DisordTorres A Lorenzo V Salido E Calcium metabolism and skeletal problemsafter transplantation J Am Soc Nephrol GonzalezRodriguez E AubryRozier B Stoll D Zaman K Lamy O Sixtyspontaneous vertebral fractures after denosumab discontinuation in women with earlystage breast cancer under aromatase inhibitorsBreast Cancer Res Treat httpsdoi101007s10549019054588Tyan A Patel SP Block S Hughes T McCowen KC Rebound VertebralFractures in a Patient with Lung Cancer After OncologyDose DenosumabDiscontinuation A Cautionary Tale Mayo Clin Proc Innov Qual OutcomesFassio A Adami G Benini C Vantaggiato E Saag KG Giollo A Lippolis IViapiana O Idolazzi L Orsolini G Rossini M Gatti D Changes in Dkk1sclerostin and RANKL serum levels following discontinuation of longtermdenosumab treatment in postmenopausal women Bone Bonewald LF The amazing osteocyte J Bone Miner Res de Groot AF AppelmanDijkstra NM van der Burg SH Kroep JR The antitumor effect of RANKL inhibition in malignant solid tumors A systematicreview Cancer Treat Rev Sahbaz NA Tutal F Aksakal N Cancer frequency in retrosternal goiterAm Surg Lamy O Stoll D AubryRozier B Rodriguez EG Stopping Denosumab CurrOsteoporos Rep Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNone for this workAvailability of data and materialsAll original data are available corresponding authorEthics approval and consent to participateInclusion in the FREEDOM protocol signedConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsWe do not have any competing interestsReceived July Accepted May ReferencesAnastasilakis AD Polyzos SA Makras P AubryRozier B Kaouri S Lamy OClinical features of patients with reboundassociated vertebral fracturesafter denosumab discontinuation systematic review and additional cases JBone Miner Res Dupont J Laurent MR Dedeyne L Luyten FP Gielen E Dejaeger MReboundassociated vertebral fractures after stopping denosumab Reportof four cases Joint Bone Spine httpsdoi101016jjbspin201907010Fernández Fernández E Benavent Núñez D Bonilla Hernán G Monjo HenryI GarcÃa Carazo S Bernad Pineda M Balsa Criado A Aguado AP MultipleVertebral Fractures Following Discontinuation of Denosumab TreatmentTen Clinical Cases Report Reumatol Clin httpsdoi101016jreuma201811002 [Epub ahead of print]Florez H RamÃrez J Monegal A Guañabens N Peris P Spontaneousvertebral fractures after denosumab discontinuation A case collection andreview of the literature Semin Arthritis Rheum Popp AW Varathan N Buffat H Senn C Perrelet R Lippuner K Bone mineraldensity changes after year of denosumab discontinuation inpostmenopausal women with longterm denosumab treatment forosteoporosis Calcif Tissue Int TriptoShkolnik L Rouach V Marcus Y RotmanPikielny P Benbassat CVered I Vertebral fractures following denosumab discontinuation inpatients with prolonged exposure to bisphosphonates Calcif Tissue IntKoldkjær Sølling AS Harsløf T Kaal A Rejnmark L Langdahl BHypercalcemia after discontinuation of longterm denosumab treatmentOsteoporos Int Kurucu N Akyuz C Ergen FB Denosumab treatment in aneurysmalbone cyst Evaluation of nine cases Pediatr Blood Cancer httpsdoi101002pbc26926 Epub Dec PubMed PMID Uday S Gaston CL Rogers L Osteonecrosis of the jaw and reboundhypercalcemia in young people treated with denosumab for giant celltumor of bone J Clin Endocrinol Metab Setsu N Kobayashi E Asano N Severe hypercalcemia followingdenosumab treatment in a juvenile patient J Bone Miner Metab Gossai N Hilgers MV Polgreen LE Greengard EG Critical hypercalcemiafollowing discontinuation of denosumab therapy for metastatic giant celltumor of bone Pediatr Blood Cancer Boyce AM Chong WH Yao J Denosumab treatment for fibrousdysplasia J Bone Miner Res Trejo P Rauch F Ward L Hypercalcemia and hypercalciuria duringdenosumab treatment in children with osteogenesis imperfecta type VI JMusculoskelet Neuronal Interact Roux S Massicotte MH Huot Daneault A BrazeauLamontagne L DufresneJ Acute hypercalcemia and excessive bone resorption following antiRANKLwithdrawal Case report and brief literature review Bone Mazokopakis EE Denosumabinduced normocalcemic hyperparathyroidismin in a woman with postmenopausal osteoporosis and normal renal 0c" | Thyroid_Cancer |
Construction of a novel prognosticpredicting modelcorrelated to ovarian cancerWeichun Tang12 Jie Li3 Xinxia Chang12 Lizhou Jia1 Qi Tang12 Ying Wang4 Yanli Zheng4 Lizhou Sun5 andZhenqing Feng121National Health Commission Key Laboratory of Antibody Technique Nanjing Medical University Nanjing Peoples Republic of China 2Department of Pathology Nanjing MedicalUniversity Nanjing Peoples Republic of China 3Department of Nursing The Second Afï¬liated Hospital of Nantong University Nantong Peoples Republic of China 4Department ofGynaecology and Obstetrics The Second Afï¬liated Hospital of Nantong University Nantong Peoples Republic of China 5Department of Obstetrics and Gynecology First Afï¬liatedHospital of Nanjing Medical University Nanjing Peoples Republic of ChinaCorrespondence Zhenqing Feng fengzhenqingnjmueducn or Lizhou Sun lizhou sun163comBackground Ovarian cancer OC is one of the most lethal gynecological cancers worldwide The pathogenesis of the disease and outcomes prediction of OC patients remainlargely unclear The present study aimed to explore the key genes and biological pathwaysin ovarian carcinoma development as well as construct a prognostic model to predict patients overall survival OSResults We identified upregulated and downregulated differentially expressedgenes DEGs associated with OC Gene Ontology GO term enrichment showed DEGsmainly correlated with spindle microtubes For Kyoto Encyclopedia of Genes and GenomesKEGG pathways cell cycle was mostly enriched for the DEGs The proteinprotein interaction PPI network yielded nodes and edges Top three modules and ten hub geneswere further filtered and analyzed Three candidiate drugs targeting for therapy were alsoselected Thirteen OSrelated genes were selected and an eightmRNA model was presentto stratify patients into high and lowrisk groups with significantly different survivalConclusions The identified DEGs and biological pathways may provide new perspective onthe pathogenesis and treatments of OC The identified eightmRNA signature has significantclinical implication for outcome prediction and tailored therapy guidance for OC patientsBackgroundOvarian cancer OC is the most lethal malignant disease in the female reproductive system with over new cases and deaths each year worldwide [] Epithelial OC accounts for ofovarian malignancies listed as the most common histological type Since the ovaries locate in the deeppelvis with mere symptoms emerging at the beginning of ovarian morbid change the early detectionfor the malignancy is truly difficult Hence when OC is detected the patient is usually at an advancedstage with invasion and metastasis accompanied [] For patients in the early stage the 5year survivalrate can reach whereas for advancedstage patients the number is mere ¼ [] Thereforeit is imperative to explore the molecular mechanisms of malignant biological behavior of OC cells andto develop more reliable molecular markers for predicting recurrence and evaluating prognosis furtherguiding clinicians for therapyAt present various highthroughput microarrays and nextgeneration sequence genomic datasetswhich were deposited in the Gene Expression Omnibus GEO [] and The Cancer Genome Atlas TCGAdatabases have been widely analyzed for identifying differentially expressed genes DEGs which couldserve as candidate diagnostic or prognostic markers further effectively improving our understanding ofthe disease from genetic perspective Whereas since the existence of tissue or sample heterogeneity inThese authors contributedequally to this workReceived April Revised July Accepted July Accepted Manuscript online July Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261each independent experiment as well as the discrepancy of different data processing methods and technology platforms the DEGs identified from a singlecohort study may generate false positives Herein the Robust Rank Aggregation RRA method which analyzes the significant probability of all elements by a probabilistic model is developedto identify statistically significant genes from multiple datasets and provide more accurate and valuable informationfor clinical use far beyond one gene list [] To date a bunch of novel prognostic markers have been discovered topotentially improve the efficacy of diagnosis and prognosis of OC [] However these identified markers were onlyeffective for partial stages or grades and were difficult to apply widely Hence a prognostic model which is basedon signature gene expression level with high discriminating power to effectively assist prognosis prediction for eachpatient is required in clinical practiceIn the present study we downloaded six primary microarray datasets from the GEO database which containeda total of samples with OC samples and normal samples The geneset and relative clinical information on ovary tissues of OC patients and healthy females from TCGA and GTEx portal were also downloadedIntegrated DEGs between cancerous and normal ovarian samples were screened using the limma R package andRRA method Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathways enrichmentof DEGs were performed for nextstep functional analysis The Search Tool for the Retrieval of Interacting GenesDatabase STRING and the Connectivity Map CMap online database were then used to analyze the association ofDEGs and explore the molecular mechanisms as well as drugs involved in tumorigenesis Through survival analysisprognostic mRNAs were also selected By performing Cox regression analysis we identified an eightmRNA signature model with the ability to predict the prognosis of OC patients and independent from clinical factors Our studyprovides reliable molecular markers and prognostic models for early detection and outcome prediction as well aseffective drug targets for treating OCMethodsData collectionThrough searching on the GEO Repository with ovarian cancer we downloaded the gene expression profiles ofGSE54388 GSE40595 GSE38666 GSE27651 GSE18520 and GSE14407 and the corresponding annotation files fromthe GPL570 [HGU133 Plus ] Affymetrix Human GenomeU133 Plus Array platform GSE54388 contains ovarian tissue samples with normal ovarian surface epithelium and tumor epithelial components from highgradeserous OC patients GSE40595 includes OCassociated stroma and epithelium samples which consist of cancerassociated stroma samples and epithelial tissues from highgrade serous OC patients along with stromal component and ovarian epitheliums from the normal ovary GSE38666 comprises stroma and matchedovarian epitheliums from healthy females as well as cancer stroma and matched cancer epitheliums from OCpatients GSE27651 incorporates normal ovarian surfaces epithelial and serous borderline ovarian tumors lowgrade serous ovarian carcinomas and highgrade serous ovarian carcinomas GSE18520 covers advancedstage highgrade primary OC specimens and normal ovarian surface epithelium tissues GSE14407 involves healthy ovarian surface epithelial samples and paired serous OC epithelium Note that all samples from these GEOdatasets are classified into the cancerous or normal part to be clear the normal stromal and surface epithelium is defined as normal ovarian tissues whereas the borderline tumors as well as cancerous stromal and epithelial tissues areconsidered as malignancies Besides we also downloaded the FPKM format gene expression data and relative clinicalinformation of OC patients samples and normal ovarian tissues from TCGA and GTEx portal respectivelyScreening for DEGs and integration of microarray dataWe used the limma R package [] to integrate the expression profiles from TCGA and GTEx portal standardize the data from the integrated TCGA and GTEx expression matrix as well as six GEO datasets and furtherscreen the DEGs between ovarian cancerous and normal samples The list of DEGs obtained from six GEO microarray datasets by limma analysis was further integrated by the RRA method to get prioritized commonly upor downregulated gene list The final overlapped DEGs for subsequent biological function analysis were the combination of prioritized jointly dysregulated genes from six GEO microarrays and the results from TCGA and GTExdatabases The cutoff criteria were set as FDR and log2fold change FC GO term and KEGG pathway enrichment analysisGO classified the known genes into three main biological progress Molecular Function MF Cellular ComponentCC and Biological Process BP [] KEGG provides researchers highlayer functions of the biological system frommolecular level information [] The Enrichr online tool amppharmmssmeduEnrichr allows for GO term The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression heatmap of the top signiï¬cantly dysregulated genes in six GEO datasetsHierarchical clustering that shows the expression profiles of mRNAs from A GSE14407 B GSE18520 C GSE27651 DGSE38666 E GSE40595 F GSE54388annotation and KEGG pathway for a cluster of genes [] We explored the biological functions of overlappedDEGs and hub modules from our proteinprotein interaction PPI network using Enrichr website Pvalue was considered as significant enrichment Likewise the functional biological pathways of the top ten hub genes fromPPI network were also analyzed by the FunRich tool version [] and the top five enriched pathways of up anddownregulated genes were displayed as bar charts respectively We set the Pvalue as statistically significant The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261PPI network construction and analysisPPI networks display the relationships of various proteins according to their physical or biochemical propertiesSTRING is a database that encompasses the interaction information between known proteins and potentially interacted proteins [] In order to explore the correlations between the DEGs we used the STRING database to constructa PPI network and visualize our results by Cytoscape software [] Confidence score was set as significantMolecular Complex Detection MCODE was utilized to select hub modules of PPI networks in Cytoscape [] Weset the degree cutoff node score cutoff kscore and max Depth was set as the criterion Thenthe significant modules were performed by GO and KEGG analyses Top ten genes were defined according to thehigh degree of connectivity in STRING network [] The coexpression analysis of ten hub genes was performed bySTRING eitherValidation of the hub genesWe downloaded the raw geneset of OC patients from TCGA to explore the expression differences of hub genes inlow and highgrade tumor tissues of OC and draw the survival plot using KaplanMeier plotter webtool [] Thegene and protein expression level of graderelated hub genes were then confirmed by Oncomine and The HumanProtein Atlas HPA database [] Meanwhile we explored the genetic alteration information of the selected tenhub genes in OC patients by plugin cBioPortal cBio Cancer Genomics Portal tool which deposits the genomicsprofiles of various cancer types and provides analysis and visualization of the genomics datasets []Identiï¬cation of candidate small molecule drugsThe CMap database was able to predict potential drugs which might reverse or induce the biological state encoded incertain gene expression signatures in OC [] The DEGs from our study were used to query the CMap databaseThe enrichment scores which represent the similarity were calculated ranging from to The positive connectivity score means an inducing influence on the input signature whereas drugs with negative connectivity score presentreversion impact on the characteristic in human cell lines and are considered as candidate therapeutic molecules After sorting all instances the connectivity score of various instances was filtered by Pvalue Next we investigatethe structures of these candidate molecular drugs from the Pubchem database pubchemncbinlmnihgovEstablishment and evaluation of the prognostic modelThe OC patients from the TCGA project were randomly classified into the training cohort n188 and the testingcohort n186 OSrelated genes were determined by performing univariate Cox regression analysis in the trainingcohort with the Survival R package and further selected for the nextstep screening Least Absolute Shrinkage andSelection Operator LASSO is a parameter selection algorithm which shrinks all highdimensional regression coefficients and generates the penalty regularization parameter λ via the crossvalidation routine by glmnet R packageTo select the optimal prognostic mRNAs we adopted LASSO regression among the selected candidate genes and further perform multivariate Cox proportional hazards regression to evaluate their independent prognostic values Theriskscore model for predicting outcomes of OC patients was the sum of each optimal prognostic mRNA expressionlevel multiplying relative regression coefficient weight calculated from the multivariate Cox regression modelRisk score patient cid2Coefficient mRNAi à Expression mRNAiiAll training cohort patients were classified into high and lowrisk groups according to the median risk score TheKaplanMeier curves of two diverse groups were plotted using survfit function and the receiver operating characteristic ROC curve was unfolded for OS prediction to estimate the sensitivity and specificity of the prognostic modelCox multivariate analysis was also performed to examine whether the risk score was independent of the clinical characters such as age tumor stage and grade Next we used the testing group to check the efficacy of the prognostic riskmodel Each individual in the testing cohort was also categorized as high or lowrisk case by comparing the patientsrisk score with the cutoff value calculated from the training cohort KaplanMeier curve analysis timedependentROC analysis and Cox multivariate analysis were performed eitherSearching tumorinï¬ltrating immune cells associated with patientsprognostic signaturesThe TIMER webtool allows for systematical evaluations of the relationship between the six immune cell types inthe tumor microenvironment which are B cell CD4 T cell CD8 T cell neutrophil macrophage as well as dendritic The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The volcano plot of all gene expression distribution in six GEO datasetsVolcano plot of differentially expressed mRNAs of A GSE14407 B GSE18520 C GSE27651 D GSE38666 E GSE40595 FGSE54388cell and clinical impact in various cancer types via a novel statistical method [] To further explore the prognosticsignature we used the TIMER online tool to search the most significant tumorinfiltrating immune cells according tothe TCGA OC gene data To be clear the relative gene expression levels of six types of immune cells for each patientin high and lowrisk groups from training and testing cohort were measuredResultsThe DEGs among six GEO microarray datasetsThe top significantly up and downregulated genes from each microarray dataset were displayed in the heatmapsFigure 1AF and the distribution of all gene expression was presented in volcano plots Figure 2AF ThroughRRA analysis of expression microarrays we identified DEGs which consisted of upregulated and downregulated genes and displayed the top dysregulated genes by pheatmap R package in Figure Next weanalyzed the expression profiles of TCGA and GTEx getting dysregulated genes Intriguingly when these DEGs were combined with the DEGs from GEO datasets we found that genes were commonly dysregulatedin these two databases with upregulated Figure 4A and downregulated genes Figure 4BGO term and KEGG pathway enrichment analysis of DEGsTo study the potential biological function of the DEGs we performed biological pathway analysis and identifiedsignificantly enriched pathways via Enrichr web tools In GO term Figure 5A for the BP group the DEGs weremostly enriched in regulation of attachment of spindle microtubes to kinetochore cellular response to laminar fluidshear stress and microtubule cytoskeleton anization involved in mitosis In MF group the dysregulated geneswere highly correlated to microtubulebinding microtubule motor activity and tubulinbinding As for CC group The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Heatmap showing the top upregulated genes and top downregulated genes according to PvalueEach row represents one gene and each column indicates one dataset Red indicates upregulation and blue represents downregulation The numbers in the heatmap indicate log FC in each dataset calculated by the limma R package Abbreviation log FClogarithmic fold changethe DEGs were closely related to condensed nuclear chromosome kinetochore and mitotic spindle KEGG pathwayanalysis showed DEGs highly enriched in cell cycle and Alanine aspartate and glutamate metabolism Figure5B The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The intersection of up and downregulated DEGs of GEO and TCGA datasetsA upregulated intersected DEGs in both datasets B downregulated intersected DEGs in both GEO and TCGA dataset Theintersected DEGs were defined as the significant DEGsPPI network construction and modules analysisUsing the STRING database and Cytoscape software a total of DEGs were mapped into the PPI network whichincluded nodes and edges Figure 6A The PPI enrichment Pvalue was 10e16 The top three key modules Figure 6CE within PPI network were then selected Module MCODE score Module MCODEscore Module MCODE score and the biological function of Module which consisted of nodesand edges was further analyzed GO analysis indicated that Module1 was mainly associated with regulation ofattachment of spindle microtubules to kinetochore condensed nuclear chromosome kinetochore and microtubulemotor activity KEGG analysis showed that cell cycle and oocyte meiosis were the most highly enriched pathwaysSupplementary Figure S1The screening of Hub genes and their characteristicsThe top ten hub genes with the highest degree of connectivity were CDC45 CDK1 TOP2A CDC20 CCNB1CEP55 UBE2C HMMR FOXM1 and TPX2 Figure 6B The coexpression analysis results of the hub genes demonstrated that these genes actively interacted with each other Supplementary Figure S2 Besides we established theinteraction network of ten hub genes with their related genes and explored the biological role Supplementary Figure S2ACF of the network by FunRich The gene alteration type and frequency as well as the most frequentlyaltered neighbor genes were also exhibited Figure Gene alteration frequency of ten hub genes among TCGAOC samples was beyond with most genes showed amplified and multiple altered Figure 7AB The top threemost frequently altered genes were FOXM1 CDC20 and CCNB1 with FOXM1 and CDC20 largely amplified whileCCNB1 deep deleted Through analysis of OC patients geneset from TCGA we found that CCNB1 UBE2C CDK1CEP55 as well as FOXM1 expressed significantly higher in highgrade tumors and predicted worse outcomes sincepatients overexpressed above genes owned lower overall survival OS and diseasefree survival DFS rates Figure The Oncomine database showed results from various studies were consistent to our finding Supplementary Figure S3 The HPA website also demonstrate that proteins translated by such five hub genes were overexpressed in OCtissues Supplementary Figure S4 HMMR and TPX2 were also negatively correlated to patients prognosis while noexpression difference was observed in diverse tumor grades and CDC20 was positively associated with tumor gradebut not correlated to patients outcomesRelated small molecule drugs screeningIn total DEGs were analyzed in CMap to screen small molecule drugs and the candidate molecules with top tenconnectivity score are listed in Table Five of these molecules showed a negative correlation and suggested potentialin clinical applications Among them Trichostatin A pyrvinium and 8azaguanine showed a significantly negativecorrelation and the stuctures of such candidate molecule drugs was found in the PubChem database and shown inSupplementary Figure S5 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure GO and KEGG functional annotation for the signiï¬cant DEGsA The top ten enriched BP of the DEGs B The top ten enriched CC of the DEGs C The top ten enriched MF of the DEGs DThe top ten enriched KEGG pathways of the DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The PPI network and top hub genes as well as top three modules were constructedA The PPI network of the DEGs B The hub genes of the DEGs CE Top three hub modules were identified by Cytoscapeplugin MCODE C module1 D module2 E module3Table The top ten OCrelated small molecules with highly signiï¬cant correlations in results of CMap analysisRankCMap nametrichostatin A8azaguaninepyrviniumisoï¬upredonequinpirolevorinostatgenisteinantimycin AheptaminolmidodrineMeanNEnrichmentP value The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The gene mutation overview of ten hub genes in TCGA OC patientsA Ten hub genes are altered in of queried patients B The summary of mutation type of ten hub genes in OC patientsC The network of hub genes and the most frequently altered neighbor genesTable Univariate cox regression identiï¬ed DEGs correlated to patients OSGene IDCCND1SYNE4CCDC80TMC4MCCFOXQ1KRTCAP3CXCR4IL4I1DEFB1CSGALNACT1KLHL14MCUR1HRAbbreviation HR hazard ratioHR95LHR95HPvalueConstruction of prognostic model and evaluation of its predictive abilityUnivariate Cox regression analysis revealed that of DEGs were significantly correlated to patients OS in thetraining cohort Table The OSrelated genes were listed as follows CCND1 SYNE4 CCDC80 TMC4 MCC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical characteristics of CCNB1 CEP55 CDK1 FOXM1 and UBE2C in OC patientsA Five genes were overexpressed in high grade G1 and G2 compared with low grade G3 and G4 in OC BC The OS time Band DFS time C of five genes in OC patients The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Identiï¬cation of prognosisrelated mRNAs using LASSO regression modelA LASSO coefficient profiles of the mRNAs associated with the OS of OC B Plots of the crossvalidation error rates Each dotrepresents a λ value along with error bars to give a confidence interval for the crossvalidated error rateTable Multivariate Cox regression selected eight DEGs correlated to patients OSGene IDTMC4KLHL14CXCR4CCDC80KRTCAP3DEFB1SYNE4FOXQ1HRHR95LHR95HPvalue845E08Abbreviation HR hazard ratioFOXQ1 KRTCAP3 CXCR4 IL4I1 DEFB1 CSGALNACT1 KLHL14 and MCUR1 Through LASSO Cox regression we narrowed the number of prognosisassociated genes to according to the minimum criteria Figure Next based on the multivariate Cox model of candidate mRNAs retained their prognostic significance and werefinally selected as independent remarkable prognostic factors which were TMC4 KLHL14 CXCR4 CCDC80 KRTCAP3 DEFB1 SYNE4 and FOXQ1 Table To predict patients outcomes we developed an individuals risk scoremodel as follows risk score à expression value of TMC4 à expression value of KLHL14 à expression value of CXCR4 à expression value of CCDC80 à expression value of KRTCAP3 à expression value of DEFB1 à expression value of SYNE4 à expression value of FOXQ1 On the basis of the median risk score patients were divided into high orlowrisk groups KaplanMeier curve analysis showed that the OS time of the lowrisk group was significantly longerthan the highrisk group P1147e07 Figure 10E ROC curve analysis revealed the area under the ROC curveAUC of the prognostic model was Figure 10D Meanwhile the risk scores Figure 10A of OC patients inthe training group were ranked for displaying their distribution and the survival status Figure 10B was marked onthe dot plot The expression pattern of eight prognostic mRNAs between high and lowrisk groups was also shown inthe heatmap Figure 10C Univariate and multivariate Cox regression analyses concerning the risk score and clinicalfactors showed that the prognostic model was able to serve as an independent prognostic indicator Figure 11ABROC curve analysis also showed that the AUC value of the model was much significantly higher than the tumor stage AUC grade AUC and patients age AUC Figure 11C Interestingly when The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA training modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E KaplanMeier survival ofthe prognostic model for the TCGA OC training cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the risk model in training cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the training model and OC patients OS status when compared with or combined with clinical factorscombined the risk score with clinical factors the ROC curve of combination model was much higher than each aloneFigure 11DAs for the testing cohort we divided the group into highrisk and lowrisk individuals based on the trainingcohort cutoff risk score The outcomes of low and highrisk groups patients of the testing cohort were also measuredand the OS time of the highrisk group was significantly shorter than the lowerrisk group P1721e02 Figure12E The AUC of the prognostic model was Figure 12D The risk scores distribution Figure 12A and survivalstatus Figure 12B of OC patients as well as the eightprognostic gene expression heatmap Figure 12C in the testinggroup were also present Meanwhile the independency of the prognostic model was confirmed in testing cohort sinceunivariate and multivariate Cox regression analyses showed the model correctly predicted high or lowrisk factroups patients outcomes without relying on any clinical factors Figure 13AB ROC curve analysis showed that theprognostic model exhibited better sensitivity and specificity when compared with tumor stage grade and patientsage for the AUC value of the model was much higher than later Figure 13C In accordance with results from trainingcohort the combination of risk score and clinical factors showed better OS prediction capability Figure 13DThe prognostic signature correlating to immune cells inï¬ltrationThrough TIMER webtool we analyzed the relative gene expression levels of six types of immune cells for each patientand found that genes concerning macrophage fraction were expressing significantly higher in the highrisk groupP005 compared with the lowrisk group in training cohort Figure Interestingly same result was also observed in the testing cohort Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA testing modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E KaplanMeier survival ofthe prognostic model for the TCGA OC testing cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the prognostic risk signature in testing cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the testing model and OC patients OS status when compared with or combined to clinical factorsDiscussionIn the present study we used the RRA methods to jointly analyze six GEO OC microarrays which contained OC and normal samples identifying DEGs and overlapped them with dysregulated genes of OC cohort fromTCGA and GTEx portal finally getting upregulated and downregulated genes Functional analysis showedthat DEGs were significantly enriched in the cell division cycle to be clear in the process of the mitotic spindleSpindle microtubules have been proved to play crucial role in physiological and pathological processes As for celldivision only when all chromosomes linked to spindle microtubules through kinetochores and the spindle assemblycheckpoint is satisfied this process could step to anaphase [] Suraokar et al found that the mitotic spindle assemblycheckpoint and microtubule network were significantly altered in malignant pleural mesothelioma MPM whileusing epothiloneB a nontaxane small molecule inhibitor targeting the microtubules could greatly decrease theviability of MPM cell lines [] Rogalska et al compared the antiproliferative capacity of epothilone B with paclitaxelon OC cell line SKOV3 found that this effect of Epo B was greater than latter [] The researches above wereconsistent with our study that the mitotic spindle process was dysregulated in OC progression playing importantroles in OC cell proliferation and tumor developmentPPI network construction of DEGs included nodes and edges among which we identified three keymodules Interestingly the top1 module was also highly associated with spindle microtubules and chromosome kinetochore confirming the role of cell cycle in OC pathogenesis The top ten hub genes from the PPI network were alsorecognized which were CDC45 CDK1 TOP2A CDC20 CCNB1 CEP55 UBE2C HMMR FOXM1 and TPX2 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression level of immune cells related genes in high and lowrisk groups of the training cohortA Bcell fraction B dendritic fraction C CD4 Tcell fraction D CD8 Tcell fract | Thyroid_Cancer |
structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These ï¬ndings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to ï¬nd newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodiï¬ed leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant antiammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] antiammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was ï¬rst measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their speciï¬ed regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the speciï¬c enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose ï¬fty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the ï¬rst group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationï¬ve mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a signiï¬cant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was conï¬rmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a ï¬avoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosigniï¬cant change in MDA level as compared to normal nonirradiated mice Fig A signiï¬cant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modiï¬es ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were signiï¬cantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a signiï¬cant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisï¬nding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were signiï¬cantly lower ascompared to vehicletreated irradiated ones but still signiï¬cantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different from control group signiï¬cantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB ï¬le 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have ï¬ve subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferronis post test signiï¬cantly different from control group signiï¬cantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test There were no signiï¬cant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different fromcontrol group signiï¬cantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 conï¬rmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientiï¬c GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSOd6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was reï¬uxed for h The solid product formed wascollected by ï¬ltration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr Ê cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSOd6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSOd6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h ï¬ltered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this ï¬gure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr Ê cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSOd6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidiï¬edatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent ï¬atbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The ï¬nal DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespeciï¬c activity of NQO1 was determined using menadione as asubstrate as described [] Brieï¬y the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t | Thyroid_Cancer |
"Oral cancer is one of the most common noncommunicable diseases worldwide This paper presentsan evaluation of the trends and geographical distributions of oral cancers in the Saudi Arabian populationMethods Data from Saudi Cancer Registry reports were used in this analysis which assessed the period between and All cancer cases are recorded in these reports as well as the age gender region and histologicalcancer sites for each patient Agestandardised and agespecific incidence rates were calculated in these reportsFor the purposes of this paper only cancers of the lips tongue and mouth were considered oral cancersResults Between and the Saudi Cancer Registry identified cancer cases in total Of these were oral cancer The mean agestandardised rate of oral cancer for the study period was per peoplefor females it was and for males it was The incidence of oral cancer varied by region with Jazan displayingthe highest agestandardised rate and Hail displaying the lowest A positive correlation was observed between oralcancer incidence and ageConclusion The overall trend of the agestandardised rate for both sexes remained constant from to However the oral cancer incidence in Saudi Arabia varies by region Studying this variation in more detail will helpto guide awareness programmes in the regions that are most in needKeywords Cancer epidemiology Cancer prevention and control Oral neoplasmBackgroundCancer is an intractable global health problem and theleading cause of death in the developed world in the developing worldit is the secondleading cause [] In the most recent year for which information fromthe International Agency for Research on Cancer IARCis available approximately million new cancer caseswere diagnosed and million people died from cancerworldwide [] In this same year new cases oflip and oral cavity cancers were reported representing of all cancer casesA review of the global prevalence of oral cancer revealsa wide variation in distribution among countries []Correspondence bmalshehrinuedusaDepartment of Clinical Laboratory Faculty of applied Medical SciencesNajran University PO Box Najran Kingdom of Saudi ArabiaTwothirds of the estimated incidence of oral cancer occurred in developing countries with up to of allnew oral cancer cases in Sri Lanka India Pakistan andBangladesh [] Converselyin France which has thehighest rate of oral cancer incidence in the EuropeanUnion only oral cancer cases were reported in representing just of all cancer cases [] Inthe USA the American Cancer Society estimated that in approximately people were diagnosed withoral cavity or oropharyngeal cancer and will dieof these cancers [] In Arab countries the prevalence oforal cancer is concentrated between western and southeast Asia [] While this type of cancer is relatively uncommon across Arab gulf countries Saudi Arabia andYemen are notable exceptions [] No studies have beenpublished discussing the epidemiological parameters andgeographic distribution of oral cancer cases or any of its The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cAlshehri World Journal of Surgical Oncology Page of subtypes in the Saudi Arabian population Thereforethis study analysed and discussed oral cancer trends inthe Saudi population by using the most recent dataavailableAccording to the International Classification of Diseases 10th revision ICD10 oral cancer is classifiedinto six sites mucosal lip ICD10 C00 tongue ICD C02 gum ICD10 C03 mouth floor ICD10C04 palate ICD10 C05 and mouth ICD10 C06However examining trends in oral cancer incidencerates that include all oral sites can be misleading Thedata analysed in this study only include cancers of thelip tongue and mouth ICD10C00C06 which formthe majority of oral cancers moreover they have severalrisk factors in common and share a similar biology []Thus those accounting for a minority of oral cancercases were excludedMaterials and methodsDataThis retrospective descriptive epidemiological study analysed oral cancer cases in a Saudi population that hadbeen diagnosed from January through December The study used a method of analysis similar tothat used by Alshehri [] Their analyses incorporated male and female data on lip tongue and mouthICD10C00C06 cancer cases to evaluate disease patterns in the Saudi population Data for the present studywere obtained from the Saudi Cancer Registry SCR apopulationbased registry established in by theMinistry of Health in Saudi Arabia This data can onlybe obtained from the reports published by the SCRSince the SCR has been publishing reports oncancer in Saudi Arabia with the primary objective of defining populationbased cancer incidences The presentstudy was conducted using these reports to derive a descriptive epidemiology of oral cancer in Saudi Arabia InSCR reports agestandardised ASR and agespecificAIR incidence rates were calculated with a focus ongenderspecific and regional differencesThe analysis included cases recorded in the SCR filesfrom January to December totalling cancer cases overall approximately of which wereoral cancerData analysisThe GraphPad Prism6 software was used to analyse thedata Descriptive analyses of epidemiological data wereconducted by calculating the mean of the percentagesand ASR stratified by age sex region and year of diagnosis The ASR was calculated in the SCR reports byadjusting all Saudi regions populations mathematicallyto have the same age structure On the other hand theAIR was calculated by summation of the number ofcancer cases occurring during the year in a regionspopulation among specific age and sex groups dividedby the midyear population of these age and sex groupsUsing these two standardised rates is important because age is a basic element of the risk of developingcancer globally [] Using summary measure tools suchas the ASR and AIR which represent the schedule ofagespecific rates in different regions and across timewill give us a more representative picture of the characteristics in question and enable comparisons of cancerincidences between several populations of Saudi regionsthat differ with respect to ageResultsIncrease in the number of oral cancer casesThe total number of cancer cases identified by the SCRfrom to was with males and females Of this total cases were oral cancer The number of registeredoral cancer cases increased gradually from MF in to a peak of MF in however only cases were reported in MF Table Table Number of oral cancer cases in Saudi Arabia for theperiod from to YearNumber of female casesNumber of male casesTotal 0cAlshehri World Journal of Surgical Oncology Page of The percentage of cases representing oral cancers was for females and for males Fig in These percentages decreased to for females and for males in Fig The percentage curve fororal cancer out of all cancer types for males and femalescorrelated with increases and decreases over the studyperiod apart from the years and Fig ASR of oral cancer fluctuated over the study periodBetween and the ASR per male casesfluctuated in it was trending downwards to alow of in and peaking at in beforedropping again to in Fig The female ASRper increased from in to a peak of in decreasing again to in Fig For bothsexes ASR curves like oral cancer percentages correlateto increases and decreases over the study period apartfrom the years and Fig and generally remained constant from to ASR of oral cancer varies by regionThe ASR data for oral cancer cases of all persons demonstrated a wide variation across Saudi regions TheASR means per people for the period from to ranged from in Hail to in Jazan with anational average of per Fig The Jazan region had the highest male ASR mean at followed by the Najran and Tabuk regions at each Fig Conversely Qassim Baha Hail and theNorthern province reported the lowest ASR averages at and per respectively Fig Male and female ASR data were generally equivalentin terms of region rankings with the Jazan region posting the highest overall ASR of as an average valueof both genders followed by the Makkah region at and the Najran region at Fig Similarly the HailBaha Qassim and Madinah regions posted the lowestASR averages at and respectively FigAIR of oral cancer increases with ageThe AIR data from to showed a positive correlation between oral cancer incidence and age withmost cancer cases occurring in the older age groups Figure shows the AIR of oral cancer increasing noticeablywith age up until age More than of cases werediagnosed after the age of Some AIR differences were found between the sexesacross age groups From ages to rates of oral cancer were higher in females than in males however thistrend had reversed to favour males in the 75andoverage group The overall AIR per showed onlyslight differences between the sexes at for femalesand for males Fig DiscussionA review of oral cancer data in Saudi Arabia for theperiod from to showed an overall increasingtrend in the numbers of oral cancer patients Despitethis rise ASR data trends for oral cancer remained constant from to Fig This curve stabilised inthe face of a substantial Saudi Arabian population increase from million in to million in [] Many accumulative factors could be contributed tothis stability First the significant increased access tohealth services in Saudi regions has contributed to thedissemination of oral health awareness and early diagnosis of some cases of metaplasia that were discovered before they could develop into cancerous tumours SecondFig Consistency in percentage curves for oral cancer out of all cancer types from to The percentage curve for oral cancer out of allcancer types for males and females are correlated with overall increases and decreases over the period from to with the exception ofyears and 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer fluctuated over the study period Between and the male ASR was per in and dropped to in The female ASR fluctuated between and per the increased level of public health in the Kingdom isusually linked to an increase in the economic level of thecountry and individuals may have contributed to thisconstancy as many infectious factors such as virusesand fungi have been linked to oral cancers Third SaudiArabia is a majority Islamic country wherein many oralcancer risk factors such as alcohol consumption andcigarette smoking are forbidden by Islamic law Islamiclaw may thus mediate the lower number of oral cancercases in Saudi Arabia compared to the rest of the world[] Thus based on the IARC data for eight ofthe nine world regions whose ASR of oral cancer isabove the global rate [ ] were located in nonMuslimcountries [] with Melanesian regions having the highest rate [] In contrast most of the regions locatedwithin Muslim countries were ranked below the globalASR [] Further investigation of this aspect could therefore be valuable to cancer prevention effortsThe ASR data revealed that more females than maleswere diagnosed with oral cancer in Saudi Arabia at for men and for women This finding is in contrastwith global data showing that men are more likely to develop oral cancer than women [] In the most recent year for which IARC information is available theglobal ASR of oral cancer was for men and forwomen [] While these rates do not match the globalFig Agespecific incidence rates AIR of oral cancer increases with age The total AIR of oral cancer increased noticeably with up until patientswere and over More than of the cases were diagnosed after the age of 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer varies by region in Saudi Arabia For all persons the ASR means per peoplefor the period from to ranged from in the Hail region to in the Jazan regionsex distribution oral cancer in Saudi Arabia has a relatively low overall ASR when compared to the globalaverage as discussed aboveResults also revealed consistency in the ASR oral cancer curve for both sexes Fig potentially due to thepresence of common risk factors for oral cancer in malesand females This finding could be used as a startingthreshold for studying the risk factors of oral cancer inthe Saudi population through studying the common factors between the sexesAs with many other types of cancer the present studyfound a correlation between the occurrence of oral cancer and age with of cases diagnosed after the age of years In the USA the average age at diagnosis of oralcancer is years and twothirds of individuals with thisdisease are over the age of [] Ageing is accompaniedby increased susceptibility to cancercausing geneticmaturations due to accumulated exposures to environmental and behavioural risk factors Avoiding these riskfactors could greatly reduce the role that ageing plays incancerThis study found a wide variation in the incidence oforal cancers among Saudi regions Such differencescould indicate that regional environmental factors andlifestyle habits affect oral cancer incidence The resultsreviewed above found that the Jazan region possessedthe highest ASR of people with oral cancer In contrastthe Northern province presented the lowest ASR Severalstudies have focused on investigating why the Jazan region has such a high incidence of oral cancer []Ibrahim and others focused on the association ofcertain eating habits and lifestyle behaviours with the development of oral cancer especially the abuse ofshamma a form of smokeless tobacco and the chewingof khat Catha edulis leaves These substances havebeen classified as carcinogens especially in relation tooral cancer Studies by these researchers found that consuming shamma increased the odds of developing oralcancer 29fold suggesting a strong link between oralcancer and diet and lifestyle choicesAccording to the above poor dietary habits related totobacco use and its derivatives are one of the main reasons for the high incidence of oral cancer in some citiesand not others Other factors such as variations in thegenetic background of the Saudi regions citizens cannotbe excluded especially because most of the populationin the Kingdoms regions is tribal so consanguineousmarriages are highly common Thus genomic sequencing can provide information on genetic variants thatmay be present in citizens of these regions and that maybe linked with increased or decreased rates of oral cancer development Populationbased genetic testing issuggestedConclusionDespite the presence of yeartoyear changes in the incidence of oral cancer in the Saudi population there wasoverall no noticeable change in the incidence of oralcancer in the Saudi Arabian population for the periodbetween and In contrast to some international findings females were somewhat more likelythan males to be diagnosed with oral cancer in SaudiArabia The positive correlation between ageing and theincidence of oral cancer for both males and femalesdemonstrates that oral cancer is mainly a disease of theelderly both in Saudi Arabia and across the globe Thewide variation in the incidence rates among Saudi regions raises an important research question concerning 0cAlshehri World Journal of Surgical Oncology Page of World Bank World Development Indicators Washington DC WorldBank Access online via httpwwwirieduarpublicaciones_irianuariocd_anuario_2014Economia4bpdf Albar MA Islamic teachings and cancer prevention J Family CommunityMed Ibrahim EM Satti MB Al Idrissi HY Higazi MM Magbool GM Al QA Oralcancer in Saudi Arabia the role of alqat and alshammah Cancer DetectPrev Alsanosy RM Mahfouz MS Gaffar AM Khat chewing among students ofhigher education in Jazan region Saudi Arabia prevalence pattern andrelated factors Biomed Res Int Quadri MFA Alharbi F Bajonaid AMS Moafa IHY Al Sharwani A AlamirAHA Oral squamous cell carcinoma and associated risk factors in JazanSaudi Arabia a hospital based case control study Asian Pacific J CancerPrev Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspotential causes that need to be investigated furtherThe knowledge produced by this study must be translated into interventions by performing indepth analysesof regional differences This will contribute to the effortsof preventing oral cancer in Saudi ArabiaAbbreviationsAIR Agestandardised incidence rates ASR Agestandardised specific ratesIARC International Agency for Research on Cancer SCR Saudi CancerRegistry ICD10 International Classification of Diseases 10th revisionAcknowledgementsI express my thanks and gratitude to the Saudi Ministry of Health forproviding me with the Saudi Cancer Registry reportsAuthors contributionsI certify that I have participated sufficiently in the intellectual contentconception and design of this work analysis and interpretation of the dataas well as the writing of the manuscript to take public responsibility for itand have agreed to have my name listed as a contributor The author readand approved the final manuscriptFundingThis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsAvailability of data and materialsThe data that support the findings of this study are available from SaudiMinistry of Health but restrictions apply to the availability of these datawhich were used under authorization for the current studyEthics approval and consent to participateThis study was approved by the Research Ethics Committee at NajranUniversity The ethical document reference No ETConsent for publicationA secondary data analysis was conducted in this retrospective study byusing a published dataCompeting interestsThe author declares that he is the only author for this work No other authorcontributed to this work He is also in agreement with the content of themanuscript He declares no conflict of interestReceived June Accepted August ReferencesJemal A Bray F Center MM Ferlay J Ward E Forman D Global cancerstatistics CA Cancer J Clin Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Warnakulasuriya S GloWarnakulasuriya S Global epidemiology of oral andoropharyngeal cancer Oral Oncology httpsdoi101016joraloncology200806002bal epidemiology of oral andoropharyngeal cancer Oral Oncol Rick A Afsaneh B Cancer Facts Figures American Cancer Society Access online via httpswwwcancercontentdamcancerresearchcancerfactsandstatisticsannualcancerfactsandfigures2017cancerfactsandfigures2017pdfAlJaber A AlNasser L ElMetwally A Epidemiology of oral cancer in Arabcountries Saudi Med J Ariyawardana A Johnson NW Trends of lip oral cavity and oropharyngealcancers in Australia overall good news but with rising rates inthe oropharynx BMC Cancer Alshehri B Descriptive epidemiological analysis of thyroid cancer in the Saudipopulation Asian Pacific J Cancer Prev Armitage P Doll R The age distribution of cancer and a multistage theoryof carcinogenesis Br J Cancer 0c" | Thyroid_Cancer |
Rapid repair of human diseasespecific singlenucleotide variants by OneSHOT genome editingYuji Yokouchi12 Shinichi Suzuki2 Noriko Ohtsuki12 Kei Yamamoto12 Satomi Noguchi12 Yumi Soejima3 Mizuki Goto34 Ken Ishioka5 Izumi Nakamura12 Satoru Suzuki6 Seiichi Takenoshita7 takumi era123Many human diseases ranging from cancer to hereditary disorders are caused by singlenucleotide mutations in critical genes Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases However current procedures for repairing deleterious singlenucleotide mutations are not straightforward requiring multiple steps and taking several months to complete In the current study we aimed to repair pathogenic allelespecific singlenucleotide mutations using a single round of genome editing Using highfidelity sitespecific nuclease AsCas12aCpf1 we attempted to repair pathogenic singlenucleotide variants SNVs in diseasespecific induced pluripotent stem cells As a result we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a singlenucleotide marker followed by absolute markerless scarless repair of the RET SNV with no detected offtarget effects The markerless method was then confirmed in human type VII collagenencoding gene COL7A1 Thus using this OneSHOT method we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one resulting in allelespecific repair that can be completed within weeks with or without a singlenucleotide marker Our findings suggest that OneSHOT can be used to repair other types of mutations with potential beyond human medicineThe human genome contains extensive variation including an estimated singlenucleotide variants SNVs that determine how we look and function as well as our specific disease tendencies1 Some SNVs are pathogenic and either directly or indirectly cause hereditary disorders4 such as multiple endocrine neoplasia type 2B MEN2B7 and dystrophic epidermolysis bullosa DEB8 MEN2B is an autosomal dominant syndrome characterised by thyroid adrenal gland and neuronal tumours and skeletal abnormalities The majority of MEN2B cases result from a single aminoacid substitution Met918Thr in the RET protooncogene which is caused by a pathogenic SNV RET c2753TC at the second base of the codon7 DEB is an inherited disease characterised by severe recurrent skin ulcers and blistering It is caused by genetic mutations in the human type VII collagenencoding gene COL7A1 the product of which is an anchoring fibril connecting the epidermis to the dermis8 To model diseases such as these in a0vitro diseasespecific induced pluripotent stem cells iPSCs carrying pathogenic SNVs or other genetic mutations can be obtained from patients9 Repairing these iPSCs to generate isogenic revertant cells is a promising strategy for genome repair and s up new avenues for drug 1Pluripotent Stem Cell Research Unit in Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Hikarigaoka Fukushima Japan 2Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Fukushima Japan 3Department of Cell Modulation Institute of Molecular Embryology and Genetics IMEG Kumamoto University Kumamoto Japan 4Department of Dermatology Faculty of Medicine Oita University Yufu Japan 5Department of Microbiology School of Medicine Fukushima Medical University Fukushima Japan 6Office of Thyroid Ultrasound Examination Promotion Radiation Medical Science Centre for the Fukushima Health Management Survey Fukushima Medical University Fukushima Japan 7Fukushima Medical University Fukushima Japan email yokouchyfmuacjpScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c discovery1314 However the repair process remains problematic and a precise and convenient genome editing procedure has not yet been developedArtificial genome repair andor modification generally starts from a targetspecific doublestrand break generated by sitespecific nucleases1516 Doublestrand breaks are then repaired by a cells own genome repair machineries1516 However most of the break sites are incorrectly repaired by nonhomologous end joining and can result in gene knockout through the generation of nonspecific insertions or deletions indels1718 If a repair template carrying a repair base is coadministrated however the cleavage sites can be accurately repaired via homologydirected repair HDR1516 Sitespecific nucleases such as transcription activatorlike effector nucleases or the Streptococcus pyogenes Sp Cas9 nuclease are typically used as genome editing tools for human iPSCs19SpCas9 is a type IIA endonuclease in the class clustered regularly interspaced short palindromic repeats CRISPRCas system that has been repurposed as a programmable sitespecific nuclease for genome engineering22 Indeed SpCas9 has become a popular genome editing tool for genetically modifying human pluripotent stem cells19 Despite its efficient cleavage activity wildtype SpCas9 has a low DNA repair rate using HDR following plasmidbased administration It recuts the repaired site because the guide RNA has a 2base mismatch tolerance during sequence recognition leading to incorrect repair by nonhomologous end joining1718 To prevent recutting a blocking mutation must be introduced into the seed sequence of the guide RNA or into the protospaceradjacent motif PAM26However for scarless genome editing repairs with wildtype SpCas92749 each method has its obvious strengths and weaknesses For example CORRECT which includes excellent tricks to prevent recutting of the edited target by the editing tool can be performed even if the target recognition ability of the genome editing tool is insufficient however the need for two consecutive editing steps27 In comparison MhAX has high genome editing efficiency but cannot achieve completely scarless editing because singlebase markers are required Further as with the CORRECT method MhAX editing requires two consecutive edits49 increasing cost and time requirementsAnother recentlyidentified bacterial programmable sitespecific nuclease CRISPRCas12aCpf1 is a type VA endonuclease belonging to the class CRISPRCas system32 Among identified Cas12a enzymes those from Acidaminococcus sp BV3L6 As and Lachnospiraceae bacterium ND2006 show strong cleavage activity in mammalian cells32 These Cas12a endonucleases have unique features that complement Cas9 and expand the genome editing range First Cas12a recognises a Trich PAM upstream of the protospacer whereas Cas9 recognises a Grich PAM downstream of the protospacer32 Second two PAMinteracting variants have been generated that expand the Cas12a target range3334 Third Cas12amediated cleavage generates a staggered cut on the PAMdistal region of the target sequence as opposed to the PAMproximal blunt ends generated by Cas932 Finally and perhaps most importantly the guide or CRISPR RNA crRNA exhibits highfidelity target recognition meaning that Cas12a can precisely distinguish the target sequence at a singlebase resolution3536 Consequently the resulting offtarget effects are kept to a background level These features suggest that Cas12a might be suitable for precise diseasespecific iPSC repair because its recut activity is lowCas12a has already been used to knock out pathogenic genes in cancer cells37 generate insertions or twonucleotide substitutions in iPSCs38 and to induce exonskipping in diseasespecific iPSCs39 Thus we investigated whether Cas12a could be used to carry out allelespecific singlenucleotide repair of iPSCs carrying the pathogenic SNVs found in MEN2B and DEB patients in a single round of genome editing To accomplish this we used an AsCas12a PAM variant and singlenucleotide mismatch detection polymerase chain reaction SNMDPCR analysis in diseasespecific iPSCs to develop a precise convenient genomeediting procedure we have called OneSHOT One allelespecific single HDR and singlestranded oligodeoxynucleotide ssODN transient drug selection with SNMDPCR screening OneSHOT provides scarless singlenucleotide substitution of a pathogenic SNV in diseasespecific iPSCs within a0weeks The final modification rate is within a practical range for handpicking cloning Our findings suggest that this simple low cost procedure could be used for genome editing in a single step drastically reducing the time currently needed for scarless SNV repairResultsPrinciples of OneSHOT repair of singlenucleotide mutations AsCas12a is a highfidelity RNAguided sitespecific nuclease that binds to the target genomic DNA site via a 20nt guide sequence in the crRNA allowing it to discriminate the target sequence at the single nucleotide level Fig a0 Following the addition of a crRNA designed for a specific target sequence containing a singlenucleotide mutation AsCas12a selectively binds to the target sequence on the mutant allele and induces a doublestrand break leaving the wildtype sequence on the alternative allele unaffected Fig a0 In the presence of a ssODN wildtype sequence template the mutant nucleotide in the target sequence can be repaired to the wildtype sequence via the cellular HDR machinery To mark the repaired allele we labelled the ssODN with a singlenucleotide marker in the vicinity of the mutant nucleotide This label allowed us to easily identify the generepaired clones by allelespecific amplification4042SNMDPCR detection of the singlenucleotide marker Fig a0 A complete outline of the OneSHOT workflow for SNV repair is provided in the Supplementary Information and in Supplementary Fig a0S1Allelespecific singlenucleotide substitution in MEN2Bspecific iPSCs Before carrying out allelespecific singlenucleotide repair of the pathogenic RET mutation we assessed whether the OneSHOT approach could be used to accomplish allelespecific singlenucleotide substitution of the wildtype alleleWe established FB414 human iPSCs from a patient with MEN2B using a Sendai viral vector protocol43 We then confirmed that the FB414 cells exhibited an embryonic stem celllike morphology and expressed pluripotent gene markers indicating that they were authentic iPSCs Supplementary Fig a0S2 and X7 To identify Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cguide seqcrRNAHigh Fidelity SiteSpeciï¬c Nuclease SSN AsCas12aCpf1target alleleDSBTYCValleleTYCVHDRTYCVTYCVby SNMDPCR ntssODN w markerFigure a0 OneSHOT principles AsCas12a pale yellow and crRNA orange and grey lines selectively bind to a target sequence containing a pathogenic SNV red triangle on the target allele Binding leads to a doublestrand break in the target sequence on the target allele left but not in the corresponding wildtype sequence containing the wildtype nucleotide blue triangle on the alternative nontarget wildtype allele right When the ssODN repair template bluegreen line with the wildtype nucleotide blue triangle and a singlenucleotide marker a silent mutation for SNMDPCR screening green triangle is cotransfected with AsCas12a into the cells the target site on the pathogenic allele is repaired using the template by the endogenous HDR machinery In this case the intended geneedited clones are easily identified by positive screening for the singlenucleotide marker because the repaired expathogenic allele now carries the singlenucleotide markerpossible target sites for AsCas12a around the SNV of interest we first searched for PAMs recognised by wildtype AsCas12a or the RR and RVR variants which recognise TYCV and TATV PAMs respectively3334 We identified two PAM sites for the RR variant TYCV Y CT V ACG TTCC located 12bp upstream of the target nucleotide on the sense strand and TTCA located 7bp upstream of the target nucleotide on the antisense strand Fig a02a magenta lines Based on this information we designed two pairs of crRNAs crRNA_RET1 and crRNA_RET1 a0m and crRNA_RET2 and crRNA_RET2 a0m which contain guide sequences that specifically recognise wildtype and mutant target sequences respectively Fig a02aTo test the cleavage activity and targetrecognition specificity of AsCas12a_RR using these crRNAs we performed a T7E1 assay using 409B2 human iPSCs carrying the wildtype RET sequence in the target site Fig a02a middle The crRNAs for the wildtype sequence crRNA_RET1 and crRNA_RET2 each exhibited significant cleavage activity towards the wildtype target sequence Fig a02b P and P respectively By contrast the crRNAs for the mutant sequence crRNA_RET1 a0m and crRNA_RET2 a0m showed extremely weak activity Fig a02b P for both A more accurate ICE analysis showed no significant activity of the crRNAs on the WT allele Supplementary Fig a0X2a These results indicate that the crRNAs for the mutant sequence do not have significant if any activity on the WT alleleHowever the observed cleavage activity of AsCas12a_RR in conjunction with crRNA_RET1 was significantly higher than that with crRNA_RET2 Fig a02b P Supplementary Fig a0X2a Puromycin treatment further promoted the cleavage activity of AsCas12a_RR with crRNA_RET1 Fig a02b P To test the applicability of the method to carry out allelespecific singlenucleotide substitution in human iPSCs we attempted to replace the wildtype nucleotide RET c2753T at the Met918 site in the wildtype allele in FB414 MEN2BiPSCs Fig a02c Following electroporation of the pY211puro vector which expresses AsCas12a_RR and crRNA_RET1 Fig a02c blue line and a ssODN modification template ssODN_RET_M918T_I913silentC carrying both a variant nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a02c red C and light green C respectively into FB414 cells we conducted SNMDPCR screening Overall clones were positive for the substitution Fig a02c d and GE1 in Table a0 Direct sequencing of the target sequence revealed that clones contained the wildtype allelespecific introduction of the mutant nucleotide at the target site T C substitution resulting in the Met918Thr substitution Fig a02e red arrow along with the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a02e blue arrow The HDR efficiency was Table a0We then searched for offtarget sequences corresponding to the target sequence using the web tool CHOPCHOP v244 and detected no indels in either of the predicted two offtarget sites by Sanger sequencing Table a0 GE1 and by AmpliSeq Supplementary Table a0X4 These results indicated that the OneSHOT method could be used to replace a single nucleotide in an allelespecific manner while minimising offtarget effects As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE1Scientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c¸Figure a0 Singlenucleotide substitution of the RET wildtype sequence in MEN2B iPSCs a Human RET locus containing the MEN2B mutation and crRNA of AsCas12a_RR for the mutation Top exon of the RET locus Middle the wildtype WT allele sequence Blue letters indicate the wildtype nucleotide at Met918 underlined Bottom the mutant allele sequence Red letters indicate the single missense mutation caused by a TC substitution producing a Met918Thr substitution underlined Coloured lines indicate the guide sequence template for the crRNA The pink line indicates the AsCas12a_RR PAM Coloured dashed lines indicate the sites cleaved by AsCas12a_RR with the corresponding crRNA b T7E1 assay using human wildtype iPSCs 409B2 electroporated with AsCas12a_RR and the different crRNAs crRNA_RET1 crRNA_RET1 a0m crRNA_RET2 or crRNA_RET2 a0m targeting exon Left the cropped gel images Arrowheads indicate cleaved bands The fulllength gels are presented in Supplementary Figure a0S7 Right statistical analysis of the cleavage activity and specificity of AsCas12a_RR with the crRNAs following selection with different concentrations of puromycin c HDRmediated editing for generating artificial homozygous MEN2B using AsCas12a_RR with crRNA_RET1 selectively targeting RET_Met918 in the wildtype allele d SNMDPCR analysis of the first round of screening The cropped gel image is shown here The arrowhead indicates positive PCR amplicon a0bp The fulllength gel is presented in Supplementary Figure a0S8 e Sequencing of the original and modified MEN2B iPSCs FB414 Top original sequence of RET exon with a T C substitution in the MEN2B mutant allele Bottom the modified RET sequence The T C substitution resulting in a homozygous Met Thr substitution Red and blue arrows indicate the positions of the pathogenic SNV and the singlenucleotide marker respectively Underlining indicates the codons affected by the editing A more detailed explanation is provided in the Extended Figure Legends in the Supplementary InformationAllelespecific singlenucleotide repair of a pathogenic RET variant To repair the pathogenic SNV RET c2753TC in the mutant allele in FB414 cells we first tested the cleavage activity and target recognition specificity of AsCas12a_RR using crRNA_RET1 a0m and crRNA_RET2 a0m Fig a02a in a homozygous MEN2B iPSC line with mutations in RET exon in both alleles GE19 genotype RETMet918ThrMet918Thr RETIle913 silentC Fig a02e bottom The T7E1 assay confirmed that the MEN2B target sequence was selectively cleaved by AsCas12a_RR with either crRNA_RET1 a0m or crRNA_RET2 a0m but not with crRNA_RET1 or crRNA_RET2 Fig a03a The ICE analysis revealed that only the AsCas12a_RR with crRNA_RET1 a0m exhibited strong cleavage activity on the target sequence Supplementary Fig a0X2b therefore we selected the crRNA_RET1 a0m for use in subsequent experimentsWe then carried out OneSHOT repair in FB414 cells using AsCas12a_RR with crRNA1m and a ssODN repair template containing a repair nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a03b T in blue and C in light green respectively Subsequent SNMDPCR screening showed that clones were positive Fig a03c GE2 in Table a0 while direct sequencing confirmed that of the positive clones contained the introduced wildtype nucleotide at the target site C T substitution leading to a Thr918Met substitution repair Fig a03d red arrow These clones also contained the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a03d blue arrow The overall HDR efficiency was Table a0 GE2 and we detected no offtarget effects by Sanger sequencing Table a0 GE2 and by AmpliSeq Supplementary Table a0X4 As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE2Allelespecific single nucleotide repair of a pathogenic variants in RET and COL7A1 without a singlenucleotide marker We next investigated whether the OneSHOT method could be used to repair the pathogenic SNV in RET without including the singlenucleotide marker which would achieve true scarless repair We therefore performed OneSHOT repair in the FB414 cells using AsCas12a_RR crRNA_RET1 a0m and the ssODN repair template with only a wildtype nucleotide at Met918 In the subsequent SNMDPCR screening for the pathogenic SNV no amplicons were obtained from repaired clones because the pathogenic SNV was lost from the mutant allele Fig a04a Overall we identified negative clones by SNMDPCR screening for the pathogenic SNV and direct sequencing revealed that carried only the wildtype nucleotide at Met918 Fig a04cd and GE4 in Table a0 In this experiment the overall HDR efficiency was Table a0 GE4 and no indels were detected in the two predicted offtarget sites by Sanger sequencing Table a0 GE4 and by AmpliSeq Supplementary Table a0X4We next attempted to perform scarless repair of a pathogenic SNV in iPSCs derived from a patient with DEB to confirm the applicability of the approach for other hereditary diseases We generated iPSCs from a patient with DEB autosomal recessive compound mutation COL7A1pGly2138Ter COL7A1c3591del13insGG and aimed to substitute the pathogenic SNV c6412G T pGly2138Ter in exon Supplementary Fig a0S4ab Scarless OneSHOT using AsCas12a_RR with crRNA_COL7A11 a0m plus the repair template scarlessly repaired the pathogenic SNV in the mutant allele Supplementary Fig a0S4cde and GE5 in Table a0 with a substitution rate of No indels were detected in the seven predicted offtarget sites Supplementary Table a0S2 Supplementary Table a0X4 Unlike the scarless OneSHOT for RET_Met918Thr in FB414 cells Fig a0 GE4 in Table a0 identical sequences within the target site were observed among the unintended geneedited clones suggesting that these clones were likely duplicated Supplementary Fig a0S3 GE5Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cabcRET locus on Chr 10q11exon M918PAM crRNA_ RET1 bpWT allelecrRNA_RET2 PAMPAM M918TcrRNA_RET1mMutant allelecrRNA_RET2m PAM M NCpuro crRNA_RET 1m 2m T7E1 assay in WT iPSC 409B2 nsns xedni ledniNCcr2mpuro cr2mpuro cr2mpuro cr2puro cr2puro cr1puro cr1puro cr1mpuro cr1mpuro cr2puro cr1puro cr1mpuro crRNApuro treatmentRETI913 C T PAMRET M918 C TcrRNA_RET1WT allele sequencessODN_RET_M918T_I913silentCModiï¬ed WT allele sequence Mutant allele sequencede1KM M1KIle913 A T T A T TCMet918 A TC G A C GIleIleMetThrThrScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0cGene editing CellGE1GE2GE3GE4GE5FB414FB414FB414FB414B1173Genotype phenotypeOriginal DestinationRETM918T RETM918TM918T I913_silentCMEN2Ba MEN2B homo with SN MarkerRETM918T RET I913_silentCMEN2Ba MEN2B revertant with SN MarkerRETM918T RET I920_silentCMEN2Ba MEN2B revertant with SN markerRETM918T RETMEN2Ba MEN2B scarless revertantCOL7A1G2138X del13 ins GG COL7A1 del13 ins GGDEBb DEB scarless revertantNo of total picked clones TCNo of 1st screening passed clones SNMD PCRNo of 2nd screening passed clonessequencing1stTC 2nd1st 2ndTC Table OneSHOT and scarless OneSHOT gene editing GE experiments After electroporation of the AsCas12a_RR expression vector and the ssODN template into the cells the crude DNA samples from the singlecell derived colonies that expanded on the master plates were subjected to SNMDPCR in the first screening round For positive screening colonies with amplifiable 200bp fragments from the SNMDPCR primer pair were the intendedclone candidates GE13 For negative screening colonies lacking PCR amplification were the intendedclone candidates GE4 and In the second screening round we directly read the sequences around the target site of the DNA fragments amplified by Tks Gflex DNA polymerase in each sample silentC a silent mutation generated by replacement with a cytidine for SN marker a Multiple endocrine neoplasia type 2B B Dystrophic epidermolysis bullosa Positive screening results Negative screening resultsSampleSiteGenomic location No of mis matchesOriginalRET exon target1chr10 GE1GE1GE2GE2GE3GE3GE4GE4Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget chr15 chr4 chr15 chr4 chr15 chr4 chr15 chr4 Sequencea including mismatchesTTCC AGT TAA ATG GAT GGC AAT TGTTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG Indel ratio b Table Offtarget effects of AsCas12a_RR in gene editing experiments GE1GE4 After amplifying the offtarget candidates predicted by CHOPCHOP v2 from the intended geneedited iPSC clones we directly read the sequences around the candidate sites after Sanger sequencing with specific primers a Underline indicates the PAM of the AsCas12a_RR variant Lower letters indicate mismatched bases in the offtarget candidates as compared with the original target sequence b Number of indel clones relative to the number of analysed clonesDiscussionMany hereditary human diseases are caused by singlenucleotide mutations These singlebase alterations have the potential to drastically alter protein structure and function Although most singlenucleotide mutations are completely harmless silent repair of pathogenic SNVs would significantly improve the quality of life and life expectancy of patients with hereditary diseases Thus in the present study we investigated whether we could achieve scarless repair of pathogenic SNVs in pluripotent stem cells from patients with two different types of hereditary disease MEN2B and DEB More importantly we aimed to carry out the repairs in a single stepUsing the OneSHOT approach developed in this study we successfully repaired a RET gene SNV in MEN2B iPSCs with the addition of a singlenucleotide selective marker in a single step We then confirmed that the same technique could be used to carry out scarless repair in MEN2B and DEBspecific iPSCs without the need for the singlenucleotide marker Scarless repair where no trace of gene editing is left around the target sequence is the goal of any gene editing technique because it safely repairs mutations in noncoding genomic regions without any secondary effects In contrast the inclusion of marker sequences during gene editing can have downstream effects Such secondary effects include the introduction of noncoding SNVs to cryptic splice sites Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0ccausing abnormal RNA splicing4546 and mutations that introduce a premature termination codon resulting in unstable mRNA46 Noncoding mutations affecting regulatory elements can also interfere with gene regulation through loss of function resulting in reduced gene expression or gain of function resulting in gene mis or overexpression4748 Therefore scarless repair is crucial for maintaining genome integrity and preventing unknown secondary effects in the target geneSeveral other methods of pathogenic SNV repair have been developed including CORRECT2627 and MhAX49 However all currently available methods have inherent obstacles to achieving scarless SNV repair in a fast and errorfree manner To overcome some of these obstacles we used the AsCas12a nuclease which has highfidelity targetrecognition3536 circumventing the need for a blocking base to inhibit recutting as is required in other methods2627 We also performed SNMDPCRbased negative screening for the pathogenic SNV which easily detects candidate clones containing the intended alteration As a result of these modifications we achieved absolute scarless editing of the RET and COL7A1 SNVs see Fig a0 Supplementary Fig a0S4 and GE4 and GE5 in Table a0 Another advantage of the AsCas12a nuclease was the ability to carry out SNV repair in a single step because only one round of HDR is required for gene editing Fig a0 The OneSHOT method was used to repair the SNVs in RET and COL7A1 within a 3week period with sufficient efficiency for handpicking In contrast other methods can take up to a0months to generate the intended geneedited clone because two rounds of HDRMMEJ may be required262749 However similar to our approach the CORRECT method can achieve scarless singlenucleotide substitution thus ensuring high sequence fidelity around the target site in geneedited cells Fig a0 and Supplementary Fig a0S4 Conversely MhAX leaves a silent single nucleotide mutation around the target site for use in screening49 Another difference is that the dsDNA template in MhAX can be randomly integrated into the genome outside of the target site by nonhomologous end joining50 whereas the ssODN templates used for OneSHOTscarlessOneSHOT and CORRECT approaches are not randomly integrated51 Thus the OneSHOT method developed for SNV repair in the current study appears to have several advantages over currently available methods see Supplementary Table a0S3In the CORRECT procedure the cuttomutation distance the distance between the CRISPRSpCas9 cleavage site and the blocking mutation is a crucial factor for HDR efficiency and zygosity determination2627 We therefore searched for more appropriate sites for the singlenucleotide markers by first comparing the efficacies of three singlenucleotide markers set in different positions around the target site using a PCRrestriction fragment length polymorphism RFLP assay52 We found that two of the markers showed similar HDRspecific cleavage activity while no cleavage activity was detected for the third marker Supplementary Information and Supplementary Fig a0S5 suggesting that Ile920 could be used as an alternative singlenucleotide marker Testing of HDR efficiency in FB414 cells following OneSHOT repair using the alternative marker again confirmed that the singlenucleotide substitutions in the geneedited clones were effectively detected by positive screening using SNMDPCR for a singlenucleotide marker Supplementary Information and Supplementary Fig a0S5 We do note however that the efficiency of identification might depend on the position of the singlenucleotide marker and the primers used for SNMDPCRDespite our success in repairing the pathogenic SNVs in a single step the study has several limitations The OneSHOT method only requires one PCR run thereby reducing the time and cost compared with standard PCRRFLP screeningbased methods which require up to three steps5253 However we found that falsepositive clones are included in the population after the first SNMDPCR screen Supplementary Fig a0X8 Therefore we are currently designing a simple way to discriminate false clones from authentic clones using a PCRbased procedure We also noted that the geneedited cell lines generated by OneSHOT are not always clonal This situation arises because high cell densities occur in the culture during puromycin selection a0days and in the recovery culture a0days prior to clonal expansion However assessment of our data suggests that a 1day recovery culture and sufficient singlecell suspension at the reseeding stage can prevent duplication and ensure clonal establishment of the geneedited cells Using the current protocol we estimate that the HDR substitution rate is While this is sufficient to permit a handpicking cloning protocol it is lower than that achieved by Cas12a in fertilised eggs from model animals3954 We hoped to improve this rate by combining OneSHOT with other procedures based on alternative principles such as introducing a blocking base into the repair template2627 andor using HDRNHEJ modification compounds3863 We have examined whether the modification compounds can promote HDR however the compounds examined in this study had no HDRpromoting effects in our experimental system Supplementary Fig a0X4It is important to emphasise though that the procedure depends on highfidelity target recognition by the sitespecific nuclease Thus the only enzymes appropriate for the OneSHOT procedure include highfidelity variants of engineered SpCas955 or naturally highfidelity Cas9 orthologues59 Finally while we confirmed the expression of pluripotency markers in the geneedited clones data not shown we next aim to carry out functional analyses to confirm the differentiation potential of the repaired cells Therefore further work is needed to finetune the protocol and to confirm differentiation potential and functionality of the proteins in the corrected cell populationsTo increase the reliability of the OneSHOT method it is important to show the robustness of OneSHOT and the fidelity of the repair In order to demonstrate these issues we performed targeted NGSbased deep AmpliSeq analysis of the target sequence With regard to repair fidelity the AmpliSeq analysis showed that accurate singlenucleotide substitutions were achieved by HDR that were faithful to the ssODN template and occurred at sufficient frequency Supplementary Fig a0X3ac These results suggest that the method has good repair fidelity With regard to the robustne | Thyroid_Cancer |
"ligandactivated transcriptional factors that belong to the nuclear receptor superfamily Among them PPAR alpha andPPAR gamma are prone to exert an antiangiogenic eï¬ect whereas PPAR betadelta has an opposite eï¬ect in physiological andpathological conditions Angiogenesis has been known as a hallmark of cancer and our recent works also demonstrate thatvascularspeciï¬c PPAR betadelta overexpression promotes tumor angiogenesis and progression in vivo In this review we willmainly focus on the role of PPAR betadelta in tumor angiogenesis linked to the tumor microenvironment to further facilitatetumor progression and metastasis Moreover the crosstalk between PPAR betadelta and its downstream key signal moleculesinvolved in tumor angiogenesis will also be discussed and the network of interplay between them will further be established inthe review IntroductionPeroxisome proliferatoractivated receptorsPPARs asligandactivated transcription factors belong to the steroidreceptor superfamily which includes three isoforms PPARalpha PPAR betadelta and PPAR gamma [] PPARs formheterodimers with retinoic X receptors and regulate theexpression of various genes upon ligand binding PPARs alsointeract with corepressors or coactivators to modulate thetranscription of its downstream target genes PPARs asimportant transcriptional regulators have been suggested tobe involved in lipid metabolism and multiple cellular functions For instance PPAR alpha also functions in fatty acidbetaoxidation and vascular ammation [] PPAR gammaacts as a regulator in adipocyte diï¬erentiation and type diabetes [] PPAR betadelta is a key player in cardiac energyproduction angiogenesis and particularly in cancer progression []PPAR alpha and PPAR gamma exert predominantly anantiangiogenic eï¬ect [] but there still exist conï¬ictingstudies showing opposite results [ ] On the contraryPPAR betadelta produces more obviously proangiogeniceï¬ects [] In this review we will focus on the promotingrole of PPAR betadelta in angiogenesis especially in tumorangiogenesis The network of interplay between PPAR betadelta and its various downstream signal molecules and alsobetween those key molecules will be further discussed andestablished Remarkably diverse important signal moleculesinvolved in tumor angiogenesis and progression and cancercell metabolism have been identiï¬ed as direct PPAR betadelta target genes AngiogenesisAngiogenesis is the physiological process through which anew capillary network forms from the preexisting vasculature[ ] whereas vasculogenesis denotes de novo bloodvessel formation mostly during embryogenesis in whichendothelial progenitor cells EPC migrate to sites of vascularization then diï¬erentiate into endothelial cells EC andcoalesce into the initial vascular plexus [ ] Besides theinteraction between proangiogenic factors and antiangiogenic factors angiogenesis is also a multiple step biologicalprocess during which a variety of molecules cooperateincluding cell adhesion molecules matrix metalloproteinases 0cPPAR ResearchMMPs extracellular matrix ECM and basement membrane componentsAngiogenesis is a physiological and vital process in development and growth An imbalance of proangiogenic andantiangiogenic factors causes angiogenesis in pathologicalconditions such as diabetic retinopathy and tumor growthThus when the imbalance comes to a point at which angiogenesis is triggered by tumor cells then an angiogenicswitch of tumor cells is turned on during tumor progression the angiogenic switch is often activated and remainson [] Inducing angiogenesis is known as a hallmarkof cancer [] and angiogenesis is also a fundamental stepby which most benign tumors transition into malignant ones Tumor Angiogenesis Tumor needs to sprout new vesselsand further develop a vascular network in order to supplynutrients and oxygen remove waste products support a continually high proliferative rate and ultimately expand neoplastic growth [ ] Hence angiogenesis is essential forhelping sustain tumor growth and facilitate tumor progression Besides being a requirement for angiogenesis an abnormal vasculature also helps to promote tumor progression andmetastasis The tumor vascular wall is imperfect and prone toleakage so it is much easier for tumor cells to directly penetrate into the blood vessels or lymphatic vessels and then proliferate at another distant site to form metastasis []Due to intensive abnormal neovascularization in tumortissues most malignant tumors grow rapidly and acquirethe ability to spread to adjacent and distant ans whichmakes them more malignant and even life threateningTherefore angiogenesis indeed plays an important role intumor progression and metastasis and to intervene with thisprocess would obviously prevent tumor development andspread Thus this has been regarded as a critical target forantitumor therapy PPAR Alpha and AngiogenesisIt was reported ï¬rstly that a selective PPAR alpha agonistWY14643 did not show any eï¬ect on angiogenesis or EC proliferation [] But some subsequent studies showed that theactivation of PPAR alpha inhibited angiogenesis in vitro byusing fenoï¬brate a clinically used PPAR alpha agonist []Moreover fenoï¬brate suppressed EC proliferation migration and tube formation through inhibition of protein kinaseB Akt and disruption of the cytoskeleton [] Furthermore PPAR alpha activation was shown to inhibit vascularendothelial growth factor VEGF induced EC migrationand basic ï¬broblast growth factor bFGFFGF2 inducedcorneal angiogenesis in vitro and in vivo [] Especiallyin vivo reduced tumor growth and microvessel numberswere observed in mice implanted with melanoma Lewis lungcarcinoma LLC ï¬brosarcoma and glioblastoma due to asystemic treatment of PPAR alpha ligand and the antiangiogenic state induced through activation of PPAR alpha withelevated thrombospondin1 TSP1 and endostatin expression []Howeverit was demonstrated inanother observation that activation of PPAR alpha stimuin that same yearlated neovascularization in vivo with increased phosphorylation of endothelial nitric oxide synthase eNOS and Akt via aVEGFdependent manner [] Furthermore Zhang andWard also suggested that PPAR alpha activation inducedproangiogenic responses in human ocular cells [] Inanother study it was shown that a new PPAR alpha agonistRK13675 had no eï¬ect on angiogenesis [] RecentlyPPAR alpha activation is further shown to have antineovascularization eï¬ects with downregulation of VEGF and angiopoietin expression in a rat alkali burn model []In summary the role of PPAR alpha in angiogenesis isstill controversial Some observations showed that ligandactivation of PPAR alpha had antiangiogenic eï¬ects mediated either through upregulation of antiangiogenic factorssuch as TSP1 and endostatin or downregulation of proangiogenic factors including VEGF FGF2 AKT and angiopoietins Others also reported opposite results showing aproangiogenic role upon PPAR alpha activation Thus thespeciï¬c molecular mechanism is still unclear and needs tobe further studied PPAR Gamma and AngiogenesisLigand activation of PPAR gamma was previously shown toinhibit human umbilical vein endothelial cell HUVEC tubeformation in collagen gels [] and VEGFinduced choroidalneovascularization in vitro and in vivo [] Another studyalso demonstrated that EC apoptosis was induced throughtreatment with the PPAR gamma ligand 15dPGJ2 [] Furthermore rosiglitazone a potent PPAR gamma agonist wasshown to inhibit primary tumor growth and metastasisthrough both direct and indirect antiangiogenic eï¬ectsin vitro and bFGFinduced corneal neovascularizationin vivo [] Moreover a similar observation also displayedthe inhibition of VEGFinduced angiogenesis in a chickchorioallantonic membrane model [] In a mouse modelwith ischemiainduced retinopathy pioglitazone a PPARgamma agonist also showed a protective eï¬ect against pathological neoangiogenesis through upregulation of antiammatory adipokine adiponectin [] Additionally thePPAR gamma antagonist GW9662 was shown to reverseOmega3 polyunsaturated fatty acidinduced reduction ofESelectin angiopoietin2 vascular cell adhesion molecule and intracellular adhesion molecule1 [] implicatingan antiangiogenic potential of PPAR gamma itself Howeveropposite results also showed that pioglitazone enhanced neovascularization and inhibited apoptosis of EPC in vitro andin vivo via a Phosphoinositide3Kinase PI3K dependentmanner []Nadra observed that PPAR gammanull embryosdisplayed a vascular structural defect at E95 Moreover disanized placental layers and an altered placental microvasculature were observed in pregnant wildtype mice treatedwith the PPAR gamma agonist rosiglitazone as well asreduced expression of proangiogenic factorsincludingVEGF proliferin and plateletendothelial cell adhesionmolecule1 PECAM1CD31 [] suggesting a crucial roleof PPAR gamma in placental vascular development The 0cPPAR Researchmajor antiangiogenic properties on PPAR gamma activationwere also reviewed here []Notablyin most cancersthe canonical Wntbetacatenin pathway is upregulated while on the contrary PPARgamma is downregulated Interestingly in numerous tissuesthe activation of PPAR gamma inhibits the betacateninpathway whereascanonicalWntbetacatenin signal cascade also inactivates PPARgamma [] implicating a negative regulatory role of PPARgamma in carcinogenesis where tumor angiogenesis mightbe a fundamental stepstimulation ofthetheIn summary PPAR gamma predominantly displays anantiangiogenic eï¬ect that may be mediated through the inhibition of VEGF or bFGFinduced neovascularization andreduction of the expression level of some proangiogenicfactors PPAR BetaDelta and AngiogenesisUnlike PPAR alpha and PPAR gamma on the contrarymany studies have explicitly shown the proangiogenic eï¬ectsof PPAR betadelta on physiological and pathological angiogenesis The ï¬rst evidence provided in a study is that activation of PPAR betadelta with GW501516 a highly selectivePPAR betadelta agonistinduces HUVEC proliferationand an increased expression of VEGF and its receptorVEGFR1 FLT1 [] Besides inducing EC proliferationPPAR betadelta activation by itsligand prostacyclinPGI2 also stimulates upregulation of alpha expression an antiapoptotic and antiammatory protein whichthereby protects ECs from H2O2induced apoptosis and oxidant injury [] Moreover a subsequent study further provides evidence that activation of PPAR betadelta withGW501516 induces angiogenesis during which VEGFrelease is considered as a major trigger factor [] ï¬rstlysuggesting the promotion for angiogenesis upon PPARbetadelta activationMüllerBrüsselbach show that PPAR betadelta mice implanted with LLC and B16 melanoma exhibit diminished blood ï¬ow and immature microvascular structurescompared with wildtype mice Moreover reexpression ofPPAR betadelta into the matrigelinvading cells triggersmicrovessel maturation and restores normal vascularization[] indicating a crucial role of PPAR betadelta in tumorvascularization Additionally another study also observedreduced levels of calcium intracellular channel protein CLIC4 but it observed enhanced expression of cellular retinol binding protein CRBP1 in migrating ECs from PPARbetadeltanull mice [] both of which play a role in tumorvascularization [ ] It was reported that PPAR betadeltawas required for placentation [] and most of the PPARbetadeltanull mutant embryos died at E95 to E105 due toabnormal celltocell communication atthe placentaldecidual interface [] However in these studies [] adefect in angiogenesis was not observed during normal development in PPAR betadeltaknockout miceSome observations also show the important role of PPARbetadelta in physiological angiogenesis For instance skeletal musclespeciï¬c PPAR betadelta overexpression leads toPPAR betadeltaan increase in the number of oxidative muscle ï¬bers andrunning endurance in adult mice [] Moreover PPARbetadelta activation promotes a rapid muscle remodelingvia a calcineurindependent manner and induces muscleangiogenesis in highly selective PPAR betadelta agonistGW0742treated animals [] Furthermore in the heartpharmacologicalstimulation withGW0742 induces rapid cardiac growth and cardiac angiogenesis through direct transcriptional activation of calcineurin [] Interestingly the same cardiac phenotype wasalso observed after treatment with the PPAR betadelta agonist GW501516implicating a response speciï¬city forPPAR betadelta stimulation [] Calcineurin activationfurther leads to the stimulation of nuclear factoractivatedT cell c3 NFATc3 and an enhanced expression of hypoxiainducible factor alpha HIF1alpha and cyclindependentkinase CDK9 [] Overall the remodeling in skeletalmuscle and heart is perfectly the same as the phenotypeobserved with exercise and both of them are mediatedthrough activation of calcineurinPPAR betadelta may act as a key regulator in mediatingpathological angiogenesis For instance PPAR betadelta wasshown to regulate retinal angiogenesis in vitro and in vivoand its inhibition reduced preretinal neovascularization possibly via an Angiopoietinlike protein Angptl4 dependent manner []implicating the potential of PPARbetadelta in modulating pathological ocular angiogenesisRecently an observation reported that PPAR betadeltaknockdown in both retinal pigment epithelial and choroidalendothelial cells caused an antiangiogenic phenotype andPPAR betadelta promoted laserinduced choroidal neovascular CNV lesions in PPAR betadelta mice [] Moreover pharmacological inhibition of PPAR betadelta with theantagonist GSK0660 also resulted in a signiï¬cantly decreasedCNV lesion size in vivo suggesting a functional role of PPARbetadelta in the development of CNV lesions [] This indicates that PPAR betadelta has an important association withpathological angiogenesisAngiotensin II Ang II the biologically active peptide ofthe reninangiotensin system RAS is a major blood pressure and cardiovascular homeostasis regulator and is alsorecognized as a potent mitogen Angiotensinconvertingenzyme inhibitors were introduced approximately yearsago as antihypertensive agents and have since become asuccessful therapeutic approach for high blood pressurecongestive heart failure and postmyocardial infarction Inexperimental systemsthe antitumor eï¬ects of diverseACE inhibitors show that these inhibit cell proliferationand possessand antiammatory eï¬ects [] It has been shown recentlythat activation of PPAR betadelta inhibits Ang IIstimulated protein synthesis in a concentrationdependentmanner and suppresses Ang IIinduced generation of reactive oxygen species ROS in vascular smooth muscle cells[] PPAR betadelta was further shown to inhibit AngIImediated atherosclerosis [] However it is not clearuntil now if PPAR betadelta activation can be consideredis foras an ACE inhibitormimicking approach as itexample the case for PPAR gamma activators[]antiangiogenicantimetastatic 0cPPAR Researchthe relevance ofFurthermorethis hypothetical PPARbetadelta feature might be limited for tumor angiogenesiswhere vascular smooth muscle hypertrophy and atherosclerosis do not contribute to the major pathologyBesides inducing angiogenesis it has been demonstratedthat PPAR betadelta directly acts on early EPC through activation of the AKT pathway and induces an enhanced vasculogenesis [] Similarlythe PPAR betadeltamediatedprovasculogenic eï¬ects are also observed on late EPC []He showed that PPAR betadelta activation withGW501516 induced EPC proliferation and tube formationwhereas EPC treated with an inhibitor of cyclooxygenaseCOX or PGI2 synthase or with PPAR betadeltaspeciï¬csiRNA also displayed an opposite eï¬ect [] Furthermoreit has been demonstrated that PPAR betadelta inducesangiogenesis and skeletal muscle regeneration throughmatrix metalloproteinase MMP 9mediated insulinlikegrowth factor1 paracrine networks upon EPC activation[] Han also observed that PPAR betadelta activationpromoted a rapid wound healing with enhanced angiogenesis in a mouse model with skin punch wound [] Overallin addition to EC PPAR betadelta is also a key regulator ofEPC or even may act as an initiator of activation of EPC tofurther induce vasculogenesis PPAR BetaDelta and Tumor AngiogenesisLinked to Tumor MicroenvironmentPPAR betadelta expression is often upregulated and promotes cancer progression in many major human cancerslung breast and gastric cancers []such as colonwhich suggests a crucial role of PPAR betadelta in cancercells even though there exist some conï¬icting studies indicating that the functional role of PPAR betadelta in tumorigenesis or carcinogenesis still remains highly controversial [] and dependent on speciï¬c tumor or cancer cell typesThus here we discuss the promotion of PPAR betadelta intumor progression through facilitating tumor angiogenesisPPAR betadelta has been suggested as a critical hubnode transcriptional factor which governs a tumor angiogenic switch [ ] In the transcriptional networkanalysis it was reported that tumor growth and tumor angiogenesis were markedly inhibited in PPAR betadeltanullmice in comparison with wildtype mice [] Moreoverthe elevated PPAR betadelta expression level was also considered to be highly correlated to pathologically advancedtumor stage and increased cancer risk for recurrence and distant metastasis in patients with pancreatic cancer [] indicating the crucial association of PPAR betadelta withtumor angiogenesis progression and cancer invasivenessPPAR betadelta may indirectly facilitate tumor angiogenesis and progression through its function on the tumormicroenvironment TME where tumor angiogenesis is fostered Moreover a tumor also releases some extracellular signals to closely communicate and constantly collaborate withTME to facilitate tumor angiogenesis in order to furtherenable tumor growth and progression For instance it wasshown that colon cancer cells with PPAR betadelta knockoutfailed to stimulate EC vascularization in response to hypoxicstress whereas wildtype cells exposed to hypoxia were ableto induce angiogenesis [ ] suggesting that PPAR betadelta is required for the promotion of angiogenesis in hypoxic stressmediated TME Moreover in the TME tumorltrating myeloid cells are considered as the most important cells for fostering tumor angiogenesis among the multiple diï¬erent kinds of stromal cells [] Besides stimulatingtumor angiogenesis tumor myeloid cells also support tumrowth by suppressing tumor immunity and promotingtumor metastasis to distinct sites [] Interestingly it hasbeen demonstrated that PPAR betadelta activation intumorltrating myeloid cells stimulates cancer cell invasion and facilitates tumor angiogenesis via an Interleukin IL10 dependent manner [] Moreover impairedtumor growth and angiogenesis were observed in PPARbetadelta KO BMT mice due to PPAR betadelta deï¬ciencyin tumor myeloid cells [] suggesting that PPAR betadeltaplays a key role in tumor angiogenesis and progression intumor myeloid cells of TMEFurthermore the endoplasmic reticulum ER an essential anelle involved in many cellular functions is implicated in TME In cancer stressors like hypoxia nutrientdeprivation and acidosis disrupt ER function and lead toaccumulation of unfolded proteins in ER a condition knownas ER stress Cells adapt to ER stress by activating an integrated signal transduction pathway called the unfolded protein response UPR UPR represents a survival response bythe cells to restore ER homeostasis and has both survivaland cell death eï¬ects The mechanisms that determine cellfate during ER stress are not well understood For instanceshort exposure to ER stress initially increases AKT signalingbut longterm ER stress suppresses AKT signaling []PPAR betadelta activation has been shown to reduce endoplasmic reticulum ER stressassociated ammation inskeletal muscle through an AMPKdependent mechanism[] and to reduce ammation in response to chronic ERstress in cardiac cells [] Furthermore it has been nicelyshown that PPAR betadelta can repress RASoncogeneinduced ER stress to promote senescence in tumors [] Thisis mediated through the decrease of pAKT activity promoting cellular senescence through upregulation of p53 and p27expression [] It would be interesting to investigate thedirect eï¬ects of PPAR betadelta on senescence of tumorendothelial cells in an in vivo setting We recently showedthat senescent endothelial cells are indispensable for ahealthy lifespan and that removal of senescent endotheliumdisrupts vascular function leading to diminished vessel densities and ï¬brotic lesions [] If PPAR betadelta mediatessenescence of tumor endothelium thereby protecting vesselintegrity this might explain the enhanced tumor growthand vascularization upon PPAR betadelta activationobserved by us and others [ ]Most recently Zuo demonstrated that PPARbetadelta in cancer cells regulates tumor angiogenesisin vivo and in vitro by promoting the secretion of proangiogenic factors including VEGF and Interleukin IL8[] Most importantly in our recent works it has beenshown that conditionalinducible vascular endotheliumspeciï¬c PPAR betadelta overexpression in vivo leads to 0cPPAR Researchenhanced tumor angiogenesis tumor growth and metastasis formationfurther indicating a vascular ECspeciï¬cPPAR betadelta action mechanism in tumor progressionindependent of some controversial observations of PPARbetadelta in speciï¬c tumor or cancer cell types [] Wagner also ï¬rstly reported the mouse model in whichrapid induction of cardiac angiogenesis and cardiac hypertrophy were observed [ ] Crosstalk between PPAR BetaDelta and SignalMolecules PPAR betadelta activation or overexpressionmay upregulate the expression of its various downstream signal molecules involved in tumor angiogenesis includingproangiogenic factors such as VEGF PDGF and FGFproinvasive matrixdegrading enzymes such as MMP9proammatory mediators such as COX2 and cytokinesand chemokines such as IL1 and CXCL8 even some ofwhich have been further identiï¬ed as PPAR betadelta directtarget genes Besides a leading role of PPAR betadelta amongthe signal molecules PPAR betadelta may function in TMElinked to diverse kinds of cells through direct or indirectmodulation of its downstream molecules Interplay between PPAR BetaDelta and Inï¬ammatoryAngiogenesis Inï¬ammatory angiogenesis is a crucial processin tumor progression For instance the proammatorymediator cyclooxygenase2 COX2 is considered as a keyregulator of angiogenesis and tumor growth through multiple downstream proangiogenic mechanisms such as production of VEGF and induction of MMPs Moreover selectiveinhibition of COX2 has also been shown to suppress angiogenesis in vivo and in vitro [] It is well known that VEGFAplays a critical role in both angiogenesis and vasculogenesis[] and it leads the directional migration of tip cells andstalk cell proliferation in microtubule branches [ ] Ithas also been demonstrated that MMP9 triggers the angiogenic switch during carcinogenesis and enhances the availability of VEGF to its receptors [] Furthermore it hasbeen reported that ammatory cell MMP9 initiates theonset of tumor neovascularization during which there existsfunctionalincludingMMP9 [] LEPTIN is shown to mediate angiogenesisin vivo and in vitro through induction of EC proliferationand expression of MMP2 and MMP9 [] and to furtherpromote EC diï¬erentiation and directional migrationthrough enhancement of COX2 activity [] LEPTIN couldalso induce angiogenesis via transactivation of VEGFR inECs [] Additionally besides inducing angiogenesisPPAR betadelta also functions in chronic ammationfacilitating tumorigenesis through induction of COX2 andits product prostaglandin E2 PGE2 in vivo [ ]Interestingly COX2 VEGF MMP9 and LEPTIN have beenidentiï¬ed as PPAR betadelta target genes via a direct transcriptional activation mechanism in hepatocellular carcinoma cells [] colorectal cancer cells [ ] EPCs[ ] and liposarcoma cells [] respectivelylinks between VEGF and MMPsIn TME tumorltrating ammatory cells also helpto induce and sustain tumor angiogenesis and further tofacilitate tissue invasion and tumor metastatic spread byreleasing some signal molecules such as proinvasive MMP9and ammatory chemokines [] Chemotaxis is alsoa crucial process for inducing angiogenesis in tumors eitherdirectly by attracting ECs towards tumor cells to form newvessels or indirectly by mediating immune ammatorycells to ltrate eventually promoting tumor angiogenesis[] Chemotaxis of tumor cells and stromal cells in TMEis also required for tumor dissemination during tumor progression and metastasis [ ]CXC chemokines such as CXCL8 encoding IL8 andCXCL5 are also involved in COX2associated angiogenesisto contribute to nonsmallcelllung cancer progression[ ] It is further shown that IL8 directly regulatesangiogenesis via recruitment of neutrophils [] whichfurther drives VEGF activation [] MoreoverIL8responding neutrophils are considered as the major sourceof angiogenesisinducing MMP9 [ ] Chemokine CC motif ligand CCL2 in addition to the promotionof angiogenesis [ ] also enhances tumor metastasis[] Furthermore myeloid monocytic cellssuch asmyeloidderivedtumorMDSCsassociated macrophages TAMs and dendritic cells arerecruited to the tumor site mainly by CCL2 and producemany proangiogenic factorssuch as VEGF CXCL8plateletderived growth factor PDGF and transforminggrowth factor beta TGF beta [] In fact bothTGF beta and hypoxia are potentinducers of VEGFexpression in tumor cells and collaborate with TME toprovide the foundation of tumor angiogenesis and cancercell invasion [] Importantly IL8 has been reported asa key target gene of PPAR betadelta to promote angiogenesis in vivo and in vitro [] and CCL2 expression isalso signiï¬cantly upregulated upon vascular PPAR betadelta overexpression in vivo []suppressorcellsCOX2 also mediates IL1 betainduced angiogenesisin vitro and in vivo [ ] IL1 beta supports neovascularization through the regulation of the expression of VEGFand its receptor VEGFR2 FLK1KDR on ECs [] IL1 actsas an upstream proammatory mediator that initiates anddisseminates the ammatory state by inducing a localinteractive network and increasing adhesion moleculeexpression on ECs and leukocytes which facilitates tumorassociated angiogenesis [] In TME ammatory IL1beta recruits myeloid cells from bone marrow and activatesthem to produce proangiogenic factors such as VEGF VEGFfurther activates ECs and myeloid cells promoting tumorinvasiveness and fostering tumor angiogenesis [] In addition IL6 also stimulates angiogenesis and vasculogenesis[ ] However Gopinathan observed an IL6induced newly forming vascular structure with defectivepericyte PC coverage ex vivo [] thus facilitating cancercell ltration and tumor metastasis through vascular leakage Interestingly IL1 and IL6 expression levels are signiï¬cantly upregulated in the PPAR betadelta overexpressionmouse model reported recently []In summary PPAR betadelta seems to act as a key leaderin ammatory mediatordriven tumor angiogenesis linkedto TME in which many proammatory mediators chemokines and proangiogenic factors closely communicate with 0cPPAR Researcheach other and also associate with tumorltrating myeloid cells such as neutrophils TAMs and MDSCs Other Key PPAR BetaDeltaMediated ProangiogenicFactors It has been demonstrated that Wilms tumor suppressor WT1 is a major regulator of tumor neovascularization andtumor progression [] E26 avian leukemia oncogene ETS1 also plays a key role in regulating vascular development and haemopoiesis particularly in angiogenesis []In addition ETS1 promotes cancer cell invasion throughupregulation of MMPs [] Consistent with this silencingof ETS1 in highly invasive breast cancer cells also reducesthe expression of MMP9 and MMP1 []ETS1 also acts as a key regulator of MMPs such asMMP1 MMP3 and MMP9 in human cancerassociatedï¬broblasts CAFs [ ] CAFs support tumor growthby secreting growth factors such as VEGF FGF PDGF andchemokines to stimulate angiogenesis and thereby promotecancer cell invasion and metastasis formation [ ]CAFs as metastatic tumor stroma are a crucial componentin tumor progression through the remodeling of the ECMstructure thus helping a tumor to acquire an aggressive phenotype [ ] PPAR betadelta in CAFs also exhibits aprotumorigenic eï¬ect It was reported that ablation of PPARbetadelta in CAFs attenuated tumor growth by altering theredox balance in TME [] suggesting that PPAR betadeltain CAFs is also an important player in tumor developmentETS1 induces the expression of VEGF VEGFR1 andVEGFR2 in ECs [] In turn VEGF is also a majorinducer of ETS1 in ECs through the activation of either thePI3KAKT pathway or the MEKERK12 signal cascade[ ] WT1 is also reported to regulate tumor angiogenesis via direct transactivation of ETS1 []SRYrelated HMGbox SOX18 has also beenreported previously to induce angiogenesis during tissuerepair and wound healing [] and cancer progression[] And most recently it was further shown that speciï¬cECderived endovascular progenitors initiated a vasculogenic process and diï¬erentiated into more mature endothelial phenotypes within the core of the growing tumorsthrough reactivation of SOX18 [] Interestingly theseimportant proangiogenic molecules including WT1 ETS1and SOX18 are also signiï¬cantly upregulated in the vascularPPAR betadelta overexpression model in vivo [] AndWT1 is also identiï¬ed as a target gene of PPAR betadeltain melanoma cells [] PPAR BetaDelta May Facilitate Cancer Progression atDiverse Cellular Levels in TME PPAR betadelta activationis shown to induce colonic cancer stem cell CSC expansionand to promote the liver metastasis of colorectal cancerin vivo via direct transactivation of the Nanog gene []NANOG as a key transcriptional factor governs the selfrenewal and pluripotency of stem cells [] and cancer cellsexpressing NANOG also often exhibit stem cell properties[] Protooncogene cKITCD117 is known as the maststem cell factor receptor and receptor tyrosine kinase andits activation in CSCs may regulate the stemness to controltumor progression and drug resistance to tyrosine kinaseinhibitors Moreover cKIT has been identiï¬ed as a potentialmarker of the cancer stemlike cells [] In addition cKITnot only functions on ECs [ ] but also belongs to thetumor angiogenesispromoting molecule [] Studiesalso suggested that activation of cKIT enhances the expression of VEGF that can be suppressed by imatinib an inhibitor of cKIT in gastrointestinal stromal tumor cells whichthereby has an impact on tumor angiogenesis [ ] cKIT is also involved in pathological ocular neovascularization [] and is regulated transcriptionally by WT1 []and PPAR betadelta []PDGFB and its receptor PDGFR beta also known asangiogenic factors are suggested to enhance angiogenesisand vasculogenesis via their function in ECs [] andEPCs [] and to regulate vascular permeability and vesselmaturation through recruitment of pericytes PCs [] and smooth muscle cells SMCs [] in newly formingvessels Moreover PDGFB and PDGFR beta also interactwith other proangiogenic factors such as FGF2 [ ]VEGFA and its receptor VEGFR2 [] FurthermorePDGFB and PDGFR beta may also aï¬ect cancer growthand progression by directly acting on TME Besides thecrosstalk with CAFs [] PDGFR beta in stromalï¬broblasts may mediate PDGFBinduced TAM recruitment[] thus implicating a role of PDGFR beta in tumorstroma to facilitate tumor progression Most recently it wasfurther shown that speciï¬c targeting of PDGFR beta kinaseactivity in TME inhibited cancer growth and vascularizationin cancers with high PDGFB expression such as LLC []Therefore this indicates the diverse role of PDGFB andPDGFR beta in facilitating tumor angiogenesis and progression at diï¬erent cellular levels in TME PDGFR beta is demonstrated as a target of telomeric repeat binding factor TRF2 that is further activated transcriptionally by WT1[] PDGFB and PDGFR beta have further been identiï¬edas critical targets of PPAR betadelta via a direct transactivation mechanism | Thyroid_Cancer |
"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ ] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ¥ mmolL onehour bloodglucose postoral sugar ¥ mmolL or twohour bloodglucose postoral sugar ¥ mmolL Preterm birth wasdefined as delivery at ¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Students ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ¥ y y ¥ y y ¥ y y ¥ y Low birth weight y ¥ y Macrosomia y ¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child NutrYun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency prevalence and risk factors among pregnant Chinesewomen Public Health Nutr Zhou J Su L Liu M Liu Y Cao X Wang Z Xiao H Associations between hydroxyvitamin D levels and pregnancy outcomes a prospectiveobservational study in southern China Eur J Clin Nutr Agarwal S Kovilam O Agrawal DK Vitamin D and its impact on maternalfetal outcomes in pregnancy a critical review Crit Rev Food Sci Nutr von Websky K Hasan AA Reichetzeder C Tsuprykov O Hocher B Impact ofvitamin D on pregnancyrelated disorders and on offspring outcome JSteroid Biochem Mol Biol Nobles CJ Markenson G ChasanTaber L Early pregnancy vitamin D statusand risk for adverse maternal and infant outcomes in a biethnic cohort thebehaviors affecting baby and you BaBY study Br J Nutr Roth DE Leung M Mesfin E Qamar H Watterworth J Papp E Vitamin Dsupplementation during pregnancy state of the evidence from a systematicreview of randomised trials BMJ 2017359j5237HautaAlus HH Viljakainen HT HolmlundSuila EM EnlundCerullo MRosendahl J Valkama SM Helve OM Hytinantti TK Makitie OM AnderssonS Maternal vitamin D status gestational diabetes and infant birth size BMCPregnancy Childbirth Khaing W Vallibhakara SA Tantrakul V Vallibhakara O Rattanasiri S McEvoyM Attia J Thakkinstian A Calcium and vitamin D supplementation forprevention of preeclampsia a systematic review and network metaanalysisNutrients 2017910E1141 Zhang Y Gong Y Xue H Xiong J Cheng G Vitamin D and gestationaldiabetes mellitus a systematic review based on data free of Hawthorneeffect BJOG Zhang MX Pan GT Guo JF Li BY Qin LQ Zhang ZL Vitamin D deficiencyincreases the risk of gestational diabetes mellitus a metaanalysis ofobservational studies Nutrients Aghajafari F Nagulesapillai T Ronksley PE Tough SC O'Beirne M Rabi DMAssociation between maternal serum 25hydroxyvitamin D level andpregnancy and neonatal outcomes systematic review and metaanalysis ofobservational studies BMJ 2013346f1169 Qin LL Lu FG Yang SH Xu HL Luo BA Does Maternal Vitamin D DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet GynecolJolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril 1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132 Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J MedLiu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
"Lung carcinoma is a prominent cause of mortality among patients with cancer Previous studies have reported the vital role of long noncoding RNAs lncRNAs in the malignant progression of lung cancer lncRNA RP11284F219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown Therefore the present study aimed to elucidate the role of lncRNA RP11284F219 in lung carcinoma progression The expression of RP11284F219 in lung cell lines and tissues was measured using reverse transcriptionquantitative PCR The endogenous expression of RP11284F219 was silenced using RNA interference and cell viabilities were measured with a Cell Counting Kit assay The invasion and apoptosis of cells were determined via Transwell assays and flow cytometry respectively The protein expression levels were measured by western blotting An increased expression of RP11284F219 was identified in both lung carcinoma tissues and cells Knockdown of RP11284F219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis The present study identified the existence of a direct interaction between RP11284F219 and microRNA miRNAmiR6273p Mechanistically it was demonstrated that RP11284F219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of miR6273p Furthermore cell division cycle and apoptosis regulator CCAR1 was identified as a target gene of miR6273p The in vivo tumor growth assay also demonstrated that the knockdown of RP11284F219 suppressed tumor growth upregulated miR6273p and downregulated Correspondence to Dr Yuan Wang Department of Medical Imaging The First Affiliated Hospital of Xi'an Jiaotong University West Yanta Road Xi'an Shaanxi PR ChinaEmail wangyuan8003126comAbbreviations CCAR1 cell division cycle and apoptosis regulator NSCLC nonsmall cell lung cancer SCLC small cell lung cancerKey words RP11284F219 lung carcinoma proliferation invasion microRNA6273p CCARCCAR1 in the xenograft model of nude mice Thus the present findings indicated the tumor promoting functions of RP11284F219 in the progression of lung carcinoma and provided a novel lncRNAmiRNA axis as a target for the management of lung cancerIntroductionPulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women Furthermore of lung cancer cases are categorized as nonsmall cell lung cancer NSCLC while the remaining are classified as SCLC Although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival Therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentialLong noncoding RNAs lncRNAs are nucleotides in length and have little or no protein coding capacity The mechanisms via which lncRNAs regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small RNAs generating different splice variants via regulating mRNA splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes In recent years previous studies have reported that various human cancer types exhibit lncRNAs dysfunction and these lncRNAs are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells In lung cancer lncRNA metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncRNA HOX transcript antisense RNA is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with NSCLC Furthermore studies have shown that the expression of lncRNA Urothelial carcinomaassociated 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONis significantly upregulated in NSCLC and may induce resistance to treatment of EGFRtyrosine kinase inhibitors by activating the AKTmTOR pathway lncRNA RP11284F219 was primarily discovered in a Pancancer transcriptomic analysis lncRNA RP11284F219 exists as a cluster of three annotated lncRNAs RP11284F219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas However the specific function and the underlying mechanism of RP11284F219 in lung carcinoma remain unknownTo the best of our knowledge the present study demonstrated for the first time that lncRNA RP11284F219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer Together with microRNA miRNAmiR6273p and cell division cycle and apoptosis regulator CCAR1 the regulatory axis of RP11284F219miR3pCCAR1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo The present study aimed to investigate RP11284F219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the RP11284F219miR3pCCAR1 axisMaterials and methodsTissue samples and cell lines Between May and Jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range years nine male patients four female patients who were diagnosed and treated in First Affiliated Hospital of Xi'an Jiaotong University The samples were dissected during the surgery and immediately flashfrozen in liquid nitrogen and transferred to ËC storage for further extraction of both RNA and protein All the tissue samples were obtained with written informed consent from the patients The protocol was approved by The First Affiliated Hospital of Xi'an Jiaotong University approval no A normal lung epithelial cell line BEAS2B and lung carcinoma cell lines NCIH460 NCIH1299 and A549 were purchased from American Type Culture Collection ATCC and cultured according to the ATCC guidelines 293T cells were purchased from Procell Life ScienceTechnology Co Ltd and cultured in DMEM supplemented with FBS cat no ATCC and 1X Penicillinstreptomycin Thermo Fisher Scientific Inc BEAS2B cells were cultured in bronchial epithelial growth medium BEGM cat no CC Clonetics Corporation according to the manufacturer's instructions NCIH460 and NCIH1299 cells were cultured in RPMI medium cat no ATCC and A549 cells in F12K medium cat no ATCC supplemented with FBS cat no ATCC and 1X Penicillinstreptomycin Thermo Fisher Scientific Inc All cells were culture at ËC with CO2RNA extraction and reverse transcriptionquantitative PCR RTqPCR Total RNA from both tissue samples and cell lines were extracted using TRIzol® reagent Invitrogen Thermo Fisher Scientific Inc For each sample ng total RNA was reverse transcribed to synthesize the firststrand cDNA using the PrimeScript RT reagent kit Takara Bio InccDNA samples were diluted times to perform the RTqPCR using SYBR Premix Ex Taq Takara Bio Inc on a CFX96 realtime PCR detection system BioRad Laboratories Inc Expression levels of mRNAs lncRNAs and miRNAs were normalized to GAPDH The primers used for RTqPCR analyses were as follows GAPDH forward 'AAC GAC CCC TTC ATT GAC C' and reverse 'TCC ACG ACA TAC TCA GCA CC' RP11284F219 forward 'AGG ATT GGC ACT CAC TTC GG' and reverse 'TCT CTC ACC ACG TCT GGT CT' and CCAR1 forward 'CTG ATG GCT AGC CCT AGT ATG GA' and reverse 'TGC CTT TCA TGC CCA CTA AAA ' The temperature protocol used to perform RT was ËC for h followed by ËC for min Thermal conditions of PCR reactions were Initial denaturation at ËC for min followed by cycles for sec at ËC and sec at ËC The mRNA expression levels were determined using the 2ÎÎCq method Oligonucleotides and cell transfection The small interfering RNA siRNA synthetic negative control siNC RP11284F219 siRNAs siRP11284F219 miRNC miR3p mimics and miR3p inhibitor were purchased from Shanghai GenePharma Co LtdAll primer sequence information is presented in Table I At a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency All of the oligonucleotides were transfected at a final concentration of nM using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instruction Cells were collected at h posttransfection for subsequent experimentsCell Counting Kit CCK assay and EdU labeling of proliferating cells A CCK was used for cell proliferation assay the cells were seeded into well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions Dojindo Molecular Technologies Inc The optical density was measured at nm using a spectrophotometer Thermo Fisher Scientific IncFor the EdU assay cells were incubated with µM EdU cat no ab219801 Abcam for h at ËC and fixed with formaldehyde at room temperature for min After a brief washing with PBS click reagent was added into each well and incubated in the dark for min at room temperature Followed by PBS washing the cells were stained with µgml DAPI at room temperature for min Images were captured using a fluorescence microscope Nikon Corporation and measured using Adobe Photoshop software Adobe Systems Inc The EdU labeled cells were analyzed with MoFlo Astrios BeckmanCoulter Inc Magnification x200Transwell assay and flow cytometry measurement of cell apoptosis Transwell assays were performed with a coating of Matrigel BD Biosciences mixed with culture medium mixed at ratio at ËC for h A total of 1x105 cells in µl serumfree medium were added to the upper layer of the Transwell chambers µm pore size Corning Inc and cultured for h The lower chamber contained the culture medium with FBS The migrated cells were fixed with 0cONCOLOGY REPORTS Table I Sequence of siRNAs and miRNA mimics and inhibitorsOligonucleotides siNC siRP11284F219 miRNC miR3p mimics miR3p inhibitor miR microRNA siRNA small interfering RNA NC negative controlSequence ''UUCUCCGAACGUGUCACGUTTUAUUGGCACCAAGGAUAGCUCGUUAAUCGGCUAUAAUACGCUCUUUUCUUUGAGACUCACUUCUUUUCUUUGAGACUCACU paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope Magnification x200Cellular apoptosis was detected using the Apoptosis Detection kit cat no KGF001 Nanjing KeyGen Biotech Co Ltd according to the manufacturer's instructions Cells were stained with fluorescein isothiocyanateconjugated annexin V and PI After incubated for min at ËC in the dark µl 1X Binding Buffer was added to each tube and stained cells were analyzed using BD FACS Canto II flow cytometry FACS Calibur BD Biosciences Data were analyzed using FlowJo software version Tree Star IncLuciferase reporter assay The RP11284F219 wildtype wt or mutant mut 'untranslated region 'UTR and CCAR1 wt or mut 'UTR sequences were cloned into the pmirGLO plasmid Youbio httpwwwyoubiocn cat no VT1439 The vectors µgml were cotransfected with miRNC or miR6273p mimic nM and Renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc Cell lysates were collected at h after transfection and the luciferase activities were detected with the DualLuciferase Reporter Assay system Promega Corporation according to the manufacturer's instructionsWestern blotting Cell were lysed using RIPA lysis buffer SigmaAldrich Merck KGaA and protein concentrations were assessed with the BCA Protein Assay kit according to the manufacturer's instructions Beyotime Institute of Biotechnology Shanghai China Equal amounts µg of cell protein lysates were loaded and separated by SDSPAGE transferred to a PVDF membrane and blocked with nonfat milk at room temperature for h The membranes were then incubated with CCAR1 primary antibody cat no ab70243 Abcam overnight at ËC followed by incubation with goat antimouse or goat antirabbit IgGhorseradish peroxidase conjugate secondary antibodies cat no ab205718 Abcam at room temperature for h GAPDH cat no ab181602 Abcam was used as loading control The signals were detected using the ECL system Protein Simple according to the manufacturer's instructionsIn vivo tumorigenicity analysis in mice Male BALBc nude mice age weeks weight g were obtained from Beijing Vital River Laboratory Animal Technology Co Ltd and housed at a room temperature of ËC with a h lightdark cycle The mice were maintained in an individually ventilated cage system under specific pathogenfree conditions temperature ËC humidity and fed with sterile food and water free access To evaluate the effect of RP11284F219 knockdown on the growth of lung carcinoma in vivo 5x106 siNC or siRP11284F219 treated NCIH1299 cells in µl serumfree medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin The animals were monitored daily and the following criteria for humane endpoint was used Severe tumor burden mm in diameter difficulty breathing significant bodyweight loss and clinical signs such as prostration hypothermia and significant abdominal distension Tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] Then weeks after inoculation the mice were euthanized by CO2 inhalation CO2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat The xenografts were imaged and weighedThe total RNA was then extracted from the xenografts as aforementioned Animal care and study were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi'an Jiaotong University approval no Target prediction Potential target miRNAs of RP11284F219 were predicted using LncBase V2 httpcarolinaimisathena innovationgrdiana_toolswebindexphprlncbasev2index The target genes of miR3p were predicted using three bioinformatics algorithms TargetScanV72 httpwwwtargetscanorgvert_72 and miRDB httpwwwmirdborgmininghtmlStatistics analysis Data were analyzed using the GraphPad Prism software GraphPad Software Inc and presented as the mean ± SD from ¥ independent experiments A twotailed unpaired Student's ttest or oneway ANOVA with Tukey's posthoc analysis were performed to evaluate the statistical significance P005 was considered to indicate a statistically significant difference\x0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11284F219 expression is upregulated in LC tissues and cell lines A Expression of RP11284F219 in LC tissues in comparison with adjacent healthy tissues was analyzed using RTqPCR P0001 vs adjacent tissues n13 B Expression of RP11284F219 in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 compared with normal human lung epithelial cell line BEAS2B was analyzed using RTqPCR P005 P0001 vs BEAS2B n3 LC lung carcinoma RTqPCR reverse transcriptionquantitative PCRResultsExpression of RP11284F219 is upregulated in lung carcinoma To investigate the potential role of RP11284F219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma The results demonstrated that the expression of RP11284F219 was significantly upregulated in tumor tissues compared with healthy tissues Fig 1A The expression of RP11284F219 was also analyzed in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 and normal human lung epithelial cell line BEAS2B Consistent with the findings in the tissue samples the expression of RP11284F219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line Fig 1B These results indicated that RP11284F219 may serve an oncogenic role in lung carcinomaKnockdown of RP11284F219 exerts antioncogenic effects in lung carcinoma cells To study the specific role of RP11284F219 in lung carcinoma cells RP11284F219 siRNA was transfected into NCIH1299 and NCIH460 cells Fig 2A After transfection the proliferation of these cells was measured using CCK and EdU assays Fig 2BD The results suggested that knocking down RP11284F219 significantly reduced the proliferation of lung carcinoma cells compared with the NC group Fig 2BD The invasiveness of siRP11284F219 transfected cells also significantly decreased as indicated by the data from the Transwell assay Fig 2F To further validate the invasive capability a RTqPCR assay was performed to detect the expression levels of invasionrelated genes and the results identified that both MMP2 and MMP9 were significantly decreased when RP11284F219 was downregulated Fig S1The results of flow cytometry measurement based apoptosis assay suggested that cells transfected with siRP11284F219 had a higher apoptotic rate compared with the siNC transfected group Fig 2E These data demonstrated the antitumor effects of RP11284F219 knockdown in lung carcinoma cells indicating an oncogenic role of RP11284F219RP11284F219 directly interacts with miR3p Based on the prediction of the online tool lncBase v2 from DIANA Prediction module httpcarolinaimisathenainnovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream miRNAs of RP11284F219 the first five miRNAs in the output list were tested Among the predicted potential targets it was found that miR6273p had the most significant upregulation in NCIH1299 cells transfected with siRP11284F219 Fig S2Using sequence alignment it was identified that miR3p was partially complementary with the 'UTR of RP11284F219 Fig 3A Subsequently 293T cells were transfected with the pmirGLORP11284F219wt or mut vector containing the wt or mut sequence of RP11284F219 'UTR with or without miR3p mimics Results from the luciferase reporter assay suggested that miR6273p mimics significantly decrease the signal of RP11284F219wt transfected cells but not the RP11284F219mut transfected cells indicating a direct interaction between the two noncoding RNAs Fig 3A Furthermore transfection of siRP11284F219 into NCIH1299 and NCIH460 cells resulted in the suppression of endogenous RP11284F219 leading to a significant increase in miR3p expression Fig 3B Thus these findings suggested an inhibitory effect of RP11284F219 on the expression of miR3p in lung carcinoma cellsThe expression of miR3p was detected in both lung carcinoma tissues and cell lines It was demonstrated that miR3p was significantly downregulated in carcinoma tissues Fig 3C and NCIH460 NCIH1299 and A549 cells Fig 3D compared with healthy tissues and cells Collectively these data suggested a direct interaction between RP11284F219 and miR6273p in which RP11284F219 suppresses the expression of miR3pRP11284F219 regulates the proliferation and invasiveness of lung carcinoma cells via miR3p To rescue the antitumor effects of siRP11284F219 in lung carcinoma cells the miR3p inhibitor which specifically downregulates the expression of miR3p was transfected into NCIH1299 and NCIH460 cells Fig 4A The results from the CCK and EdU assays demonstrated that treatment with siRP11284F219 0cONCOLOGY REPORTS Figure RP11284F219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis A RP11284F219 knockdown was achieved via RP11284F219 siRNA and the knockdown efficiency was verified using reverse transcriptionquantitative PCR n3 Cell Counting Kit assay was performed to measure the proliferation of B NCIH1299 and C NCIH460 cells after transfection with siRP11284F219 compared with the siNC group n5 D An EdU assay was performed to measure the proliferation of NCIH1299 and NCIH460 cells after transfection with siNC and siRP11284F219 Magnification x200 E Flow cytometry analysis was performed to determine the effects of RP11284F219 knockdown on apoptotic rates in NCIH1299 and NCIH460 cells n3 F Transwell assay was performed to determine the effects of RP11284F219 knockdown on NCIH1299 and NCIH460 cell invasion n3 Magnification x200 P005 P001 vs control group NC negative control siRNA small interfering RNA OD optical density and miRNC significantly decrease the proliferation of both NCIH1299 and NCIH460 cells Fig 4BD However the administration of miR3p inhibitor partially reversed the antiproliferative effect of siRP11284F219 indicating that RP11284F219 regulates the proliferation of lung carcinoma cells partially via miR6273p Fig 4BD In addition the 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11284F219 directly interacts with miR3p A Binding site between RP11284F219 and miR3p that was identified using the DIANA tools and a luciferase reporter assay was conducted in pmirGLORP11284F219wt or mut treated cells in the presence of miR6273p mimics or miRNC n3 P005 vs miRNC B Expression of miR3p in NCIH1299 and NCIH460 cells transfected with siRP11284F219 was analyzed using RTqPCR P001 vs siNC n3 miR3p expression in C LC tissues and D NCIH460 NCIH1299 and A549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using RTqPCR n3 P005 P001 vs adjacent tissue or BEAS2B cells NC negative control siRNA small interfering RNA wt wildtype mut mutant miR microRNA LC lung carcinoma miR3p inhibitor restored the reduction in the number of NCIH1299 and NCIH460 cells that migrated through the Transwell membrane induced by siRP11284F219 treatment Fig 4F These data indicated the participation of miR6273p in the RP11284F219mediated invasive effectThe qPCR assay results identified that both MMP2 and MMP9 expression levels were restored in RP11284F219downregulated cells when miR6273p was inhibited compared with the miRNC group Fig S3 In addition transfection with miR3p inhibitor also diminished the proapoptosis effect of siRP11284F219 in both NCIH1299 and NCIH460 cells Fig 4E Therefore it was suggested that RP11284F219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of miR3pRP11284F219 regulates CCAR1 via targeting miR3p To further evaluate how RP11284F219 exerts an oncogenic role via miR3p the publicly available algorithms of TargetScan httpwwwtargetscanorg and miRDB were used which identified CCAR1 as a potential target for miR6273p Fig 5A In order to validate this prediction miR6273p mimic was transfected into cells and the transfection efficiency was assessed The results demonstrated that transfection of miR6273p mimic increased the expression of miR3p by times compared with cells transfected with miRNC Fig S4After validating the upregulation of miR6273p mimic a CCAR1wt vector was constructed which contained the wt binding site between miR3p and the CCAR1 'UTR and CCAR1mut vector containing the mut sequence Fig 5A The results from luciferase reporter assays indicated that compared with the miRNC group the miR6273p mimic significantly decreased the luciferase activity of CCAR1wt treated cells but not the CCAR1mut treated cells suggesting a direct binding of miR3p to the 'UTR of CCAR1 Fig 5B Increased expression levels of CCAR1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues Fig 5C Moreover a significant decrease in both mRNA and protein expression levels of CCAR1 was detected upon transfecting NCIH1299 and NCIH460 cells with miR6273p mimics Fig 5D and E Thus CCAR1 may be a direct target of miR3p in lung carcinoma cells and tissuesRP11284F219 knockdown inhibits tumor growth and the expression of CCAR1 in vivo In order to investigate the effect of RP11284F219 on in vivo tumorigenicity NCIH1299 cells were transfected with siNC or siRP11284F219 and injected into the nude mice After weeks a significantly 0cONCOLOGY REPORTS Figure RP11284F219 regulates proliferation and invasiveness in lung carcinoma cells via miR3p A Expression of miR3p in NCIH1299 and NCIH460 cells transfected with miRNC or miR3p inhibitor was detected using RTqPCR analysis n3 P005 vs miRNC Cell Counting Kit assay was performed in B NCIH1299 and C NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor n5 D EdU assay was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor Magnification x200 E Flow cytometry analysis was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor n3 F Transwell assay was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor Magnification x200 n3 P005 vs siNC NC negative control siRNA small interfering RNA OD optical density miR microRNA 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure miR3p directly targets CCAR1 A Bioinformatic analysis of the predicted binding sites between the CCAR1 'untranslated region and miR3p B Luciferase reporter assay in CCAR1wt or CCAR1mut treated cells in the presence of miRNC or miR3p mimic n3 P005 vs miRNC C CCAR1 expression in LC tissues compared with adjacent healthy tissues was analyzed using RTqPCR n13 P001 vs adjacent tissue Expression of CCAR1 in NCIH1299 and NCIH460 cells transfected with miRNC or miR3p mimics was detected using D RTqPCR and E western blotting n3 P005 vs miRNC miR microRNA NC negative control wt wildtype mut mutant RTqPCR reverse transcriptionquantitative PCR CCAR1 cell division cycle and apoptosis regulator LC lung carcinoma slower proliferative rate of the tumors was observed in the siRP11284F219 group compared with the siNC group Fig 6A and B Furthermore the tumor volume and weight were significantly decreased in the siRP11284F219 group compared with the control group Fig 6A and B RTqPCR analysis also demonstrated that compared with the siNC group the tumors in the siRP11284F219 group expressed higher levels of miR6273p Fig 6C and lower levels of CCAR1 Fig 6D providing further evidence to the existence of the RP11284F219miR3pCCAR1 regulatory axis in lung carcinoma tumor tissuesDiscussionThe present study investigated the function of RP11284F219 in lung carcinoma It was initially found that RP11284F219 was significantly upregulated in both lung cancer tissues and cell lines Following the deduction of a potential oncogenic role of this lncRNA siRP11284F219 was transfected into NCIH460 and NCIH1299 cells and it was demonstrated that knockdown of RP11284F219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells In the mechanistic studies using online prediction tools and in vitro assays the results indicated that miR3p directly interacts with RP11284F219 by binding to its 'UTRThe function of miR627 was initially reported in colorectal cancer CRC Padi found that when upregulated by calcitriol miR627 targets the histone demethylase Jumonji domain containing 1A to increase methylation of histone H3K9 and suppresses the proliferative factors of CRC cells thus inhibiting the proliferation of CRC both in vitro and in vivo Moreover in CRC Sun discovered the role of miR in vitamin Denhanced efficacy of irinotecan via inhibition of the cytochrome P450 enzymemediated intratumoral drug metabolism miR is also reported to be a potential noninvasive diagnostic marker in gastric and breast cancer types In pulmonary diseases miR627 is downregulated in patients with chronic obstructive pulmonary disease and targets the highmobility group box protein to inhibit its expression thus improving transforming growth factorβ1induced pulmonary fibrosis The present results demonstrated the inhibitory effect of RP11284F219 on the expression of miR3p In addition it was identified that the miR3p inhibitor can neutralize the antitumor effects of RP11284F219 knockdown indicating that RP11284F219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating miR3p This antitumor role of miR6273p under the regulation of RP11284F219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human CRC gastric and breast cancer types\x0cONCOLOGY REPORTS Figure RP11284F219 knockdown inhibits tumor growth in vivo A Macroscopic image of xenografted tumors B Tumor volume in nude mice injected with NCIH1299 cells transfected with siNC or siRP11284F219 measured over weeks n5 C Weight of tumors in nude mice at weeks after injection of NCIH1299 cells transfected with siNC or siRP11284F219 n5 Expression levels of D miR3p and E CCAR1 in the tumor tissues from nude mice injected with NCIH1299 cells transfected with siNC or siRP11284F219 for weeks were detected using reverse transcriptionquantitative PCR n5 P005 P001 P0001 vs siNC miR microRNA NC negative control sh short hairpin RNA CCAR1 cell division cycle and apoptosis regulator Using the publicly available RNA interaction prediction algorithms the current study identified that CCAR1 which was initially shown as the target gene of miR6273p is also regulated by RP11284F219 Furthermore the regulatory axis of RP11284F219miR3pCCAR1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model The interaction between RP11284F219 and miR3p and the interaction between miR3p and CCAR1 were demonstrated by the dualluciferase assay Although this method has been used to validate RNARNA interactions in previous studies other assays such as RNA pulldown and RNA binding protein immunoprecipitation that would provide more direct evidence for the RNARNA and RNAprotein interactions should be performedCCAR1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as Adriamycin and etoposide Subsequently Kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors In human breast cancer cells as CCAR1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells In CRC cells Ou reported that CCAR1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor CCAR1 is essential for the expression of Wnt target genes as well as the neoplastic transformation of CRC cells In gastric cancer cells researchers have revealed the cooperation between CCAR1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells In lung cancer CCAR1 was reported to be an effector of Doxorubicininduced apoptosis Moreover Muthu demonstrated that certain chemical compounds that bind with CCAR1 can increase the expression of CCAR1 and induce apoptosis However a contradictory conclusion was reported in a recent study which observed that CCAR1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate Corroborating this finding in the current study via the targeting of miR3p the expression of CCA | Thyroid_Cancer |
FAM83HAS1 is a potential modulator of cancer driver genes across different tumors and a prognostic marker for ERPR BRCA patientsMagdalena RosRomero13 Alberto CedroTanda Mnica Pe±aLuna1 Marco Antonio ManceraRodrguez1 Lizbett HidalgoPrez1 Mireya CisnerosVillanueva1 Fredy Omar Beltr¡nAnaya Roco ArellanoLlamas1 Silvia JimnezMorales1 Luis Alberto AlfaroRuz1 Alberto TenorioTorres2 Carlos DomnguezReyes2 Felipe VillegasCarlos2 Elsa OchoaMendoza1 Alfredo HidalgoMiranda Breast cancer BRCA is a serious public health problem as it is the most frequent malignant tumor in women worldwide BRCA is a molecularly heterogenic disease particularly at gene expression mRNAs level Recent evidence shows that coding RNAs represent only of the total transcriptome in a human cell The rest of the of RNAs are noncoding so we might be missing relevant biological clinical or regulatory information In this report we identified nine novel tumor types from TCGA with FAM83HAS1 deregulation We used survival analysis to demonstrate that FAM83HAS1 expression is a marker for poor survival in IHCdetected ER and PR positive BRCA patients and found a significant correlation between FAM83HAS1 overexpression and tamoxifen resistance Estrogen and Progesterone receptor expression levels interact with FAM83HAS1 to potentiate its effect in OS prediction FAM83HAS1 silencing impairs two important breast cancer related pathways cell migration and cell death Among the most relevant potential FAM83HAS1 gene targets we found p63 and claudin CLDN1 to be deregulated after FAM83HAS1 knockdown Using correlation analysis we show that FAM83HAS1 can regulate a plethora of cancerrelated genes across multiple tumor types including BRCA This evidence suggests that FAM83HAS1 is a master regulator in different cancer types and BRCA in particularBreast cancer BRCA is a serious public health problem as it is the most frequent malignant tumor in women worldwide According to GLOBOCAN1 at least million new cases and a total of deaths are reported globallyBRCA is a phenotypically heterogenic disease with welldefined histological types and protein markers such as Estrogen receptor ER Progesterone Receptor PR and membrane receptor HER2 The physical and phenotypical BRCA heterogeneity is also reflected at the molecular particularly at gene expression mRNAs level This heterogeneity has been extensively studied and evidence shows that breast cancer comprises four intrinsic groups Luminal A Luminal B HER2 enriched and Basallike tumors23Molecular classification has been an important milestone in BRCA biology as it has been used to differentiate aggressive and nonaggressive tumors metastatic potential clinical prognosis and survival among other relevant cancerrelated features4 Additionally therapy responseassociated expression profiles are now available This expression profiles are able to predict if a patient can benefit from chemotherapy or antihormonal therapy5 1Laboratorio de Genmica del C¡ncer Instituto Nacional de Medicina Genmica Periferico Sur Tlalpan Arenal Tepepan Ciudad de Mxico CDMX Mexico 2Fundacin de C¡ncer de Mama FUCAM Ciudad de Mxico Mexico 3Programa de Doctorado de Ciencias Biolgicas Universidad Nacional Autnoma de Mxico Ciudad de Mxico Mexico email ahidalgoinmegengobmxScientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cThese useful clinical advances are focused on coding RNAs profiles only however recent evidence show that coding messenger RNAs represent only of the total transcriptome in a human cell6 The rest of the of RNAs are noncoding so we might be missing relevant biological clinical or regulatory information if we only focus on messenger RNAIn this regard recent papers have focused on the role of long non coding RNAs lncRNAs in cancer biology7 and in the role of specific lncRNAs in breast cancerFAM83HAS1 is a lncRNA whose expression impairs important cancerrelated pathways such as cell proliferation migration invasion and cell death in lung colorectal glial bladder ovarian and cervical cancer cells11 At the molecular level one report showed that METEGFR signaling is regulated by FAM83HAS1 and16 showed that FAM83HAS1 epigenetically silenced CDKN1A by binding to EZH2 in glioma cellsIn addition two reports showed that FAM83H antisense RNA FAM83HAS1 a0is deregulated BRCA samples High expression of FAM83HAS1 indicated an unfavorable prognosis in luminal type BRCA and earlystage BRCA Altogether this evidence shows that FAM83HAS1 is an important actor in cancer biology In this paper we identified nine novel tumor types from TCGA with FAM83HAS deregulation and used a multivariate Cox regression analysis to demonstrate that FAM83HAS1 expression is a marker for poor survival in Progesterone receptor PR positive BRCA We found a significant correlation between FAM83HAS1 overexpression and tamoxifen resistance in luminal BRCA patients Using KaplanMeier and Cox regression analysis we found that estrogen and progesterone receptor expression levels interact with FAM83HAS1 to potentiate its effect in OS prediction Using FAM83HAS1 short hairpin knockdown coupled with microarray analysis we demonstrate that FAM83HAS1 silencing impairs two important breast cancer related pathways cell migration and cell death We further validate this phenotypic effect with in a0vitro migration and caspase assays Among the most relevant potential FAM83HAS1 gene targets we found p63 and claudin CLDN1 to be deregulated after FAM83HAS1 knockdown Using correlation analysis we show that FAM83HAS1 can regulate a plethora of cancerrelated genes across multiple tumor types including BRCAResultsFAM83HAS1 is deregulated in multiple tumor types Multiple studies have related FAM83HAS1 high expression levels with different tumors including luminal breast cancer11 These findings suggest an important role for FAM83HAS1 in cancer tumor biology We therefore screened FAM83HAS1 expression levels in the TCGA database which comprises data from different tumor types and the correspondent normal tissues As expected we found significant FAM83HAS1 expression deregulation in different tumor types Fig a01A Log2FC p Some of these FAM83HAS1 expression deregulation data has been reported previously111216 but we have also found significant deregulation of FAM83HAS1 in nine additional tumor types Fig a01B Log2FC p Interestingly FAM83HAS1 was upregulated in different tumor types but downregulated in acute myeloid leukemia LAML suggesting a different mechanism for this particular malignancy Fig a01BFAM83HAS1 expression level is enriched in BRCA locallyadvanced tumors It was reported that FAM83HAS1 expression is a prognostic marker for luminal breast cancers12 We were interested to see if FAM83HAS1 expression was more widely associated with BRCA tumors since we and others found alterations for this lncRNA in a large number of malignancies As shown in Fig a01B FAM83HAS1 is significantly upregulated in all BRCA patients not only in the luminal subtype BRCA FAM83HAS1 overexpression is also marginally associated with BRCA locally advanced II and III clinical stages oneway ANOVA p Fig a01CFAM83HAS1 is a prognostic marker for ER and PR positive BRCA and its expression is related with tamoxifen resistance Altogether these widespread alterations in FAM83HAS1 expression suggested that its expression could be a prognostic biomarker for all BRCA subtypes but as mentioned above FAM83HAS1 was previously reported to have particular prognostic association with the BRCA luminal subtype1216 To test if FAM83HAS1 is a widespread or a luminal specific prognostic marker in BRCA we first screened FAM83HAS1 expression as a prognostic marker for all BRCA tumors We did not find significant association with poor OS in the Cox regression model n CI [] Cox p value however we observed a clear tendency in poor survival prognosis in the FAM83HAS1 high expression group see Fig a02A We further validated these results in an independent Mexican patient cohort Fig a02B with all the BRCA subtypes The general clinical features of this cohort are listed in Table a0 We then tested if this effect was due to FAM83HAS1 interacting with other significant clinical and survivalrelated variables in particular luminal typerelated FAM83HAS1 predictive value was significant when interacting with Immunohistochemistry IHCdetected Progesterone receptor n HR CI [] Cox p value supplementary Table a0 Marginal but not significant association was observed with ER status supplementary Table a0 KaplanMeier analysis of PR logrank p or ER positive patients logrank p showed significant association poor OS when FAM83HAS1 was overexpressed Fig a02C D No effect in survival rate was seen when FAM83HAS1 was overexpressed in PR and ER negative patients supplementary Figs a0 and This data strongly suggest that FAM83HAS1 is an independent prognostic marker for OS in PR positive BRCA subtype and confirms with further statistical analyses previous findings made by12We found a significant association with IHCdetected PR and ER status in the survival context Fig a0 C D supplementary Table a0 We then analyzed tamoxifen treatment resistance or sensitivity in our independent cohort Fig a02E and we found that FAM83HAS1 overexpression was significantly related with poor tamoxifen initial response n OR onetailed Fexact test p Scientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 expression is altered in multiple human tumors A FAM83HAS1 expression levels TPM in tumors from the TCGA database In green are shown normal tissue samples in blue tumor samples B FAM83HAS1 is aberrantly expressed in nine not previously reported tumors Color code as in A C FAM83HAS1 is enriched in locally advanced BRCA clinical stages II and IIIWe did not find a significant prognosis association for unreported tumors shown above in the Gene expression Profiling Interactive Analysis GPIA database see methods We could determine however a strong correlation between high FAM83HAS1 expression and poor OS in skin cutaneous melanoma SKCM patients n HR logrank test p Supplementary Fig a0ER and PR expression levels potentiate FAM83HAS1 prediction of survival in BRCA patients In order to further characterize our previous finding regarding ER and PR status and its association with FAM83HAS1 in BRCA prognosis we built a risk model taking into account ER PR and FAM83HAS1 expression levels in the same analysis This model fundamentally displays ER PR and FAM83HAS1 interaction and potentiation of the poor OS prediction in BRCAWe first calculated the Allred score17 to identify IHC ER and PR level of positivity ieER and PR expression levels from TCGA data We then obtained FAM83HAS1 expression levels from the TCGA cohort and divided it in four strata quartiles We then multiplied these two values Allred score values and FAM83HAS1 quartile values and the product was a new risk score We obtained four risk groups with the above described method shown in Fig a0 As shown here combination of very high FAM83HAS1 and ERPR expression levels potentiates Scientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 overexpression is a marker for poor prognosis in ERPR BRCA patients A Overall survival analysis Cox Regression for the BRCA TCGA cohort B KaplanMeier analysis for our independent BRCA cohort C KaplanMeier analysis for PR and D ER positive BRCA patients from the TCGA cohort E FAM83HAS1 expression is associated with tamoxifen resistance in BRCA patientsCharacteristicAgeTumor gradeClin stageERPRHER2Lymph NodesRecurrenceMetastasisClassIIIIIISV IA IIA IIBIIIA IIIB IIICPositiveNegativePositiveNegativePositiveNegativePositiveNegativeNAPositiveNegativePositiveNegativeFrequencyPercentTable Clinicalpathological characteristics of population n In this Mexican cohort none of these clinical variables were significantly correlated with FAM83HAS1 expression levelScientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 ERPR expression potentiates FAM83HAS1 death risk prediction Expression levels from ER and PR were calculated using Allread scores FAM83HAS1 expression levels were calculated from the quartile distribution of the log2 values TANRIC We then multiplied Allread scores and FAM83HAS1 expression levels in order to define risk groups see methods and main texthigh risk of decease in the TCGA cohort KaplanMeier model one tailed logrank p Cox hazard proportional risks for decease in these four groups were for low risk for moderate risk for high and for very high risk This data confirms a strong interaction between ER PR and FAM83HAS1 expression levels in BRCA Interaction between these three variables potentiates poor OS prediction in BRCA patients and possibly other hormonerelated human tumorsFAM83HAS1 potentially regulates a plethora of cancerrelated genes In order to better understand FAM83HAS1 role in BRCA we performed a differential expression analysis using the TCGA BRCA cohort We compared high versus low FAM83HAS1 RNA expression level samples see methods We found differentially expressed genes between these two groups Log2FC and padjvalue The vast majority of these transcripts RNAs were found to be downregulated in this analysis These results might suggest candidate target genes for FAM83HAS1 Fig a04AAmong the downregulated RNAs we identified several cancerrelated transcripts such as fibroblast growth factor FGF4 fibroblast growth factor FGF21 leptin LEP Claudin CLDN17 cadherin CDH9 Tumor Necrosis Factor receptor TNFRSF11B BCL2associated X BAX Tumor protein p53 TP53 and Phosphatase and tensin homolog PTEN see Table a0After pathway enrichment analysis we found a significant downregulation of cellular migration synthesis of steroids and lipid metabolism Fig a04B Gene set enrichment analysis GSEA also showed alterations in apoptosis and p53signalling pathways Fig a04C Taken together this evidence suggests an important regulatory role for FAM83HAS1 in cancerrelated pathwaysFAM83HAS1 is present both in nucleus and cytoplasm in ERPR BRCA cells To further characterize FAM83HAS1 functional role in BRCA we measured its expression in nine breast cancer cell lines including MDAMB231 HCC1187 MCF7 SKBR3 BT20 Hs578 ZR75 and one nontransformed cell line MCF10 Fig a05A As expected FAM83HAS1 was upregulated in transformed cell lines We then performed cellular fractionation assays in MCF7 cells and detected its enrichment in the cytoplasmic fraction of total input RNA Fig a05B LncATLAS screening further confirmed this observation as MCF7 cells display both FAM83HAS1 cytoplasmic and nuclear localization Supplementary Fig a0FAM83HAS1 knockdown deregulates transcripts expression in MCF7 cells In order to gain further insight into the potential FAM83HAS1 targets we performed shmediated FAM83HAS1 silencing experiments in MCF7 cells As shown in Fig a05C we obtained of silencing efficiency after a0h of plasmid transfection After knockdown we performed microarray experiments in MCF7 cells We identified differentiallyexpressed genes in the FAM83HAS1silenced cells FC and p value Fig a05D Key cancerrelated transcripts were identified such as CLDN1 TP63 FGF14 DDX60 and DRAM see Table a0 transcripts were found to be downregulated whereas were upregulated Fig a05D Table a0 Among the most upregulated RNAs we found TP63 and CLDN1 These genes can also be potential candidate target genes for FAM83HAS1 activityFAM83HAS1 silencing impairs cellular migration and apoptosis Pathway enrichment analysis showed that the most activated cellular processes in the shFAM83HAS1 condition are migration and cellular Scientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cDescriptionMyosin Light chain Myosin heavy chain Actin alpha LeptinMicroRNA Nuclear receptor H4Fibroblast Growth Factor Fibroblast Growth Factor Claudin Insulin like growth factor binding protein Cadherin Hydroxydelta5steroid dehydrogenaseUDP glucuronosyltransferase family member A1Alcohol dehydrogenase 1A class I alpha polypeptideCytochrome P450 family subfamily A member Deathassociated protein kinase Tumor protein p53TNF receptor superfamily member 11bBCL2 associated X apoptosis regulatorBH3like motif containing cell death inducerPhosphate and tensin homologPathwayTight junction signalingWNT signaling pathwayWNT signaling pathwayCell migrationCell migrationCell migrationMAPK signalingFocal adhesionMAPK signalingFocal adhesionEpithelial to mesenchymal transitionPI3K pathwayCadherin signalingSteroid synthesisSteroid synthesisDrug metabolismDrug metabolismCell death regulationCell death regulationCell death regulationApoptosis regulationCell death regulationCell survival signalingFold change log2 padjvalue338E951E106E110E807E147E978E374E175E244E157E405E313E115E500E422E 664E107E146ETable Examples of differentially expressed genes in the FAM83HAS1 high versus low BRCA samplemotility altered molecules p value range and cellular death pathways molecules p value range Fig a05E These data are in accordance with our previous differential expression results in BRCA samples Fig a0 further supporting a master regulatory role for FAM83HAS1 in breast cancer cellsWe then aimed to functionally validate that both cellular processes are altered after FAM83HAS1 silencing We thus performed Transwell migration assays and caspase activity assays in MCF7 cells As shown in Fig a06A MCF7 cells migration significantly increases after shFAM83HAS1 transfection one tailed Ttest p We then performed matrigelinvasion assays and observed an increase a0h after transfection but this observation did not reach statistical significance Supplementary Fig a0 Furthermore we did not find significantly altered invasion related genes or pathways in the microarray assays nor the differential expression analysis in the TCGA cohort further suggesting that FAM83HAS1 is not involved in invasion in this modelCaspase assays identified a significantly increase in enzymatic activity Ttest p after a0h of FAM83HAS1 silencing which corroborates its role in apoptosis mediated cell death Caspase is the primary activator of apoptotic DNA fragmentation18 Significant increase in caspase activity in the shFAM83HAS1 condition suggests FAM83HAS1 regulation of late stage apoptosis Fig a06BMigration and cell death alterations are enriched in the FAM83HAS1 low expression group in BRCA samples To further validate our previous in a0vitro results we performed single sample GSEA ssGSEA in a BRCA independent cohort Gene Expression Omnibus dataset GSE115577 see methods In this approach gene sets are ranked according to absolute expression values in every sample rather than by a comparison with another sample We first stratified the GSE115577 cohort onto two groups samples with high levels of FAM83HAS1 RNA and samples with low FAM83HAS1 levels using the quartile approach described above We then aimed to know if the gene sets corresponding to migration apoptosis and other cell death processes like necrosis were significantly enriched in any of these two FAM83HAS1 expression groups As expected we found a significant enrichment of the migration Normalized Mutual Index [NMI] score AUC p value 918e and apoptosis NMI score AUC p value processes in the FAM83HAS1low expression group Fig a0 B Interestingly we also found a strong enrichment of the necrosis pathway in this BRCA cohort NMI score AUC p value 13eFAM83HAS1 and its potential target genes are coderegulated across multiple tumor types We found that FAM83HAS1 is upregulated not only in BRCA but also in other tumors see Fig a0 We reasoned that if upregulated FAM83HAS1 may be exerting similar regulatory roles in other tumors as well In order to show this we correlated FAM83HAS1 expression levels with its potential target genes BAX CLDN1 CLDN17 DRAM DDX60 FGF4 FGF14 LEP PTEN TNFRSF11B TP53 and TP63 As shown in Fig a0 7A FAM83HAS1 is strongly correlated with these coding genes across multiple tumor types namely BLCA BRCA CESC COAD LAML LUAD LUSC OV PAAD PRAD READ SKCM STAD TGCT UCEC and UCS We then calculated the hazard ratio HR of decease event related to FAM83HAS1 and its potential targets expression in different tumor types from TCGA Fig a07B We found a significant association logrank Scientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 High FAM83HAS1 levels in BRCA samples are correlated with downregulation of cancerrelated inhibitors A Volcano plot depicting differentially expressed transcripts in high versus low FAM83HAS1 levels Points in grey nonsignificant blue p value significant red fold change and p value significant B Migrationrelated and steroid metabolism genes are significantly downregulated when FAM83HAS1 is highly expressed in BRCA C GSEA analysis showed enrichment for apoptosis cell death and p53signalling pathways in the FAM83HAS1high samplestest p of FAM83HAS1 overexpression and a high decease HR in BRCA PAAD and SKCM Fig a07B The expression of FAM83HAS1 potential targets is also associated with high or low HR in these tumors Some of the coding genes that we either found in the differential expression analysis Fig a0 or after FAM83HAS1 knockdown in MCF7 cells Fig a05D are also coderegulated in other tumor types Fig a07C Altogether this evidence suggests a wide master regulatory role for FAM83HAS1 not only in ER PRBRCA but in other tumor types such as PAAD and SKCMDiscussionLong noncoding RNAs are molecules that exert numerous roles in human cancers as their biological activities involve regulation of cell proliferation cell death differentiation migration and invasion Deregulation in lncRNAs expression has also been associated with clinical outcome LncRNAs can affect expression of thousands of genes so they are regarded as key master regulators7In this work our aim was to investigate a wider deregulation for FAM83HAS1 expression in tumors focusing on its functional and clinical role in breast cancer and the identification of potential FAM83HAS1 targets We found that FAM83HAS1 was overexpressed in nine different tumor types in the TCGA database In particular FAM83HAS1 is overexpressed and significantly correlated with a worse clinical outcome in PR positive detected by immunohistochemistry BRCA subtypes in the TCGA breast cancer cohort One previous report12 shows that FAM83HAS1 high expression indicated unfavorable prognosis in luminal breast cancer and was an independent prognostic indicator To the best of our knowledge this is the first report that suggests variable interaction between FAM83HAS1 and IHCdetected PR and ER in the clinical outcome context Furthermore we demonstrate that ER and PR expression levels can act as potentiators of FAM83HAS1 poor OS prediction In particular ER and PR high expression levels together with FAM83HAS1 overexpression confers a very high risk HR of decease in BRCA patients This data suggest an important clinical role for FAM83HAS1 in ERPR positive breast cancer It is currently unknown however if these statistical and clinical interactions are reflected at the biological or molecular level and future studies must address this issueScientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 knockdown in MCF7 cells is associated with deregulation of multiple cancerrelated genes A FAM83HAS1 expression profile in breast cancer cell lines B FAM83HAS1 is localized both in nucleus and cytoplasm but is enriched in cytoplasm in MCF7 cells C short hairpin RNA silencing of FAM83HAS1 in MCF7 cells D shmediated silencing of FAM83HAS1 induces differential expression in genes in MCF7 cells Grey nonsignificant blue p value significant green fold change significant red p value and fold change significant E Cellular migration and cell death are two significantly enriched pathways after FAM83HAS1 silencing in MCF7 cellsGene symbolDescriptionCLDN1TP63H3F3CFGF18DDX60IFI6DRAM1GPER1PARP9GSTM3Claudin Tumor protein p63H3 histone family 3CFibroblast growth factor DEAD AspGluAlaAsp box polypeptide Interferon alphainducible protein DNAdamage regulated autophagy modulator Gprotein coupled estrogen receptor PolyADPribose polymerase family member Glutathione S S transferase mu brainPathwayEpithelial to mesenchymal transitionApoptosis signalingDNA replicationMAPK signalingFocal adhesionCell death regulationInterferon signalingCell death regulationEndocrine resistanceDNA repairDrug metabolismFold change shFAM83HAS1 vs shRANDOM p valueTable Examples of differentially expressed genes after FAM83HAS1 knockdown in MCF7 cellsScientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cFigure a0 FAM83HAS1 knockdown impairs cellular migration and induces cell death in breast cancer cells A Transwell in a0vitro migration assays in MCF7 cells B Caspase activity assays in MCF7 cells C Heatmap of the gene sets enriched in the high FAM83HAS1 expression group blue compared with that of FAM83HAS1 low expression samples green D Constellation Map of the gene sets Three connected clusters of gene sets migration apoptosis and cell death and necrosis pathways are detected in the lowFAM83HAS1 groupWe were also able to find a significant correlation between FAM83HAS1 high expression and poor tamoxifen response in BRCA patients This association could partially explain the reduced clinical response in FAM83HAS1 high expression groupWe also report that FAM83HAS1 overexpression in TCGA breast cancer samples is associated with downregulation of migration and cell deathrelated transcripts like FGF4 FGF21 LEP CLDN17 TP53 BAX and TNFRSF11B Accordingly we also found that FAM83HAS1 knockdown significantly deregulates migration and apoptosisrelated genes such as TP63 and CLND1 Transwell migration assays showed that indeed cellular migration increases after FAM83HAS1 silencing LEP and CLDN1 had been both shown to induce cellular migration and epithelial to mesenchymal transition EMT in breast cancer cells19 further suggesting a FAM83HAS1 role in the early steps of migration Taken together this data might explain the underlying mechanisms related to FAM83HAS1 cell migration impairment in breast cancer cellsFAM83HAS1 might play a dual role probably due to cellular context FAM83HAS1 was involved in regulation of cell proliferation migration and invasion processes that were decreased after FAM83HAS1 knockdown in lung cancer cells Further analysis indicated the cell cycle was arrested at the G2 phase after FAM83HAS1 knockdown11 In the same report they found that METEGFR signaling was regulated by a0FAM83HAS1 These conflicting results may be due to cellular context or specific regulation mechanisms and henceforth specific molecular targetsWe also identified that FAM83HAS1 overexpression is associated with downregulation of cellular deathrelated transcripts like BAX TNFRSF11B and P53 In a0vitro assays also show that FAM83HAS1 silencing increases cellular death possibly by up regulating genes like p63 One previous report14 showed that cell death was markedly increased after with a0FAM83HAS1 a0knockdown in colorectal cell lines FAM83HAS1 Notch1 and Hes1 were significantly increased in colorectal cancer samples and cell lines Cell proliferation was inhibited with FAM83HAS1 knockdown and this effect mediated by a0FAM83HAS1 a0could be reversed by Notch1 regulators14It is currently not clear however if FAM83HAS1 has a direct or an indirect effect in gene regulation In this regard it has been shown that FAM83HAS1 epigenetically silenced CDKN1A by binding to EZH2 in glioma cells24 In our differential expression analysis we demonstrate that FAM83HAS1 is mostly downregulating gene expression This data might suggest an inhibitory regulation role for FAM83HAS1 Future studies must address Scientific RepoRtS 101038s41598020710622Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The expression of master regulators of cancer such as p53 and p63 are dependent of FAM83HAS1 A FAM83HAS1 expression levels are strongly correlated with potential target genes TP53 TP63 BAX CLDN1 CLDN17 CDH9 TNFRSF11B PTEN FGF4 FGF14 DRAM DDX60 across different tumors BLCA BRCA CESC COAD LUAD LUSC PAAD PRAD READ OV STAD UCEC UCS SKCM B Overall survival heatmap depicting that overexpression of FAM83HAS1 confers high risk of death in BRCA PAAD and SKCM patients C FAM83HAS1 and its potential target genes are deregulated in different tumorsthese mechanisms as we cannot discard that FAM83HAS1 may regulate master gene expression via recruiting epigenetic complexes eg EZH2 In addition the exact role for FAM83HAS1 in upregulated genes remains obscure We cannot discard a subtle alternative role for this lncRNA in gene activation and future studies must address this issueOur results also show that FAM83HAS1 is present both in nucleus and cytoplasm of breast cancer cells It is possible that this lncRNA is playing a different role in cytoplasm and future studies must focus on this questionIn conclusion FAM83HAS1 is a lncRNA that is deregulated in multiple cancers and is a promising molecule that can perform as an independent prognostic factor in ER PR positive breast cancer FAM83HAS1 deregulation is associated with migration and cell death impairment in BRCA samples and breast cancer cells and may regulate a plethora of cancerrelated gene targets such as p63 BAX LEP and CLDN1MethodsThe Cancer Genome Atlas TCGA and Gene expression omnibus GEO datasets FAM83HAS1 expression levels were screened in the tumor datasets see supplementary Table a0 for details from TCGA and correspondent normal tissues using the Gene expression Profiling Interactive Analysis GPIA platform gepia cance rpkucn The tumors included are enlisted as follows Acute myeloid leukemia LAML Adrenocortical carcinoma ACC Bladder Urothelial Carcinoma BLCA Brain Lower Grade Glioma LGG Breast invasive carcinoma BRCA Cervical squamous cell carcinoma and endocervical adenocarcinoma CESC Cholangiocarcinoma CHOL Chronic Myelogenous Leukemia LMCL Colon adenocarcinoma COAD Esophageal carcinoma ESCA Glioblastoma multiforme GBM Head and Neck squamous cell carcinoma HNSC Kidney Chromophobe KICH Kidney renal clear cell carcinoma KIRC Kidney renal papillary cell carcinoma KIRP Liver hepatocellular carcinoma LIHC Lung adenocarcinomaLUADLung squamous cell carcinoma LUSC Mesothelioma MESO Ovarian serous cystadenocarcinoma OV Pancreatic adenocarcinoma PAAD Prostate adenocarcinoma PRAD Rectum adenocarcinoma READ Sarcoma SARC Skin Cutaneous Melanoma SKCM Stomach adenocarcinoma STAD Testicular Germ Cell Tumors TGCT Thyroid carcinoma THCAUterine Carcinosarcoma UCUterine Corpus Endometrial Carcinoma UCEC Uveal Melanoma UVMScientific RepoRtS 101038s41598020710622Vol1234567890wwwnaturecomscientificreports 0cPotential target genes expression correlation Hazard ratio HR map coexpression map and BRCA stage plots were also generated in the GPIA platform FAM83HAS1 expression levels were considered significantly correlated with tumors when log2FoldChange and p value Microarray generated expression data was downloaded from the GEO dataset GSE115577 This dataset includes RNA levels from BRCA samples analyzed with the Affymetrix HTA platform Downstream analysis is described belowIHCdetected hormonal receptors and FAM83HAS1 risk model Clinical information of the BRCA patients was downloaded from the TCGA database porta lgdccance rgov FAM83HAS1 expression levels were downloaded from the TANRIC tool iblmdand erson tanri c_desig nbasic mainhtml We first searched for the ER and PR status and the numerical value for percent stained cells also available in the clinical data We then calculated the Allred score17 which measures the stain intensity and stain pattern for ER and PR positivity | Thyroid_Cancer |
New diseaseCase reportAtypical manifestations of COVID19 in general practice a case of gastrointestinal a0symptomsSardam Faraidon Wahab1 Brian Bridal L¸gstrup2 SUMMARYDuring the previous months we have seen the rapid pandemic spread of SARS CoV2 Despite being considered a respiratory virus it has become clear that other clinical presentations are possible and some of these are quite frequent In this paper a case of a man in his late 70s showing atypical symptoms in general practice is presented Apart from fever the patient complained of diarrhoea borborygmus loss of appetite and nausea He developed no respiratory symptoms during his disease Due to his symptoms malignant disease was suspected and he was referred for further testing which revealed typical COVID19 findings on a chest CT scan The occurrence of atypical symptoms is discussed including the importance of recognising these in an ongoing pandemicBACKGROUNDIn December a cumulation of patients with pneumonia of unidentified cause was registered in Wuhan Hubei Province China Prior to this six strains of human pathogenic coronaviruses had been identified In February the WHO and the International Committee on Taxonomy of Viruses established the classification of the seventh human pathogenic virus SARS CoV2 as the disease causative agent of COVID191 While the name of the virus indicates respiratory disease the ongoing pandemic has shown that COVID19 is capable of causing symptoms from several an systems either concomitant with respiratory illness or as the only manifestation Recognising the clinical characteristics of COVID19 typical as well as atypical is of high importance in the prevention of further spread of the virus In this report an atypical case of COVID19 in general practice is presented Furthermore the significance of alternative manifestations is discussedCASE PRESENTATIONThe case of a man in his late 70s is presented The patient had a medical history of herpes zoster and stroke without sequelae He first contacted his general practitioner in mid March due to days of fever which was gradually decreasing Rectal temperature was °C The patients complaints were musculoskeletal body aches influenza like symptoms and headache These symptoms had subsided at the time of initial contact He also experienced rumbling of the stomach diarrhoea a general feeling of malaise and unease weight loss and night sweats He denied shortness of breath cough or other airway related symptomsAt the objective examination the patient appeared tired and exhausted but with normal awareness contact and cerebral condition There were no signs of respiratory distress and respiratory frequency was per minute Examination of the eyes and oral cavity as well as auscultation of the heart and lungs were normal There was no palpable lymphadenopathy in the head neck and periclavicular regions Abdominal inspection palpation percussion and auscultation were with normal findings Laboratory results are presented in table Blood pressure was mm Hg and pulse was estimated to be approximately beats per minute Urine dipstick showed trace of protein The condition was diagnosed as a viral infection and blood samples were collected for further testing He was given a control appointment days laterAt his control appointment the patient reported poor appetite and nausea and food intake was sparse Otherwise symptoms had remained the same with a continuous feeling of malaise There was no stomach pain but he had abdominal discomfort His headache had diminishedThe objective examination was with unchanged general condition The patient still appeared exhausted but otherwise his respiratory condition and appearance were natural Digital rectal examination revealed an enlarged and hard prostate with normal lateral boundaries No faeces or blood was seen on the examination glove and there was no pain on examination There was an increase in C reactive protein CRP to with a normal white and red blood cell count Due to continuous diffuse symptoms primarily abdominal discomfort and weight loss the patient was sent for further investigations This included occult cancer screening with thorough blood work and a CT scan of the throat chest abdomen and pelvisTwo weeks after the onset of symptoms the CT scan showed bilateral ground glass opacities GGO in the lungs raising the suspicion of infectious or postinfectious foci As a result of these findings combined with the short history fever and an increase in CRP COVID19 was thought of as a possible diagnosis The patient was tested for SARS CoV2 with materials obtained from tracheal suctioning The test came out positive for SARS CoV2 and negative for influenza virus and respiratory syncytial virus In the mean time the patients condition had deteriorated and he 1General Medicine Hospitalsenhed Midt Vib Denmark2Cardiology Aarhus University Hospital Aarhus N DenmarkCorrespondence toSardam Faraidon Wahab sardam w hotmail comAccepted August BMJ Publishing Group Limited No commercial re use See rights and permissions Published by BMJTo cite Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020Wahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseaseTable Biochemical profileTestCRP mgLAlanine aminotransferase ULMonocytes 109LLeucocytes 109LNeutrophils 109LEosinophils 109LBasophils 109LLymphocytes 109LThrombocytes 109LHaemoglobin gLErythrocyte sedimentation rate mmhourReticulocytes 1012LHaptoglobin gLFerritin µgLTransferrin µmolLPlasma iron µmolLBlood glucose mmolLPlasma creatinine µmolLeGFR mLminPlasma kappa chain Ig free mgLPlasma lambda chain Ig free mgLPlasma kappa chain to lambda chain ratio Ig freeIgM gLHigh density lipoprotein mmolLValue day day day Reference rangeDay indicates first contact to general practice CRP development is denoted and the rest are results from day The following tests were normal HbA1c INR potassium sodium alkaline phosphatase total bilirubin TSH antinuclear antibodies IgA urate alpha fetoprotein HBsAg anti HCV HAV IgM folate amylase M component IgG cobalamin B12 PSA albumin and human choriogonadotropinCRP C reactive protein eGFR estimated glomerular filtration rate HAV hepatitis A virus HbA1c Haemoglobin A1c HBsAg hepatitis B surface antigen HCV hepatitis C virus INR international normalized ratio PSA prostate specific antigen TSH thyroid stimulating hormonefelt unwell Therefore the doctor admitted the patient to the hospital at the COVID19 isolation floorAt admission oxygen saturation was without oxygen therapy Furthermore he had a respiratory frequency of per minute temperature of °C blood pressure of mm Hg and otherwise stable vital parameters During the first days of admission he developed an oxygen demand of up to Lmin to uphold an oxygen saturation of The patient was hospitalised for a total of days Throughout the course of the disease he did not complain of regular sore throat stuffy nose sneezing cough dyspnoea or other airway related symptomsINVESTIGATIONSThe suspicion of COVID19 in our patient was raised by the radiology department The chest CT scan was performed with arterial phase contrast It mainly showed peripheral but also central peribronchovascular GGO Streaky consolidations were seen in the right upper lobe Multiple swollen lymph nodes were detected in the mediastinum and both lung hila The key findings are presented in figures There were insignificant laminar subsegmental atelectasis in the posterobasal areas of both lower Figure CT scan in axial plane showing streaky consolidations in the right upper lobe arrowslobes No pericardial or pleural effusions were seen Apart from minimal mucosal thickening and polyp like changes in the right maxillary sinus there was no other pathology detected in the airwaysOUTCOME AND FOLLOWUPThe patient was seen for a follow up appointment months after discharge He reported complete recovery without sequelae On discharge he felt fatigue which gradually subsided in the following weeks There were no complications during Figure CT scan in axial plane showing ground glass opacities arrowsWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseaseTable Biochemical profile at follow up weeks after dischargeTestReference rangeValueFigure CT scan in axial plane showing ground glass opacities extending to the pleura arrowshospitalisation He particularly mentioned a pronounced positive effect of oxygen therapy which increased his appetite and general condition within hours He was slowly regaining the kg of weight that he had lost CRP alanine aminotransferase and blood monocytes had all normalised weeks after initial contact Follow up blood tests are shown in table DISCUSSIONSARS CoV and the Middle East respiratory syndrome coronavirus are among the previously identified human pathogenic coronaviruses Both are known to cause respiratory and enteric illness Gastrointestinal GI symptoms appear to be less common C reactive protein mgLAlanine aminotransferase ULMonocytes 109LLeucocytes 109LNeutrophils 109LLymphocytes 109LThrombocytes 109LHaemoglobin gLTotal cholesterol mmolLTriglycerides mmolLCreatine kinase ULLow density lipoprotein mmolLHigh density lipoprotein mmolLin SARS CoV2 compared with SARS CoV3 but studies are continually published Since COVID19 is still a novel disease it might be too early to conclude which symptoms are typical and which are not A recent comprehensive systematic review and meta analysis reported a pooled prevalence of digestive symptoms of The three most common GI manifestations were loss of appetite diarrhoea and nausea or vomiting While fever was the most frequent manifestation and relatively constant across the studies shown in table diarrhoea ranged from to In the meta analysis by Mao et al4 the frequency of diarrhoea ranged from to It is also important to note that symptoms from different an systems may present at different times in the course of the disease For instance diarrhoea and cough may occur after fever has abated5 These circumstances which may complicate the diagnostic process are important to recogniseThe cause of these different presentations is an interesting matter Factors such as genetics age immunological response and viral properties could be relevant explanations As a result of a synonymous mutation variations in viral components responsible for antigenicity and immunogenicity were found in a recent study This indicates the possible existence of strains with either increased or decreased virulence6 The founder effect must be taken into consideration though Further studies are needed to validate the significance of these findings and contribute to the understanding of the evolutionary trends of SARS CoV2 Studies of the already known coronaviruses have shown that the virions resemble a wreath or a crown due to the conformation of the surface glycoproteins This explains the Latin name corona In contrast to most enveloped viruses these glycoproteins enable the virus to withstand the conditions of the GI tract and thus spread faeco orally7 Xiao et al8 found that of patients had SARS CoV2 RNA in stool Among these still tested positive in faecal samples after converting to negative in respiratory samples Similar to SARS CoV virological studies Table Prevalence of symptoms Sore throatDyspnoeaFeverCough Diarrhoea MyalgiaFigure CT scan in coronal plane showing mediastinal lymph node enlargement arrowZhang et al21Liu et al22Xu et al23Chen et al24Myalgia or fatigueWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0cNew diseasehave pointed towards the ACE2 receptor as the site of cell entry of SARS CoV29 Previous studies have shown that ACE2 is an enzyme which physiologically counters the activity of the renin angiotensin aldosterone system and thus decreases blood pressure10 It is a membrane bound receptor which is expressed in the vascular endothelia cardiovascular and renal tissue epithelium of the small GI tract and testes ACE2 is abundantly present in the alveolar epithelium of the lungs and the small intestinal tract including the duodenum jejunum and ileum11 The mechanism by which GI manifestations occur is presumably multifactorial Positive immunofluorescent staining of ACE2 and intracellular viral capsid protein in GI tissue has been reported8 Combined with the presence of viral RNA in faecal samples this suggests secretion of infectious virions in faecal matter These findings indicate that direct virus mediated tissue damage may be a possible pathophysiological explanation The endothelial expression of ACE2 facilitates the involvement of vascular beds across several ans Histopathological examination of the small intestine has shown endotheliitis of the submucosal vessels with apoptotic bodies and accumulation of inflammatory cells This may cause endothelial dysfunction vasoconstriction and mesenteric ischaemia13 Evidence of interstitial oedema with infiltration of plasma cells and lymphocytes in GI tissue has also been reported8 These findings contribute to the understanding of GI manifestations of COVID19It is of high importance that healthcare workers are familiar with typical as well as atypical presentations and the course of this novel disease Due to the focus on respiratory symptoms in the early stages of the pandemic our patient was not initially suspected for COVID19 and thus he was sent for further testing without isolation In our case the main suspicion was influenza disease or occult cancer Among the different blood tests in table the elevated ferritin level particularly stands out It is known that ferritin levels may increase during inflammatory infectious and malignant disease among others Zhou et al14 found that ferritin levels were clearly elevated in cases of COVID19 with fatal outcome compared with survivors Another recent study found significantly elevated ferritin levels in patients with severe COVID19 and it was the last blood parameter to normalise A decrease in ferritin levels was not seen along with patient improvement Therefore it was suggested that it is a sensitive marker of severe COVID19 but it cannot be used for disease assessment15 Moreover it has been found that elevated ferritin erythrocyte sedimentation rate CRP fibrinogen and procalcitonin were higher in patients with thrombotic complications16 The present knowledge thus suggests that elevated ferritin level is a marker of severe COVID19 and is associated with thrombotic complicationsAfter the CT scan the main differential diagnoses were pneumonia eosinophilic pneumonia and COVID19 A systematic review of chest CT results in patients showed that GGO was the most frequent finding and was present in of the patients while had bilateral lung involvement17 Radiological findings of GGO have also been reported in asymptomatic patients18 This demonstrates the importance of guidelines being continually updated and communicated to clinicians As already mentioned a significant number of patients are tested positive for virus RNA in stool samples Although it is still not clear to what extent SARS CoV2 spreads faeco orally testing of stool should be further investigated in order to optimise disease controlAs an additional note related to atypical symptoms a recent study found that patients might present with conjunctivitis as the only symptom of COVID1919 Finally some patients experience Patients perspectiveIt all began when I came home from tennis and felt slightly cold with a mild fever In the following days the fever continued and fluctuated between °C and °C and my appetite was decreasing At first I could not eat anything sweet Later this came to include ordinary food as well I developed a feeling of unease in my body and I was prescribed paracetamol for my fever and general condition I experienced severe sweating every time I took paracetamol My wife is a former nurse and we were both convinced that I was having influenza The Danish Health Authorities had encouraged citizens to stay home in the presence of cough fever shortness of breath or musculoskeletal aches Approximately a week after onset I went to my physician and my blood tests showed signs of mild inflammation After three days I came back for a check up appointment which showed a further increase in the inflammatory count I had no appetite and felt very ill tired and disheartened I experienced severe unease and slept very poorly After a thorough examination the doctor said At this time I dont know the cause of your condition I would like to refer you for further examination in order to rule out severe illness and malignant disease and to find the underlying cause I was given an appointment the following day for further blood tests and imaging The imaging of my lungs showed signs of possible coronavirus infection and I was admitted to hospital in isolation I was tested with a throat swab and some fluid was obtained from my airways through a tube in my nasal cavity I was then discharged and told that we would be informed about the test results Late in the evening close to midnight we were contacted with the results I had coronavirus Half an hour later an ambulance arrived and I was admitted to an isolated COVID19 unit at another hospital nearby Further tests were conducted and I was given nasal oxygen therapy because of a low oxygen level in my blood Shortly after I felt significantly better The only therapy I received during my eight days at the hospital was oxygen therapy For every day that passed I felt better and my appetite returned When I was discharged I was told that I had to be symptom free for at least hours before I could consider myself recovered Did I have any remnants of the disease I was very tired but this receded in the following three to four weeks While I am writing this it has been seven weeks since I was discharged I have recovered completely and my condition is now as good as it was before I fell sick with coronavirus I now play tennis three times a week again and I am looking forward to the reopening of the gym Throughout my illness I have received fantastic treatment by all parts of the health care service and I am deeply gratefulLearning points º Our understanding of typical and atypical symptoms of COVID19 is still under development º The existing evidence suggests that gastrointestinal manifestations are present º This calls for increased focus on atypical non respiratory symptoms of COVID19 for optimal disease control º It is important to recognise typical as well as atypical presentations of COVID19 in general practice and other places with initial contactWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0colfactory and gustatory disturbances such as hyposmia anosmia and dysgeusia in the absence of other symptoms20Acknowledgements Maria Tudb was actively involved in clinical decision making as senior consultant in general practiceContributors SFW was the lead author of the manuscript and was involved in initial care and management case identification manuscript write up and drafting editing and literature review BBL was senior supervisor and was involved in acquisition of data and imaging and performed critical revision and editing of the manuscript for important intellectual content SFW and BBL were equally involved in the design and interpretation of dataFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewedThis article is made freely available for use in accordance with BMJs website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedREFERENCES Has¶ks¼z M Kili§ S Sara§ F Coronaviruses and SARS COV2 Turk J Med Sci Jin Y Yang H Ji W et a0al Virology epidemiology pathogenesis and control of COVID19 Viruses Cipriano M Ruberti E Giacalone A Gastrointestinal infection could be new focus for coronavirus diagnosis Cureus 202012e7422 Mao R Qiu Y He J S et a0al Manifestations and prognosis of gastrointestinal and liver involvement in patients with COVID19 a systematic review and meta analysis Lancet Gastroenterol Hepatol Kobayashi K I Kaki T Mizuno S et a0al Clinical characteristics of patients with coronavirus disease in Japan a single center case series J Infect Dis Kim S J Nguyen V G Park Y H et a0al A novel synonymous mutation of SARS CoV2 is this possible to affect their antigenicity and immunogenicity Vaccines 103390vaccines8020220 [Epub ahead of print May ]New disease Patrick R Murray KSR Michael A Pfaller medical microbiology ELSEVIER Xiao F Tang M Zheng X et a0al Evidence for gastrointestinal infection of SARS CoV2 Gastroenterology Wan Y Shang J Graham R et a0al Receptor recognition by the novel coronavirus from Wuhan an analysis based on decade long structural studies of SARS coronavirus J Virol 101128JVI0012720 [Epub ahead of print Mar ] Vaduganathan M Vardeny O Michel T et a0al Renin Angiotensin Aldosterone system inhibitors in patients with Covid19 N Engl J Med Donoghue M Hsieh F Baronas E et a0al A novel angiotensin converting enzyme related carboxypeptidase ACE2 converts angiotensin I to angiotensin Circ Res 200087E1 Jia HP Look DC Shi L et a0al Ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia J Virol Varga Z Flammer AJ Steiger P et a0al Endothelial cell infection and endotheliitis in COVID19 Lancet Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet Li Y Hu Y Yu J et a0al Retrospective analysis of laboratory testing in patients with severe or critical type novel coronavirus pneumonia Lab Invest Al Samkari H Karp Leaf RS Dzik WH et a0al COVID19 and coagulation bleeding and thrombotic manifestations of SARS CoV2 infection Blood Salehi S Abedi A Balakrishnan S et a0al Coronavirus disease COVID19 a systematic review of imaging findings in patients AJR Am J Roentgenol Pan Y Yu X Du X et a0al Epidemiological and clinical characteristics of asymptomatic SARS CoV2 carriers J Infect Dis Scalinci SZ Trovato Battagliola E Conjunctivitis can be the only presenting sign and symptom of COVID19 IDCases 202020e00774 Pellegrino R Cooper KW Di Pizio A et a0al Corona viruses and the chemical senses past present and future Chem Senses 2020101093chemsebjaa031 [Epub ahead of print May ] Zhang H Shang W Liu Q et a0al Clinical characteristics of cases of COVID19 in Huanggang and Taian China Infection 101007s15010020014405 [Epub ahead of print May ] Liu K Fang Y Y Deng Y et a0al Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province Chin Med J Xu X Yu C Qu J et a0al Imaging and clinical features of patients with novel coronavirus SARS CoV2 Eur J Nucl Med Mol Imaging Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet Copyright BMJ Publishing Group All rights reserved For permission to reuse any of this content visithttpswwwbmjcomcompanyproductsservicesrightsandlicensingpermissionsBMJ Case Report Fellows may reuse this article for personal use and teaching without any further permissionBecome a Fellow of BMJ Case Reports today and you can º Submit as many cases as you like º Enjoy fast sympathetic peer review and rapid publication of accepted articles º Access all the published articles º Reuse any of the published material for personal use and teaching without further permissionCustomer ServiceIf you have any further queries about your subscription please contact our customer services team on or via email at supportbmjcomVisit casereportsbmjcom for more articles like this and to become a FellowWahab a0SF L¸gstrup a0BB BMJ Case Rep 202013e237520 101136bcr2020237520 0c' | Thyroid_Cancer |
Purpose of Review Recognize which are the elements that predict why a person is aging faster or slower and which interventionwe can arrange to slow down the process which permits to prevent or delay the progression of multimorbidity and disabilityRecent Findings Aging is a complex process that leads to changes in all the systems of the body and all the functions of theperson however aging develops at different rates in different people and chronological age is not always consistent withbiological ageSummary Gerontologists are focused not only on finding the best theory able to explain aging but also on identifying one or moremarkers which are able to describe aging processes These biomarkers are necessary to better define the agingrelated pathologies manage multimorbidity and improve the quality of life The aim of this paper is to review the most recent evidence onaging biomarkers and the clusters related to them for personalization of treatmentsKeywords Biomarker of aging Frailty syndrome Aging phenotype Quality of life Multimorbidity Life expectancy SocialneedsIntroductionMost people dont grow up Most people age They findparking spaces honour their credit cards get married havechildren and call that maturity What that is is agingMaya Angelou One of the biggest megatrends impactingthe world today is population aging Aging is a topic thathas captivated both scientists and philosophers throughouthistory but aging as a population scenario emerged on aThis is part of the Topical Collection on Geriatric Oncology Beatrice Di Capuabeatricedicapuagmailcom UOC di Radioterapia Oncologica Dipartimento Diagnostica perImmagini Radioterapia Oncologica e Ematologia FondazionePoliclinico Universitario A Gemelli IRCCS Rome Italy Dipartimento di Scienze dellinvecchiamento neurologicheortopediche e della testacollo Fondazione Policlinico UniversitarioA Gemelli IRCCS Rome Italy Moffitt Cancer Center Tampa FL USAworldwide scale for the first time in the last century Thus itis hard to really identify a definition of aging It is a decrease infitness with chronological age it is a developmental phasebeyond the normal life trajectory and it is a time of the increased risk of physical and psychological disabilities testingthe limits of resilienceAging occurs at a different rate in varying geographic regions of the worldEurope is currently the oldest region with of thetotal population aged and older However the Asia andLatin America older population is growing fast with Asiasolder population almost tripling in size from million in to million in []All these data do not consider aging as an epiphenomenonbut an individual data of the global population just a chronological number Aging is intrinsically a complex scenariocharacterized by changes that take place at different levels ofbiological systems Biological age is of course influenced bychronological age but chronological age is by itself not representative of biological age biological age is determined byphysiological reserve and functional status Assessing biological age is essential to predict life expectancy and resilience to 0c Page of Curr Oncol Rep stressors [] If any definition of aging may appear incompleteand insufficient much more difficult and complex is to findthe marker or biomarker that can identify itMany theories currently trying to explain aging processesand many biomarkers are identified to measure aging and itsevolutionary stages Theories and biomarkers are not studiedto extend life span but to guide therapeutic choices and optimize patient management and personalization of careThe purpose of this paper is not purely to list which biomarkers are able to identify the various stages of aging ratherexplain how an epiphenomenon natural and physiological isso complex [] how many factors are protagonists in its development and how many actors and characters play in maximizing its individual features taking into account social andmorbidity biomarker These factors such as frailty loss ofautonomy essential needs and comorbidities influence theaging process and are able to justify why the biological ageof a person living in a country does not correspond to the ageof another person living in a country with better sociosanitaryconditionsClinical and Biological Aging PhenotypesThe aging phenotype can be described as a complex mosaicresulting from the interaction of a variety of environmentalstochastic and geneticepigenetic eventsstimuli impinginglifelong on our body [ ]There is no clear evidence which molecular cellular orphysiological changes are the most important drivers of theaging process andor how they influence one another [] In itsbroadest sense aging merely refers to the changes that occurduring an anisms life span though the rate at which thesetake place varies widely [] Despite its enormous complexityinvolving combinations of these variables a small number ofbasic molecular mechanisms underpin the aging process including a set of evolutionary highly conserved basic biological mechanisms responsible for body maintenance and repairOne of the key mechanisms is inflammation a typical featureof the aging process is the development of a chronic lowgrade inflammatory status named inflammaging [8cid129] whichemerged as critical in the pathogenesis of major agerelatedchronic diseases such as atherosclerosis type diabetes and neuro degeneration Inflammaging plays a pivotal role in themost important geriatric conditions such as sarc ia [9cid129cid129]osteoporosis [] frailty and disability thus contributing tomortality [] Interestingly a variety of tissues adipose tissue muscle ans brain liver systems immune systemand ecosystems gut microbiota of the body indicated assubsystems can contribute to the onset and progressionof such a systemic inflammatory state [] by increasing theproduction of several proinflammatory mediators or loweringthat of the antiinflammatory ones [8cid129]To differentiate the innocuous changes from those leadingto increased risk of disease disability or death biogerontologists tend to use a more precise termsenescencewhen describing aging [] Senescence is thereforethe progressive deterioration of bodily functions over time andnormal human aging has been associated with a loss of complexity in a wide range of physiological processes and anatomic structures [] including blood pressure [] strideintervals [] respiratory cycles [] and vision [] amongothers such as postural dynamics [] ultimately leading todecreased fertility and increased risk or mortality []Systemic consequences of aging are widespread but theycan be clustered into four domains Fig Changing in body compositionThe balance between energy availability and energydemandSignaling networks that maintain homeostasis NeurodegenerationThese changes develop in parallel and affect each otherthrough many feedforward and feedback loopThe phenotype that results from the aging process is characterized by increased susceptibility to disease high risk ofmultiple coexisting diseases impaired response to stress theemergence of geriatric syndromes altered response to treatment high risk of disability and loss of personal autonomywith all its psychological and social consequences On theother hand all these factors influence aging itself in a dynamic and parallel way so that they can be considered as not onlya consequence of aging but also an integral part of the agingprocessTheories of AgingHuman aging is currently defined as a dynamic process involving the continual adaptation of the body to lifelong exposure tointernal and external damaging as conceptualized in the remodelling theory of aging [21cid129cid129] Theories of aging are generallyclassified as either program or damage theories Programmedaging theories suggest that there is a deliberate deterioration withage because a limited life span results in evolutionary benefits[] This plan could be a result of aging genes The firstdescribed mutation to yield a significant extension in the life spanof Caenorhabditis elegans was in the ageI gene which wasshown to result in a increase in mean life span and a increase in maximum life span of this anism []Evolutionary biologists may argue that aging occurs due to theabsence of natural selection at the postreproductive stage of life[] Although such aging theories are subjectively appealing asthey convey a cure for aging the accumulation of damage is aspontaneous entropydriven process [] Among the damage 0cCurr Oncol Rep Fig Systemic consequences ofagingPage of theories a prevailing idea is that of oxidative damage Reactiveoxygen species ROS are generated during metabolism throughseveral interrelated reactions The supposition that aging may becaused by ROS has been further substantiated by studies involving transgenic animals for genes encoding antioxidants The lifespan of Drosophila melanogaster has been extended by overexpression of both superoxide dismutase SOD and catalase bothantioxidant enzymes [] Since mitochondria are the major producer of ROS in mammalian cells mitochondrial DNAmtDNA is therefore particularly susceptible to oxidative damage [] Mitochondrial maintenance is therefore essential topreserve cellular homeostasis and impaired mitochondrial maintenance has been described as a shared hallmark of numeroushuman pathologies and aging [] Mitochondrial DNA varieswith age and it is commonly considered that DNA hypomethylation is a typical aspect of the aging process [] ROS are activeintermediates of DNA methylation as well as histone modification These reactive oxygen species may play a role in epigeneticprocesses physiological phenotypic variations caused by external or environmental factors that switch genes onoff throughreactions of nucleophilic substitution at the DNA levelConsequently it has been suggested that better preservation ofDNA methylation levels slower cell metabolism and improvedcontrol in signal transmission through epigenetic mechanismscould be key processes involved in human longevity Oxidativedamage to proteins is irreversible and irreparable [] and mustbe degraded by the proteasome The proteasome is the mostimportant proteolytic machinery in eukaryotic cells largely responsible for the removal of oxidized proteins and the preventionof its aggregation [] However it has been shown that theactivity of proteasome is impaired during aging leading to theaccumulation of oxidizing proteins aggresome and lipofuscinsocalled the age pigment Similarly to oxidative damage nitrosamine damagethat caused by reactive nitrogen speciesRNS such as nitric oxidehas been suggested to also contribute to agerelated diseases namely hepatic steatosis and apoptosis [] as well as functional and structural changes in the cardiovascular system [ ] sleep homeostasis [] psychological disorders [] and dementia []Most supporters of the genomic instability theory of agingrefer to telomere shortening [] and mutation in DNA mitochondrial Telomeres are the repeated DNA sequences at theends of linear chromosomes which are unable to be fullyreplicated by DNA polymerasesMutations in mtDNA cause a wide range of human mitochondrial diseases and have been implicated in agerelateddiseases and agingBiomarker FeaturesFinding the biomarker of aging is one of the most importantgoals of medicine The National Institutes of HealthBiomarkers Definitions Working Group defined a biomarkeras a characteristic that is objectively measured and evaluatedas an indicator of normal biological processes pathogenicprocesses or pharmacologic responses to a therapeutic intervention []The American Federation for Aging Research AFAR recommends the following criteria for biomarkers of aging [39cid129]It must predict a persons physiological cognitive andphysical function in an agerelated way independentlyof chronological age 0c Page of Curr Oncol Rep It must be testable and not harmful to test subjects forexample a blood test or an imaging technique it mustalso be technically simple to perform and it must be accurate and reproducibly without the need for specializedequipment or techniquesIt should work in laboratory animals as well as humanssince preliminary testing is always done in nonhumansubjectsFerrucci et al reviewed the biomarkers proposed as elements of a theory based on the balance between resiliencemechanisms and accumulated damages where biomarkersact in reducing resilience mechanisms or increasing damages[40cid129] Tables and The pathways eligible to become biomarkers are thefollowingGenomic Instability Endogenous and exogenous agents continuously challenge the integrity of DNA when DNA repairmechanisms cannot manage the repeated damage the result isan accumulation of DNA somatic mutations This phenomenon causes dysregulation of gene expression and the production of altered proteins that lead to cellular damage Somaticmutation accumulation has been observed in skeletal musclecells neurons and lymphocytes B related to aging []nevertheless quantification of DNA repair capacity in humanshas yet to be finalized []Telomere Attrition Telomeres are the DNA sequences that areplaced at the end of the DNA chain and protect theTable Biological changesunderlying agingGenomic instabilityTelomere attritionEpigenetic alterationscid129 DNA methylationcid129 Histone modificationcid129 Noncoding RNALoss of proteostasisMitochondrial dysfunctionCellular senescenceDeregulated nutrientsensingSteam cell exhaustionAltered intercellular communicationchromosome ends from damage During each replicationtelomeres are reproduced but not completely so with agingthey become shorter and contribute to cellular senescence[] To date different techniques are available to detecttelomere length in circulating cells however no techniqueshave been validated for evaluating aging because of the heterogeneity between different cells between individuals andhigh measurement errors that make these techniques not yetvalid in clinical practice []Epigenetic Alterations Epigenetics refers to those mechanismsexternal to DNA that modulate gene expression in cells theregulation of gene expression determines the phenotypic characteristics of the different cells and tissues The main mechanismsare DNA methylation histone modification and noncodingRNA While DNA methylation is easily measured in circulatingcells and seems to be correlated to aging [ ] measuringhistone modification or noncoding RNA is difficult and expensive Recent evidence correlates DNA methylation with agingand agerelated chronic diseases in humans [ ]Individuals with higher levels of DNA methylation have a higherrisk of developing several agerelated diseases and prematuremortality for all causes and cardiovascular diseases [] as wellas physical and cognitive functions [ ]Loss of Proteostasis The repair of damaged structures or theirelimination is fundamental to maintain cell integrity and function [] Studies suggest that proteostasis becomes defectivewith aging and contributes to immunosenescence [] and thatautophagy appears to be more functional in longlived peopleAccumulation of DNA somatic mutationsDysregulation of gene expressionAltered proteins productionTelomere shortening contribute tocellular senescenceAltered gene expressionRelated to agerelated chronic diseasesAccumulation of damaged structuresAltered energy productionIncreased ROS productionApoptosisprogrammed cell deathActivation of pathways leading to apoptosisProduction of SASPIncrease of life span in dietary restrictionDecline of regenerative potentialInflammagingDysfunction of endocrine neuronaland immune systems 0cCurr Oncol Rep Page of Table Measurable biomarkers classified by respective hallmarksPathways measuredMeasurable biomarkersHallmarkGenomic instabilityTelomere shorteningCellular senescencecid129 DNA repair mechanismscid129 DNA modificationscid129 Telomere lengthcid129 Markers of DNA damage responsecid129 Telomerase activitycid129 Senescent markers in blood and tissueEpigenetic changes or epigenetic clockcid129 DNA methylationcid129 Histone acetylationcid129 Noncoding RNAMitochondrialDecreased autophagy proteostasiscid129 Mitochondrial volumenumbershapecid129 Mito respirationcid129 Markers of biogenesiscid129 mtDNA copy number and haplotypescid129 Autophagy markerscid129 Chaperon proteinsStem cell exhaustionDeregulated nutrientsensingAltered intercellular communicationcid129 Proliferative capacity in vitrocid129 Resistance to stresscid129 Growth hormone GH axiscid129 Metabolism alterationscid129 Measures of inflammationcid129 yH2AX immunohistochemistrycid129Leukocyte telomere lengthcid129MIR31HGcid129 p16INK4acid129 Senescenceassociated secretoryphenotype SASP proteinscid129 Measures of DNA methylationcid129 SIRT1 SIRT2 SIRT3 SIRT6 SIRT7cid129 Dosage of circulating microRNAs miR34aMiR21 miR1263p miR151a3pmiR181a5p miR1248cid129 p31 MRI spectroscopycid129 Growth differentiating factor GDF15cid129 NADcid129 Target of rapamycin TORcid129 Protein carbamylationcid129 Advanced glycation end productscid129 Insulinlike growth factor IGF1cid129 HGBA1ccid129 IL6cid129 TNFαcid129 CRP Creactive proteincid129 TNFRII tumor necrosis factorα RII[] Measuring the loss of proteostasis mechanism could be agood biomarker but to date there are no valid techniques forthis purposeMitochondrial Dysfunction The main role of mitochondria isto guarantee energy for the cell through the production ofATP They are also involved in signaling by the productionof ROS and in apoptosisprogrammed cell deathMitochondrial dysfunction is a good biomarker of aging andis associated with disability in older persons through the reduction of muscle strength [65cid129]Many techniques are measuring oxidative phosphorylationand ROS generation that have been associated with chronicdisease [ ] nevertheless the relation with aging is notcompletely validatedCellular Senescence Genomic instability telomere shorteningand other endogenous and exogenous mechanisms can inducethe cell to activate specific pathways that lead to apoptosis[] This process is called cellular senescence and is characterized by structural and functional changes in the cell []Senescent cells produce proinflammatory cytokines andchemokines growth factors and matrix proteases called senescenceassociated secretory phenotype SASP [ ]which may induce some agerelated diseases [] Thedetection of SASP has been proposed as a biomarker of aging[]Deregulated NutrientSensing Genetic mutations in growthhormone and the insulinlike growth factor have been linkedto longevity [] Moreover dietary restriction showed to increase life span in primates [ ] For these reasons thispathway has been proposed as biomarkers of agingSteam Cell Exhaustion The decline in the regenerative potential is one of the elements at the base of aging [] Despitepharmacological interventions being explored to counteractthis phenomenon [] evidences are still poor 0c Page of Curr Oncol Rep Altered Intercellular Communication With aging we also observe changes in intercellular communication as inflammatory reaction increases the other communication ways becomedysfunctional endocrine neuronal immune system []As we discussed earlier inflammation can be inappropriately increased in aging and this has been related to agerelated disease [ ]Indeed the pathways described as potential biomarkers ofaging are strongly related to inflammation for this reasonmeasuring circulating levels of cytokines is considered anew field of research [ 84cid129 ]Aging and Life ExpectancyAging and life expectancy are closely related In a broadsense determining an individuals life expectancy is also away of schematizing his or her aging process Life expectancyis a statistical measure of the average time an anism isexpected to live based on the year of its birth LEB itscurrent age and demographic factors including gender []In the last decades life expectancy has increased in high income country the rise in human life expectancy has involveddeclines in intrinsic and extrinsic mortality processes associated respectively with senescence and environmental challenges []In association to this increased longevity there are diseasescalled agerelated that increase quadratically with age andcause a progressive loss of physical mental and cognitiveintegrities leading to impaired function and increased vulnerability to morbidity mortality [] and disability in additionto increasing care needs and agerelated burden measuredthrough the sum of disabilityadjusted life years DALYs ofthese diseases among these adults Fig Ninetytwo of the of the Global Burden of Disease causes were identified asagerelated diseases In particular cardiovascular diseaseneoplasm and chronic respiratory disorders are those withhigher agerelated disease burden []Determinants of Frailty Syndrome as AgingBiomarkerFrailty can be defined as a state of increased vulnerabilityto stressors or a loss of capacity to resolve homeostasisperturbation Frailty condition is closely related to aging[88cid129cid129] and the frailty indexes can consequently be considered biomarkers of aging themselves In frail individuals it is possible to find both changing in body composition and balance between energy availability and energydemand Moreover in the definition of frailty it is welldescribed how signaling networks maintain homeostasisand association with neurodegeneration These fouraspects all refer to the hallmarks of aging Frailty is associated with adverse clinical outcomes including falls institutionalization and death [88cid129cid129]Two principal models emerged in the last decades that areable to conceptualize and consequently measure frailty in everyday clinical practice and research the frailty phenotypemodel and the cumulative deficits modelThe frailty phenotype was first described by Fried and colleagues in analyzing data from the CardiovascularHealth Study CHS involving men and women aged years and older In this study it was investigated whichcharacteristics of the population were predictive of falls disability hospitalization and death Their operational definitionof frailty included a cluster of at least three of the followingvariables unintentional weight loss selfreported exhaustionlow energy expenditure slow gait speed and weak gripstrength This model does not take into consideration cognitive impairment as a cause of increased vulnerability as thiscould contribute to functional decline and adverse events inolder people [ ]The cumulative deficits model was developed byRockwood and colleagues as part of the prospectiveCanadian Study of Health and Aging CSHA involvinga cohort of older adults [] The authors identified parameters including diseases disabilities signssymptoms and laboratory values which were defined asdeficits The sum of the deficits in a single individualallowed for the calculation of a frailty index ie thenumber of deficits divided by Frailty in this modelis not considered as a cluster of symptoms but is conceptualized as a gradable syndrome with a higher number ofdeficits implying an increased vulnerability state The twomodels of frailty show significant overlap although theycapture slightly different sides of the same problem It isimportant to notice that physical frailty is frequently associated with multimorbidity [ 93cid129 ]It has been observed that the frailty phenotype construct is intrinsically related to mobility issues Indeedin older adults physical performance measures are a robust and consistent predictor for disability hospitalization institutionalization and death both in the researchand in the clinical setting Lower physical performance isfrequently associated with loss of skeletal muscle massand quality causing reduced strength and functional impairment [95cid129cid129] This process has been called sarc iaEven though sarc ia has been long associated withaging it has to be acknowledged that it can develop muchearlier in life [] Different definitions exist for this condition for the operational definition of sarc ia both inthe clinic and for research purposes that prioritize theassessment of muscle strength over muscle mass to identity sarc ic patients Strength is more closely related tosurvival and functional decline compared with muscle 0cCurr Oncol Rep Page of CARDIOVASCULAR DISEASESAtrial ï¬brillaÆon and ï¬uÆ©er endocardiÆs hypertensive heart disease intracerebralhaemorrhage ischaemic heart disease ischaemic stroke myocardiÆs nonrheumaÆc valve disease other cardiomyopathy other cardiovascular and circulatory diseases peripheralartery diseaseNEOPLASMSLeukaemia lymphoma mulÆple myeloma myelodysplasÆc syndroms and other hematopoieÆc neoplasms brain and nervous system cancer breastcancer prostate cancer larynx cancer lip and oral cavity cancer oesophagealcancer stomach cancer colon and rectum cancer liver cancer gallbladder and biliary tract cancer pancreaÆc cancer kidney cancer bladder cancer melanoma and nonmelanoma skin cancer ovarian cancer uterine cancer thyroid cancer tracheal bronchus and lung cancer mesothelioma othermalignant neoplasms other benign and insitu neoplasmsGASTROINTESTINAL ENDOCRINE AND KIDNEY DISEASESChronic kidney disease type diabetes mellitus cirrhosis due to nonalcoholicsteatohepaÆÆs pancreaÆÆs paralyÆc ileus and intesÆnal obstrucÆon pepÆculcer disease vascular intesÆnal disorders diarrhoeal diseasesSKIN AND SUBCUTANEOUS DISEASESCelluliÆs decubitus ulcer fungal skin diseases pyoderma other skin and subcutaneousdiseasesFig Agerelated diseases adapted from Chang et al []mass [95cid129cid129] According to EWGSOP criteria sarc iais defined by the presence of low muscle strength criterion and either or low muscle quantity or quality criterion or low physical performance criterion [95cid129cid129]The physical performance parameters used in the identification of frailty syndrome both integrated eg SPPB andalone walking speed handgrip strength can be used as agingperformance biomarkersDetermination of Medical and Social NeedsWhy consider medical and social needs aging biomarkersIn Robert J Havighurst said In considering theneeds of older people it is well first to remember that olderpeople have the needs that are common to all people andsecond that they have special needs due to the fact that theyare old people This sentence describes everything there is toknow about the need for the elderly and answers the questionbeforeIn every society and age there is what is meant by normality An elderly person in this scenario needs what is needed tomaintain this level of normalcy Activity of daily living andinstrumental activity of daily living ADL and IADL aloneremodelled according to the context and gender can identifythe minimum necessary Conducting needs assessment various areas must be considered including physical health mental health emotional care social cultural economic nutritional service security legal and educationalCHRONIC RESPIRATORY DISEASESAsbestosis chronic obstrucÆve pulmonary disease coal worker pneumoconiosis intersÆÆallung disease and pulmonary sarcoidosis other pneumoconiosis silicosis lower respiratoryinfecÆonsNEUROLOGICAL DISORDERSAlzheimers disease and other demenÆas motor neuron disease Parkinsonsdisease encephaliÆs pneumococcal meningiÆsAGE RELATED DISEASESENSE AN DISEASESHearing loss vision loss ex agerelated macular degeneraÆon cataract glaucoma other sense an diseases refracÆon disorders trachomaINJURIESDrowning environmental heat and cold exposure falls foreign body in other body part other transport injuries other unintenÆonal injuriesOTHER DISEASESCongenital musculoskeletal and limb anomalies digesÆve congenital anomalies endocrine metabolic blood and immune disorders other haemoglobinopathies and haemolyÆcanaemiasMany tools are used to evaluate peoples needs The majority of these tools are focused on physical performance ableto maintain autonomy few studies focus on social needs andthe costs of care In the West World of patients accountfor of total health care expenditures This is represented by older people individuals with multiple chronic conditions many medications frequent hospitalizations and limitations on their ability to perform basic daily functions due tophysical mental or psychosocial challenge []Since the health care and social needs of older adults differfrom that of other adults it is necessary to identify the needs ofthe elderly to make proper plans that will promote their healthCurrently most of the conducted studies had mainly focused on the elderly physical health needs and had neglectedto take into account other needs such as social and health careneeds Furthermore in addition to quantitative studies discovering the older adults perceptions of their own health needsis also necessaryConclusionThere is a large interest of researchers in biomarkers of agingand despite some of them seem to be very promising biological biomarkers are still far from a clinical application to datethere is no technique that meets the mentioned criteria of theideal biomarker [40cid129] Moreover we know that the biologicalpathways are the final agents of aging but on one side theycan be influenced by social economic and environmental factors and on the other side they express in various disease and 0c Page of Curr Oncol Rep disabilities of the person physical and cognitive impairmentsagerelated disease systems functions sensory functions etcFig To date more than a single biomarker to assess agingwe should consider a cluster of biomarkers that comprisethe various elements that we analyzed social and educational aspects economic factors country of origin presence of agerelated disease presence of dependence indaily activities physical capability cognitive functionlung and cardiovascular function and presence of sensorydysfunctions In Table we propose several clinical andlaboratory biomarkers that can be used in clinical practiceand researchThe geriatric assessment GA can currently be considered a system capable of monitoring multiple biomarkersclinical and laboratory of aging and at the same timeable to relate them to each other Through the GA it ispossible to make a prediction of the risk of toxicity of atreatment of life expectancy of social needs and of compliance with the treatments GA is composed indeed byseveral evaluations made through standardized toolswhich examine various aspects of the person a multidimensional assessmentAlthough it seems difficult to imagine a geriatric assessment as a biomarker currently for its characteristicsand for the high predictivity it has it can be consideredthe gold standard in the management of the older individual and instrumenttoward which other biomarkersshould be evaluatedThe purpose of this paper was to evaluate the multipleaspects that distinguish the aging process Aging must noFig Mechanisms connectingdifferent clusters of biomarkerslonger be described as a simple demographic event butas a complex mosaic in which several tesserae relate toeach other some in a very evident way others often in amore subdued but all fundamental way Each aging theory has attempted to justify this process effectively however there is no single biomarker to date that has beenfound able to identify the stage of this process At thesame time clinical clusters have been added to purelybiological markers and social ones should certainly beconsidered It therefore becomes important not to consider biomarkers only as life span but to try to overcomethis link and focus on the set of factors that influencingeach other are able to guide aging in good health andgood quality of life towards a lived aging as a slowdecline At the time we are writing this paper COVID infection is reaping victims especially in Italy Thehighest mortality is observed among the older adultsbut surprisingly it seems to maintain similar values between the youngest and oldest old over yearsCurrently no plausible justification is provided for thesedata In frailty the number of comorbidities the reducedfunctional reserve was the most used reasons Indirectlythis infection is highlighting the need to use parametersthat can more easily identify the aging process regardlessof chronological ageThe studies analyzed in the literature show that if on theone hand there are physiological biomarkers able ofhighlighting some features of aging other functionalmarkers performance social and economic status somepathologies and the presence of addiction are able ofspeed it up or slow it | Thyroid_Cancer |
Comparison of different calculationtechniques for absorbed dose assessment inpatient specific peptide receptor radionuclidetherapyDomenico Finocchiaro12 Salvatore Berenato3 Valentina Bertolini1 Gastone Castellani2Nico Lanconelli2 Annibale Versari4 Emiliano Spezi35 Mauro Iori1 Federica FioroniID1Elisa Grassi1 Azienda Unit Sanitaria Locale di Reggio EmiliaIRCCS Medical Physics Unit Reggio Emilia Italy Department of Physics and Astronomy University of Bologna Bologna Italy Department of MedicalPhysics Velindre Cancer Centre Cardiff United Kingdom Azienda Unit Sanitaria Locale di Reggio EmiliaIRCCS Nuclear Medicine Unit Reggio Emilia Italy School of Engineering Cardiff University CardiffUnited Kingdom federicafioroniauslreita1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSAbstractCitation Finocchiaro D Berenato S Bertolini VCastellani G Lanconelli N Versari A Comparison of different calculation techniques forabsorbed dose assessment in patient specificpeptide receptor radionuclide therapy e0236466 101371journalpone0236466Editor Choonsik Lee National Institute of HealthUNITED STATESReceived July Accepted July Published August Copyright Finocchiaro This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement All relevant data arewithin the manuscriptFunding This work was supported by theEuropean Metrology Programme For InnovationAnd Research EMPIR joint research project15HLT06 Metrology for clinical implementation ofdosimetry in molecular radiotherapyMRTDosimetry which has received funding fromthe European Union The EMPIR initiative is cofunded by the European Unions Horizon AimThe present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation anlevel dosimetry voxellevel dose kernel convolution and Monte Carlo simulations The aim was toassess the importance of the choice of the most adequate calculation modality providingrecommendations about the choice of the computation toolMethodsThe performances were evaluated both on phantoms and patients in a multilevel approachDifferent phantoms filled with a 177Luradioactive solution were used a homogeneous cylindrical phantom a phantom with anshaped inserts and two cylindrical phantoms withinserts different for shape and volume A total of patients with NETs treated by PRRTwith 177LuDOTATOC were retrospectively analysedResultsThe comparisons were performed mainly between the mean values of the absorbed dose inthe regions of interest A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two andanlevel dosimetry both for phantoms and patients Phantoms measurements alsoshowed the discrepancies mainly depend on the geometry of the inserts eg shape and volume For patients differences were more pronounced than phantoms and higher interintrapatient variability was observedPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTresearch and innovation programme and theEMPIR Participating States SB acknowledgesfunding from Cancer Research Wales throughgrant No Competing interests The authors have declaredthat no competing interests existConclusionThis study suggests that voxellevel techniques for dosimetry calculation are potentiallymore accurate and personalized than anlevel methods In particular a voxelconvolution method provides good results in a short time of calculation while Monte Carlo basedcomputation should be conducted with very fast calculation systems for a possible use inclinics despite its intrinsic higher accuracy Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from sphericalgeometry in which Monte Carlo seems to be more accurate than voxelconvolutionmethodsIntroductionRadiopharmaceutical therapy RPT as defined in ICRP [] is based on the use of specificpharmaceuticals labelled with radionuclides to deliver a lethal dose of radiation to tumourareas Radiopharmaceuticals are specifically designed to have high affinity with given tumoursites so that ionizing radiations such as ps and photons emitted by the isotopes maydeposit energy inside or close to unhealthy tissues saving surrounding healthy tissues Thisapproach produced very encouraging results in the treatment of neuroendocrine tumoursNET in the last decades in particular in therapies which make use of somatostatin analogueslabelled with 90Y or 177Lu [] such as the recently registered Lutathera [] Different responserates and a large interpatient variability of the outcome were however reported by someauthors eg Campana D and Vinjamuri S [ ]The wellestablished experience with external beam radiation therapy EBRT has providedstrong evidence that tumour response and normal an toxicity is related to absorbed dosesFor this reason it was supposed that the treatment outcome correlates with the absorbed dosedelivered to tumours even in RPT [ ] Yet a dosimetry as more accurate and personalized aspossible is needed to this purpose to provide clinicians with reliable resultsDespite the general demand for a more individualized treatment based on pretherapeuticdosimetry study in NET dosimetry is not conducted always in the clinical routine This ismostly because dosimetry is often considered time consuming a lot of time required for imaging expensive costs for every image scan and every measurement and sometimes inaccuratefor the lack of standardization and harmonization mainly At present a standard procedurefor calculating the absorbed dose is not well defined for every kind of radionuclide therapy Inrelation to NET the evidence of prolonged survival has been demonstrated only recently [] ina subgroup of NETDifferent methods have been developed to perform dosimetry since its beginnings Techniques based on standardized reference models were first developed thanks to their simplicityof implementation and have been used for many years These models assume uniform activityie homogeneous uptake in the source regions However evidence indicates that deterministic biological effects including tumour response and normal tissue toxicity may not be wellpredicted by the mean absorbed dose in the region and may be significantly influenced bynonuniform doses [] To take into account this aspect voxelbased techniques were considered similarly to those which have been also used for decades as standard of care in EBRT [] Contrary to what happens for EBRT however in which plenty of software for therapyplanning are available on the market in RPT only few systems which are adequate toPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdosimetry for Peptide Receptor Radionuclide Therapy PRRT and which can work with multiple 3D imaging have been officially released in the last few years [] For this reasonmany dosimetry software and tools are in use worldwide but only some of them are commercially available Several of them are homemade tools which were developed before the commercial software were finalized [] and have been fully customized by clinical users in themeantimeAt present standardization and harmonization of the calculation systems are importantTherefore it is essential to compare the various results obtained with the most advanced existing homemadecommercial software and other less advanced still used worldwide methodsYet both categories should be tested on a larger sample of cases than ever done before Thesetests should provide an example of the most accurate methodology for 3D dosimetry in RPTthanks to the gained experience in the last decades giving recommendations about the appropriate use and the limitations of each methodA few studies presenting some comparisons have already been published [] Howeverthese works either did not report dosimetry studies performed completely at the voxel level[] or considered a limited number of clinical cases [] or showed a comparison based onthe dose factors and not based on absorbed doses [] Therefore more studies are needed tofully evaluate calculation performances in clinically relevant conditions considering a highnumber of casesIn this context the main objective of the present work is to compare different modalitiesfor absorbed dose calculation to point out the pros and the cons in each modality and to provide recommendations about the choice of the most adequate computation technique for thesingle clinical or research centres approaching the methodology The modalities here considered include the most used techniques in this field worldwide ranging from the less advancedand less personalised to the most accurate and patient specificThe considered modalities listed in growing complexity are an level dosimetry based onstandardized reference models such as OLINDA version [] which has been used fordecades before the recent release of the new updated commercial version OLINDA version [] voxellevel dosimetry based on dose kernel convolution VoxelMed20 [] and voxellevel dosimetry based on Monte Carlo MC simulations RAYDOSE [] OLINDA11 waschosen because it is still widely used for RPT dosimetry VoxelMed20 was chosen because itwas designed to achieve a good compromise between calculation accuracy and easy applicability in clinical practice RAYDOSE was considered because MC techniques are considered toprovide the most accurate approach to dose estimate []The comparison was performed on 3D images of specifically designed phantoms and onmultiple 3D dataset of images of a high number of clinical cases This multiapproach methodbased both on phantoms and patients allowed to investigate the differences of performancebetween the calculation modalities depending on the shape and the volume of the activity distribution and to provide a valuable comparison based on a conspicuous number of clinicalcasesMaterials and methodsThis study involves human participants All participants were enrolled in a clinical trialEUDRACT at Azienda USLIRCCS of Reggio Emilia Italy The study wasapproved by the ethics committee of Azienda USLIRCCS of Reggio Emilia Italy and eachpatient gave written informed consent for the study conductionThe following sections describe in detail the specific phantoms the image set the softwareand the data elaboration approachPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTPreparation of phantomsThree different phantoms filled with 177Lu radiolabelled peptides leftover from the clinicalapplication were used¢ a Cylindrical phantom filled with a homogeneous radioactive solution Jaszczak Data Spectrum Corporation USA shown in Fig 1A Details are included in Table ¢ a cylindrical phantom and a set of fillable plastic inserts arranged in two different configurations to originate a couple of Geometrical phantoms The inserts different for shapetoroidal pearshaped tubular and ellipsoidal and volume are depicted in Fig 1C Insertstake the name from the shape and the equivalent diameter ie the diameter for a spherewith the same volume as shown in Table Each insert was filled with the same activityconcentration and placed in a nonradioactive water background Details of volume andactivity concentration are shown in Table ¢ an Anthropomorphic phantom with an shaped inserts LiquiPhill The Phantom Laboratory Greenwich NY shown in Fig 1B Details are included in Table Every insert wasfilled with an activity concentration typical of real ans in clinical cases and placed in aradioactive water backgroundTo accurately measure the volumes the weight of the phantoms and of the insertsbefore and after refilling was taken with a calibrated scale The density of the waterbasedsolution was of 1gml HCl M was used as a carrier solution to prevent radioactive177Lu deposition on the phantom walls and to guarantee a homogenous radionuclidesolutionEvery phantom was scanned once and the timeactivity curve was generated using the physical decay of the isotope All specific data regarding the volumes of inserts and phantoms andthe activity used are reported in Table Fig CT scans of phantoms used in this study a Cylindrical phantom b Anthropomorphic phantom c Insertswith different shapes placed in the Geometrical phantom101371journalpone0236466g001PLOS ONE 101371journalpone0236466 August PLOS ONE 0cTable Description of phantoms used to test the dosimetry toolsPhantomPhantom volumeInsert nameInsert volumeInsert activity concentration MBqBackground activity concentration MBqComparison of different absorbed dose calculation methods in MRTCylindricalGeometricalmlAnthropomorphicNATo17aTo26E20E30E38To17bP38P39aP39bTu38aTu38bLesionPancreasLeft kidneyRightkidneySpleenLivermlNA101371journalpone0236466t001Clinical trialmlmlNANAThe clinical cases considered in the present work were all extracted from a preexisting clinicalPRRT trial including patients and conducted by Azienda USLIRCCS of Reggio EmiliaItalyAll considered patients were previously enrolled in the trial EUDRACT between and The clinical trial design established that every patient had to besequentially administered with either 177Lu labelled radiopeptides 177LuDOTATOC or 90Ylabelled radiopeptides 90YDOTATOC up to a maximum of infusions or cycles Dosimetry was mandatory in the clinical trial and was to schedule during the first cycle of therapyafter a therapeutic administration of 177LuDOTATOC Each patient underwent SPECTCT scans at h post injection According to the trial design clinical absorbedTable Legend of the insert acronyms for the Geometrical phantomInsert geometryEquivalent diameter mmInsert nameTorusTorusTorusEllipsoidEllipsoidEllipsoidPearPearPearTubeTube101371journalpone0236466t002To17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTdoses for 177Lu and 90Y labelled radiopeptides for liver spleen and kidneys were calculatedEach an was manually contoured and absorbed doses were calculated in compliance withthe MIRD scheme [] at anlevel from images The number of cycles the isotope and theactivity chosen for every injection were planned by an expert physician on the basis of thedosimetry results The activity prescription had to be determined based on the BiologicalEffective Dose BED delivered to kidneys Kidneys are regarded as the principal ans at riskin PRRT [ ] As suggested by different works [] in this clinical trial the cumulativedose limit to kidneys was set to Gy of BED for patients with no risk factors hypertensiondiabetes renal failure are considered risk factors for this therapy and at 28Gy for patients withrisk factorsIn the present work absorbed doses to kidneys spleen and liver were calculated to comparethe three dosimetric methodsImage acquisition and reconstructionAll activity measurements were performed with an accurate activity calibrator for 177Lu Aktivimeter Isomed Nuklear Medizintechnik Germany and all image acquisitions were performed through a SPECTCT scanner Symbia T2 Siemens Medical Germany NaITldetector previously calibrated [] The standard clinical protocol for body studies was usedboth for phantoms and patients with the following SPECT settings MEHR collimators matrix x zoom views x timeview s step and shoot mode degree of rotation Ë noncircular orbit detector configuration ËThe first CT acquisition per patient was performed with the following parameters 130kVand max mAs using tube current modulation The subsequent CT images were acquiredwith kV and 40mAs for radiation protection safety of patients The CT reconstructed slicethickness was mm and a smooth reconstruction kernel was used B08s Siemens MedicalSolution Germany The higher image quality of the first CT scan is necessary for contouringvolumes of interest more accuratelyThe SPECT projections were reconstructed by an iterative algorithm including CT attenuation correction scatter correction and full collimatordetector response in Siemens ESoftworkstation Syngo MI Application version 32B Siemens Medical Solution Germany withFlash 3D iterative algorithm iterations subsets Gaussian filter cutoff mm mmcubic voxel []All cases of Sample A were rigidly registered to the first CT image of the sequence in Siemens Esoft workstation Images of patients included in Sample B were registered using adeformable multipass algorithm with the Velocity Advanced Imaging workstation Varian Medical Systems Palo Alto USA [] The registration procedures rescaled the originalvoxel size to 39x39x35 mm3The Volumes Of Interest VOI for each phantom and each patient were manually drawnon the reference CT image as recommended by Uribe [] using the VelocityworkstationSoftware for image processing and dosimetry calculationsOLINDA11 OLINDA version [] is an an level dosimetry software based on theMIRD methodology [] for internal dose estimation This is the method adopted in the clinical trial the clinical cases of this work are extracted from Absorbed doses to ans and tolesions can be calculated by using different models in the software human phantom modelsie mathematical representations of the human body to represent ans and whole body andsphere models ie mathematical representations of spheres to represent lesions [ ]PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTUnlike VoxelMed and RAYDOSE OLINDA needs timeintegrated activity A values ofVOIs as input parameters [] which were calculated with VoxelMed20 which will bedescribed in the next section and then inserted in OLINDAOLINDA sphere model commonly used to calculate doses to lesions was used to generatethe results for the inserts placed in the Geometrical phantom and for the dummy lesion housedin the anthropomorphic phantom while OLINDA an model adult male was used for thedummy ans placed in the anthropomorphic phantom Real insert volumes were used forcalculationsThe human models adult male or adult female were used to calculate dosimetry of thecohort of patients Doses were scaled using the true patient weight and the true an massesVoxelMed20 VoxelMed is a homemade software for dose calculation developed atAzienda USLIRCCS research hospital Reggio Emilia Italy It was developed in the MatlabThe Mathworks Natick MA programming environment and designed on the CERR platform wwwcerrinfo It performs voxellevel dosimetry based on the MIRD guidelines []The first version of the software along with the S value matrices for voxel dosimetry used incalculations were described in detail elsewhere []VoxelMed version includes a graphical user interface the possibility to export the resultsof calculations to Microsoft Excel file the visualization of the fitting curves both mono andbiexponential a module for renal BED calculation following the model suggested by StrigariL [] and the possibility to correct activity for partial volume effect PVE as presentedin [] Moreover VoxelMed20 provides the user with the timeintegrated activity A at VOIlevel which can be used for dosimetry with OLINDA version both for ans and lesionsTo calculate the number of disintegrations VoxelMed integrates the timeactivity curvewith the trapezoidal method in the time interval between the first and the last acquisitionBeyond this timeinterval the integration is performed analytically and the timeactivity curveis extrapolated using the effective halflife or the physical halflife it is chosen by the userThe effective halflife of the an or lesion is derived with a biexponential fit of the activitiesin the VOI the physical halflife is known from the selected isotope Timeintegrated activityis calculated in each voxel or in the whole an depending on the modality of dose calculationselected ie voxel level or an levelRAYDOSE RAYDOSE is a software package developed at Cardiff University School ofEngineering Cardiff University UK and designed to carry out 3D patientspecific imagebased dosimetry for RPT RAYDOSE provides personalized 3D dose map performing MonteCarlo simulations on radiation transport based on the Geant4 MC toolkit CERN Switzerland Geant4 is the stateoftheart package for the simulation of the transport of psthrough matter [] RAYDOSE generates voxellevel dose maps using anatomical and physiological data taken from morphologic and functional images []In order to obtain the area under the timeactivity curve RAYDOSE allows to use differentfitting modalities monoexponential decay linear uptake plus monoexponential decay or thetrapezoidal method In this study for the dose calculation of the clinical cases we used thetrapezoidal method at the voxel level up to the last time acquisition point while the timeactivity curve beyond the last scan time was extrapolated from the monoexponential curve fittingof the whole an activities in the VOI For dose calculation in phantoms we used the physical halflife of the isotope to extrapolate the activity from the scan time upwardsData and statistical analysisTwo groups of patients were considered for the purpose of this workPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Demographic and baselines clinical characteristics of all patients�CharacteristicSample A N Sample B N Gender NoMaleFemaleAge yHeight cmWeight kgPrimary tumour site NoIleumPancreasLungThyroidRectumOthers177Lu activity for dosimetry MBq ± ± ± ± ± ± ± N\\AN\\AN\\A ± � Plusminus values are means ± standard deviation Injected activity at the first cycle of therapy Dosimetry was performed after the first injection101371journalpone0236466t003A first subgroup of cases named as Sample A was extracted by random samplingfrom the original clinical trial to adequately represent the whole population The number ofcases was calculated safely adopting a margin of error of and a standard deviation of A second independent subgroup of patients named as Sample B was extracted toofrom the original clinical trial similarly to Sample A A sample of cases was considered adequate in relation to the aim of the experiment conducted on Sample BPatient baseline characteristics for Sample A and Sample B are reported in Table The study type the image registration and the software used for the dose calculations of theclinical cases in sample A and sample B are summarised in Table Absorbed doses were calculated separately with OLINDA11 VoxelMed20 and RAYDOSEusing the same set of imagesKidney liver and spleen absorbed doses were calculated for each patient Two differentcomparison studies were performed The first study involved only comparison between VoxelMed and OLINDA based on absorbed dose calculations of patients in Sample A The secondstudy involved comparison between all the three software VoxelMed OLINDA and RAYDOSE based on absorbed dose calculations of patients in Sample B Furthermore in order toreduce the contribution of the fitting of the activitytime curves in the comparison of the software the VoxelMed dosimetry calculations for Sample B were repeated using the same effective halflife applied in RAYDOSE RAYDOSE estimates the effective halflife by fitting theTable Summary of phantom and patient studies performedStudy typePhantomObject of studyImage registrationSoftwareHomogeneous phantomNo registration only scanOLINDA11VoxelMedRAYDOSEGeometrical phantomNo registration only scanOLINDA11VoxelMedRAYDOSEAnthropomorphic phantomNo registration only scanOLINDA11VoxelMedRAYDOSEClinicalSample A patientsRigid registrationOLINDA11VoxelMedSample B patientsDeformable registrationOLINDA11VoxelMed VoxelMedλ RDRAYDOSE101371journalpone0236466t004PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Clinical comparison study workflow Procedure flow of absorbed dose calculation for each patient of sample A top of the image and sample B lowerpart of the image101371journalpone0236466g002an activities against time as previously described Flow chart in Fig illustrates methodology in clinical studyDosevolume histograms DVH were evaluated to compare spatial dose distribution atvoxellevelMean values of absorbed dose were used to compare an level and voxel level techniquesComparison between the different dosimetry methods was statistically evaluated using theLins concordance correlation coefficient CCC and the BlandAltman plot [] The CCCsymbolized by Ïc allows to evaluate the degree of concordance between two measures whilethe BlandAltman plot is used to analyse the agreement between two quantities The CCC wascalculated using SAS SAS Institute Cary NC USA A value of Ïc equal to denotes perfect concordance a value equal to perfect discordance while a value of no correlationResultsPhysical phantom studyThe values of mean absorbed dose for the physical phantoms calculated with OLINDA11VoxelMed and RAYDOSE are reported in Table Visual representation of the same data isprovided in Fig Similar DVH curves were generated with VoxelMed and RAYDOSE both for the Cylindrical phantom Fig and for the other two phantoms Fig Fig shows DHVs only for theinserts in the Geometrical and the Anthropomorphic phantoms with the smallest and the largest relative differences of mean absorbed dose respectivelyClinical studyAbsorbed dose for kidneys liver and spleen of the sample A of patients calculated withOLINDA11 and VoxelMed are shown in Table The absorbed doses to liver and to spleenwere found to be highly correlated while lower correlation was found for kidneys The CCCPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTTable Mean absorbed dose Gy calculated with OLINDA11 VoxelMed and RAYDOSE for all of the three phantoms The absorbed dose calculated withOLINDA11 was performed using either the an model and the Sphere model Absorbed doses to Pancreas Kidneys Spleen and Liver were calculated using the anmodel otherwise the Sphere model was usedPhantomCylindricalGeometricalAnthropomorphicInsert nameOLINDA11VoxelMedRAYDOSENATo17aTo17bTo26E20E30E38P38P39aP39bTu38aTu39bLesionPancreasKidneysSpleenLiver101371journalpone0236466t005[ confidence interval] values were Ïc liver [ ] Ïc spleen [ ]Ïc kidneys [ ] The BlandAltman plot is shown in Fig OLINDA11 VoxelMed VoxelMedλ RD and RAYDOSE calculated mean absorbed dosefor patients of Sample B are shown in Table while the BlandAltman plot is shown in Fig The absorbed doses calculated with VoxelMed and RAYDOSE were highly correlated withÏc kidneys [ ] Ïc liver [ ] and Ïc spleen [ ] andalmost complete agreement were found between VoxelMedλ RD and RAYDOSE withÏc kidneys [ ] Ïc liver [ ] and Ïc spleen [ ]DiscussionIn this study we compared the performances of three tools for dosimetry calculationsOLINDA11 VoxelMed20 and RAYDOSE with the primary aim to evaluate the influence ofthe calculation modality on absorbed dose assessment anlevel based voxellevel dose kernel convolution based and Monte Carlo simulations based respectively The secondary aimwas to give some recommendations about the choice of the adequate technique for dosimetrycalculation to be implemented in a hospital in a research centre or in an academic instituteclinical or research purpose small or large number of patients clinical trials only or standardprocedures This analysis was performed in standard conditions by acquisition and processing of radioactive phantoms provided with inserts of specific volume and geometry and inclinical conditions over a large cohort of patients a selection of clinical cases taken from a clinical trial in which dosimetry had already been calculated Clinical conditions are indeed quitedistant from and more complicated than the standard conditions achievable in a phantom forseveral reasons biological kinetics in place of only physical decay of activity serial acquisitionsof functional images and associated issues related to image registration [] motion of thepatient that creates artefacts in images irregular shape of volumes of interest inhomogeneousactivity distributionPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of mean absorbed dose Gy calculated using OLINDA11 VoxelMed and RAYDOSE a Homogeneous phantom b Anthropomorphicphantom and cf Geometrical phantom Note the Lesion insert in the Anthropomorphic phantom is missing because beyond the range of dose visualized in thegraph101371journalpone0236466g003Therefore to consider a large sample of clinical cases was of great interest since many studies about methods for dosimetry calculation are based on smaller groups of patients []and in a small group the inter patient variability cannot be properly investigatedThe quantitative intercomparison between all the three software was performed betweenthe mean values of absorbed doses In fact OLINDA provides only mean values while RAYDOSE and VoxelMed20 voxelbased tools provide the dose distribution that can be represented with DVHs from which the mean dose values can be derived To compare thetechniques of calculation the relative differences and the correlation between data pairs wereevaluatedFor standard conditions we evaluated discrepancies of calculated absorbed dose in a cylindrical phantom and in differently shaped inserts filled with a homogeneous radioactive solution Table shows the values of absorbed dose obtained with OLINDA11 VoxelMed andRAYDOSE in each of the phantoms These values are also plotted in Fig Lower values ofabsorbed dose were generally calculated using OLINDA in comparison with the dose calculated with other voxel modalities In the case of the cylindrical phantom a good agreementwas obtained between VoxelMed20 and RAYDOSE discrepancy equal to while largerdifference was observed between VoxelMed20 and OLINDA Absorbed dose map provided by VoxelMed and RAYDOSE showed similar spatial distribution close values of standard deviation across voxels around and analogues slope in DVHs Fig PLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calculation methods in MRTFig Comparison of DVHs calculated using VoxelMed continuous line and RAYDOSE dotted line for the Cylindrical phantom101371journalpone0236466g004To compare the calculation techniques in different conditions of volume and geometry theGeometrical phantoms were acquired Relative differences in absorbed dose depend on theshape and on the volume of the inserts smaller is the volume and further from a regular sphereis the shape more the relative difference is higher In the Geometrical phantom the toroidalinserts provided the greatest discordance relative difference with VoxelMed dose rangingfrom to for OLINDA and from to for RAYDOSE while in the otherinserts differences ranged between [ ] for OLINDA11 and [ ] for RAYDOSEOn one hand the insert dose calculations in OLINDA were performed using the spheremodel since OLINDA only allows to perform dosimetry calculation for specified models ieans or spheres This approximation might explain the huge discrepancies obtained withthe voxelbased methods On the other hand a reason for the difference between RAYDOSEand VoxelMed is that the latter applies a mask before the convolution while RAYDOSE doesnot This contribution affects calculations in so far as the geometry and the volume of theinsert may influence the activity distribution and leave empty spaces around or inside theobjects This effect is especially pronounced for example in the case of the toroid The application of a mask also implies the lack of photon cross irradiation contribution between insertswhich has an impact on dose calculation contribution around [] Discrepancies ofPLOS ONE 101371journalpone0236466 August PLOS ONE 0cComparison of different absorbed dose calc | Thyroid_Cancer |
Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learnings groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center DiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patients life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images []Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined []Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the identityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called BONENAVI version The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed CADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing à convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the networks weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physicians scores Taking a closer lookat the results it can be concluded that networks performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physicians further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patients whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center Diagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatients cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution à pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center DiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patientscategory for example malignant_ and healthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value was assigned for malignant_ prefixes whereas the value for healthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Pythons randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po | Thyroid_Cancer |
"Pluripotent stem cellsDirected differentiationIn vitro disease modellingLungAirwayMechanical cuesContentsChronic lung diseases remain major healthcare burdens for which the only curative treatment is lung transplantation In vitro human models are promising platforms for identifying and testing novel compounds to potentiallydecrease this burden Directed differentiation of pluripotent stem cells is an important strategy to generate lungcells to create such models Current lung directed differentiation protocols are limited as they do not recapitulate the diversity of respiratory epithelium generate consistent or sufï¬cient cell numbers for drug discoveryplatforms and establish the histologic tissuelevel anization critical for modeling lung function In this review we describe how lung development has formed the basis for directed differentiation protocols and discussthe utility of available protocols for lung epithelial cell generation and drug development We further highlighttissue engineering strategies for manipulating biophysical signals during directed differentiation such that futureprotocols can recapitulate both chemical and physical cues present during lung development Elsevier BV All rights reservedOverview of key developmental stages Lung anogenesis molecularly deï¬ning lung fate in the embryo Branching morphogenesis and other mechanical cues generated during lung development Introduction Human embryology as a blueprint for lung directed differentiation Directed differentiation of lung epithelia inspired by embryology Mouse embryonic stem cell derived lung epithelia Modeling airway and lung diseases for drug discovery Opportunities to exploit mechanical cues for improving directed differentiation protocols in the future Micropatterning in 2D Stem cell behaviour on substrate topographies Micropatterning in 3D anoid systemsSubstrate textureHuman pluripotent stem cellderived lung epithelia Creation of human proximal lung epithelia Comparisons of proximal airway directed differentiation protocols Creation of human distal lung epitheliaComparisons of distal lung directed differentiation protocols Limitations of current directed differentiation protocols Correspondence to G Karoubi Latner Thoracic Surgery Research Laboratories Toronto General Hospital College St Toronto ON M5G 1L7 Canada Correspondence to A P McGuigan Institute for Biomaterials and Biomedical Engineering University of Toronto College Street Toronto ON M5S 3G9 CanadaEmail addresses golnazkaroubiuhnresearchca G Karoubi alisonmcguiganutorontoca AP McGuigan101016jaddr2020080050169409X Elsevier BV All rights reservedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxAcknowledgementsReferencesFuture outlook IntroductionEndstage lung disease is the third leading cause of morbidity andmortality worldwide [] and produces a significant burden onhealthcare systems due to extensive resource expenditures for diseasemanagement and as lung transplantation is the only curative treatmentoption Such diseases include acute respiratory distress syndromechronic obstructive pulmonary disease cystic ï¬brosis and pulmonaryï¬brosis Chronic pulmonary diseases result in million global deathsper year [] Patients who receive transplants face continued complications associated with chronic immunosuppression and graft rejectionwith the transplant survival rates at and years being and respectively [] Furthermore since lungs function as an important barrier between the internal and the external environments they are a critical site for bacterial and viral infections and disease transmissionparticularly relevant given the current COVID19 pandemic There istherefore a critical need to better elucidate the mechanisms of infectiondisease progression host response and cellular repair in the lung to enable the development of novel targeted therapeutics for lung diseaseTissueengineered models have emerged as a technology to addressthis challenge and shown some success in drug identiï¬cation and toxicology studies For example commercially available airway epithelialmodels such as EpiAirwayTM MatTek Life Sciences serve as convenientplatforms with airliquid interface culture capabilities for assessing theeffect of chemical and physical stimuli [] Other examples includethe Alveolus LungChip and Airway LungChip systems Emulate Incoriginally developed in the Ingber laboratory which mimic theepithelialendothelial interface of the airway and provide a more dynamic platform for testing new antiammatory compounds inasthma [] and new small molecule targets to decrease cancerassociated pulmonary edema [] More complex models have alsobeen reported which involve selfassembly of heterogeneous progenitor cells into 3D structures termed anoids [] These anoidmodels can recapitulate aspects of human lung development in termsof tissue structures and protein expression and therefore present apromising opportunity for drug screening []A challenge in developing such human in vitro lung models to screenfor drugs however is the requirement for large batches of similarhuman cells as a starting population for tissue manufacturing to ensureminimal heterogeneity between test wells [] Achieving this is especially challenging when using primary human lung cells which exhibitconsiderable heterogeneity across donors and have a limited ability togrow and differentiate reliably [] Furthermore primary cells areoften extracted from diseased donors which is not ideal for conductingcontrolled studies due to the wide range of therapeutic and environmental factors these cells have already been exposed to Directed differentiation of pluripotent populations has the potential to create vastnumbers of cells from either healthy or diseased patients It allows introduction of speciï¬c diseaseassociated mutations via CRISPRCas9gene editing to recapitulate and understand pathologies in a controlledmanner As such directed differentiation enables the generation of anattractive cell source for drug screening platforms and personalized disease models that may provide insight into tissue regeneration mechanisms []Directed differentiation protocols to manufacture speciï¬c cell populations from pluripotent stem cells PSCs have been developed to meetthe need for a homogeneous human cell source Older lung directed differentiation protocols from the late 2000s have been proven inefï¬cientdue to the nonstandardized methods through which they derive lungendoderm from embryoid bodies [] A series of more standardizedstepwise protocols have since emerged in the last decade that provideavenues for developing airway and lung epithelia albeit with variableefï¬ciencies [] The ï¬rst uential directed differentiation protocol to produce lung epithelia used human PSCs in [] whichwas further supported by two prominent studies conducted usingmouse PSCs in [] These protocols have continued to be enhanced through adaptations related to the selection of growth factorsand small molecules the chronology of morphogen delivery as wellas innovations in enabling platforms such as cell sorting 3D cultureand singlecell analyses to efï¬ciently derive normal and diseased lungepithelia from human PSCs [] Despite such advancements limitations pertaining to heterogeneity in the resultingpopulations still exist which are likely attributed to variability across directed differentiation trials PSC cell lines or the persistence of contaminating cell populations belonging to other lineages While protocolshave progressed to some degree in differentiating proximal airwayand distal alveolar epithelia they remain limited Overall many unanswered questions remain with regards to the identity maturity andfunctionality of resulting cell types as well as their utility for tissue engineering and drug testing approaches Therefore these protocols mustbe optimized further to reliably produce large numbers of spatially relevant and functional lung and airway epithelial cells that appropriatelyrespond to both chemical and mechanical stimuli in the context of disease modeling and drug discoveryIn this review we discuss the directed differentiation protocols thatattempt to recapitulate lung development and disease and highlightpossible opportunities to enhance these protocols in the future Weï¬rst describe development of native lung tissue and the patterningevents that occur that differentiation models attempt to mimic andhighlight how human lung embryology has served as the blueprint tocreate the common pathway of lung directed differentiation protocolsWe then discuss the evolution of directed differentiation protocols toï¬nd opportunities for creating speciï¬c populations of airway and lungepithelia through targeted manipulation of key signaling pathways in2D and 3D models We further describe how these models have beenused to recapitulate different airway and lung diseases Finally we discuss how tissue engineering and biophysical cues using biomaterialscan be utilized during lung directed differentiation to mimic patterningcues present in development to augment current differentiationprotocols Human embryology as a blueprintdifferentiationforlung directed Overview of key developmental stagesDirected differentiation protocols have been designed to mimicin vivo human lung development [] Indeed in vitro models of lungdevelopment have provided unique insight into human lung development [] As human lung development has been described at greatlength in earlier reviews [] we provide a brief overview as followsschematically represented in Fig During early embryogenesis at days post fertilization a process called gastrulation begins with the appearance of a structure called primitive streak through which cells migrate to form the primary embryonic germ layers deï¬nitive endodermmesoderm and ectoderm [] Deï¬nitive endoderm expandsthereby forming the primitive gut tube comprised of three endodermalregions foregut midgut and hindgut [] This is when lungPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxFig Schematic of human lung development from an epithelial perspectivedevelopment begins at approximately four weeks into embryonic lifewith the outgrowth of foregut endoderm [] and continues througheight years of postnatal life [] There are ï¬ve stages to lungdevelopment Embryonic weeks The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surroundingmesenchyme and develop into embryonic lung buds with early trachea and bronchi [] Pseudoglandular weeks Branching of the airway continuesleading to formation of conducting and terminal bronchioles whilethe proximal airway epithelium begins to develop [] Canalicular weeks Development of the respiratory or gasexchanging airways is initiated primitive alveoli form and the future distal epithelium begins to thin as distal epithelial markers areexpressed [] Saccular weeks Emergence of sacshaped distal airwayswhich develop crests with muscle and elastin to create indentationsThese distal airways extend to form alveoli by weeks [] The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange andfurther differentiation of alveolar epithelial cells AEC I and II takesplace Alveolar periods week years True alveoli are seen in week and the majority of alveolarization takes place through sacculeseptation a process by which the sacshaped distal airways changetheir internal architecture and create thin walls intraluminallySeptation leads to an increase in surface area of the gas exchangingportion of the developing lung and prepares the fetus to breath airduring this stage [] Lung anogenesis molecularly deï¬ning lung fate in the embryoDuring the embryonic period early lung is genetically deï¬ned by theexpression of transcription factor NK2 Homeobox NKX21 and Srybox SOX2 [] During human lung development it has beenfound that the lung buds and branches given off during thepseudoglandular period are mostly SOX2SOX9 [] BothSOX2 and SOX9 are individual markers of the early proximal or distal lineage respectively [] Over the course of the canalicular and saccular periods of development weeks these double positivepopulations downregulate one SOX protein and maintain expressionof the other as these cells mature towards proximal or distal lineages[] The proximal airway closer to the mouth is comprised of apseudostratiï¬ed columnar epithelium that is responsible for theconducting airway function debris and pathogen removal ciliatedcells mucus production goblet cells prevention of airway ammation club cells and humidiï¬cation of air as it passes through to the distal lung compartment [] The squamous distal epitheliumcomposed of alveolar epithelial cells AEC I and II facilitates the respiratory function of the lung as air in the epithelial compartment is broughtinto close apposition to blood from the pulmonary vasculature it alsosecretes surfactants which play an immunologic role and decrease thesurface tension present at the airliquid interface thereby preventing alveolar collapse [] In humans a number of cell types are found in theproximal airway each identiï¬ed with speciï¬c markers Table This includes basal cells tumor protein p63P63 keratinKRT5 nerve growthfactor receptorNGFR integrin α6ITGA6 integrin β4ITGB4 ciliatedcells Forkhead BoxJ1FOXJ1 acetylated tubulinAcTUB goblet cellsmucin 5ACMUC5AC mucin 5BMUC5B club cells club cell secretoryproteinCCSP or SCGB1A1 and pulmonary neuroendocrine cellsPNECs synaptophysinSYP chromogranin ACHGA On the otherhand homeodomainonly protein HOPX identiï¬es the distal lungalong with AEC I cells T1α podoplaninPDPN aquaporin 5AQP5while AEC II cells are recognized via surfactant protein B SPC prosurfactant protein C proSPC or SPC and HT2280 []One mechanism by which lung epithelia begin to mature is based onchemokine secretions from the surrounding mesenchyme and the developing heart ï¬eld which are well reviewed here [] Key players including ï¬broblast growth factors FGFs [] WNTs []and bone morphogenetic proteins BMPs [] are known to inducethe differentiation of early lung progenitors in a controlled manner Forexample in mouse it has been found that FGF10 plays a role in bud outgrowth [] and drives lung progenitors towards a distal fate []through canonical WNT signaling [] Proximal epithelia developbecause they are located further away from distally located FGF reservoirs in the mesenchyme in a mechanism that appears dependent onconcentration gradients [] BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventralback to front and proximodistal top to bottom patterning in the nascent lung [] BMP4 is also present at high levels in distal bud tips andepithelia including AEC II cells [] however its inhibition promotesa proximal fate and along with BMP2 inhibition ciliated cell development [] Branching morphogenesis and other mechanical cues generated duringlung developmentWhile the cell fate of early proximal and distal lineages is directedthrough chemical signals the lung epithelium itself undergoes markedPlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxTable Epithelial populations in native human airways and lungsRegionProximal AirwayDistal LungCell TypeAssociated Markers for Cell CharacterizationCiliated CellGoblet CellClub CellBasal CellAlveolar epithelial cell type I AEC IAlveolar epithelial cell type II AEC IIFOXJ1 AcTUBMUC5AC MUC5BCCSP SCGB1A1 SCGB3A2P63 KRT5 NGFR ITGA6 ITGB4HOPX PDPN AQP5SPB SPC HT2280Cell Proportions in Native Lung [] [] [] [] [] []changes in architecture a process known as branching morphogenesis[] From the simple tube of the anterior foregut endoderm to thecomplex tubular structure of the adult a highly stereotyped mechanismof branching morphogenesis facilitates the outgrowth division placement and structure of lung airway [] Branching morphogenesis ofthe lung is driven by three simple and iteratively used processes domain branching planar and orthogonal bifurcation [] The ï¬rst formof branching is domain branching along a primary branch buds formin a linear and sequential fashion from proximal to distal The nextform of branching is planar bifurcation in which the tip of the formingtube bifurcates to create two new tips which subsequently elongateand bifurcate again creating four tips The last process of branching isknown as orthogonal bifurcation In this process the initial planar bifurcation is followed by a rotation around the planar axis which createstwo new tips through bifurcation A critical gene in this processSprouty has been found to attenuate Erk12 signaling thereby alteringthe orientation of cell division and future tube elongation [] Othercritical genes and regulatory networks associated with FGF signalingalso contribute to controlling the periodicity of the branched network[] Although elements such as domain speciï¬cation bifurcation rotation and branch generation remain largely undetermined [] newtechnologies involving highresolution live imaging tension sensingand forcemapping are opening paths to further explore and explainthe branching morphogenesis phenomenon []The early structure of the lung gives rise to a striking architecturalseparation of future SOX2 proximal lineages and SOX9 distal lineages at least in mice [] The diameter of tube generated duringbranching morphogenesis in the pseudoglandular and canalicularstages has a small degree of variance within each stage as measuredfrom electron micrograph sources of fetal human tissue [] This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on thedeveloping epithelia Once the basic an structure has formed thelung continues to be exposed to mechanical cues as it continues to mature In several cases these cues have been shown to be essential forcorrect an function In utero the fetal lung is a secretory an thatonly converts to an absorptive one to prepare for breathing afterbirth through a change in the activity of chloride and sodium channelslate in development Fetal lung secretions result in a static ï¬uid pressureof around cmH2O in the developing terminal sacs of the fetus whichpropels branching morphogenesis outwards into the developing thoracic cavity [] Lack of amniotic ï¬uid in the developing lung alters the expression of distal epithelial markers and consequentlyresults in the creation of smaller than normal lungs pulmonary hypoplasia [] highlighting the importance of this mechanical pressureduring lung development In addition cyclic strain is generated fromfetal breathing movements FBM in utero that prime the airway foruse after birth FBM are detectable from the tenth week of pregnancyand begin as infrequent and erratic activity with long quiescent periodsAs development continues these quiescent periods decrease andsustained periods of fetal breathing occur These breathing movementsvary with the fetal sleep cycle and can be chemically tuned [] andalter the volume of terminal sacs by around [] againhighlighting the importance of mechanical signals uencing lung development Finally a novel FGF10FGFR2dependenttensionalmechanism has been shown by which distal epithelial cells in the lungaccumulate motor proteins at the apex of the cell thereby becoming resistant to compression from increasing ï¬uid pressure within the tubelumen Cells under this tension are more likely to become AEC II cellswhile those under compression become AEC I cells [] Interestinglywhile the above examples highlight the importance of speciï¬c mechanical signals in the growth development and differentiation of the lungPSC directed differentiation protocols of the lung are primarily based onmimicking the sequential chemical changes that occur during lungdevelopment Directed differentiation of lung epithelia inspired by embryologyEarly attempts to create lung epithelia from PSCs began in mouseand did not attempt to mimic the stepwise changes in chemical signaling that occur during development Rather groups focused on applyinglunglike physical cues such as airliquid interface [] These protocols while successful in generating NKX21 positive populationsalso produced contaminating cells expressing pluripotency markersOCT4 NANOG SSEA4 TRA160 TRA181 These early attempts solidiï¬ed that further optimization particularly related to the chemical cuesapplied was needed to reliably create lung progenitors from pluripotentsources without remnant pluripotent contaminating cells More successful directed differentiation protocols were rationalized from the detailed understanding of the chemical changes during lung embryologyIn this section we describe in detail the different differentiation protocols currently available that evolved from this approach Mouse embryonic stem cell derived lung epitheliaAlthough mouse models do not fully recapitulate human lung development they have served as guides for earlier iterations of PSC directeddifferentiation protocols and have identiï¬ed critical chemical cues forlung anogenesis Broadly speaking these protocols begin by drivingstem cells towards a deï¬nitive endoderm fate SOX17 and FOXA2mimicking the preembryonic period of human lung developmentweeks through high doses of the nodal activating molecule ActivinA [] Foregut endoderm is then induced via transforminggrowth factor beta TGFβ inhibition either alone [] or with BMP inhibition [] for a short period a process called anteriorization as duringthe embryonic period of human lung development weeks Thisforegut endoderm FOXA2SOX2 is subsequently induced to generate NKX21 cells putative lung progenitors by stimulating theretinoic acid RA BMP WNT and FGF signaling pathways []These lung progenitors are further matured as demonstrated by increased NKX21 expression through application of corticosteroids[] In brief each protocol begins with PSCs guided through deï¬nitiveendoderm followed by anteriorization to foregut endoderm and subsequent ventralization to generate NKX21 cells [] These protocolsformed the basis and backbone for the creation of human lung epitheliafrom PSCsGiven the structural and cellular complexity of the lung it is reasonable that the earliest protocols focused on mouse However there aresubtle differences that highlight how human models are different interms of structure patterning and differentiation For example thePlease cite this as R Varma JP Soleas TK Waddell Current strategies and opportunities to manufacture cells for modelinghuman lungs Adv Drug Deliv Rev 101016jaddr202008005 0cR Varma Advanced Drug Delivery Reviews xxx xxxentire human conducting airway is comprised of a pseudostratiï¬ed epithelium even at diameters less than 05mm [] In contrastconducting airways in mice only exhibit pseudostratiï¬ed epitheliumwith accompanying submucosal glands and cartilage in the most proximal portion of the airway and transition directly into alveolar sacs []This difference in histology affects the residing cell populations as evidenced by the lack of basal cells in the lower portion of the proximal airways of mice [] Similarly mouse models suggest thatSOX2SOX9 progenitors are quite rare and their cell fate is ambiguous [] However evidence from directed differentiation of humanlung epithelia [] which has been conï¬rmed in vivo []reveals that SOX2SOX9 progenitors are common in the developinglung buds and that branch tips of the pseudoglandular staged lunggive rise to both proximal and distal epithelia [] Moreover speciï¬cprotein markers have been found to differ in both timing and locationof expression between human and mouse models proSPC in mouseis expressed early and throughout the developing mouse epithelium[] while in human proSPC is rarely detected early in development and is only robustly found later in distal epithelia [] These examples highlight that while there are similarities development andpatterning of mouse and human lungs is different and these differencesrequire human models to be fully appreciated Human pluripotent stem cellderived lung epitheliaHuman PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation wherein deï¬nitive endoderm anterior foregut endoderm and NKX21 lungprogenitors are produced sequentiallyDifferent groups have adhered to their own methods of generatingdeï¬nitive endoderm which primarily involves exposing PSCs to highconcentrations of Activin A Slight variations such as introducing WNTagonism through WNT3a or CHIR99021 prior to [] or alongside[] Activin A or additional exposure to BMP4 and FGF2[] during this stage exist across protocols for differentially inducing primitive streak and its anteriorization towards producing deï¬nitive endoderm In addition the use of embryoid bodies which arelimited by user experience and technique has resulted in a widerange of production efï¬ciencies for achieving this stage from CKITCXCR4EPCAM cells [] to CKIT CXCR4 cells []Recent advances in commercial products have led to development ofstandardized 2D culturebased media STEMdiff Deï¬nitive EndodermKit STEMCELL Technologies which allow reliable derivation of of deï¬nitive endoderm []Similarly generation of both anterior foregut endoderm andtoventralized lung progenitor populations has been subjectmuch investigation and modiï¬cation Earlier work suggested thatSOX2FOXA2 ± in their case anterior foregut endoderm canonly be induced by subjecting deï¬nitive endoderm to TGFβ and BMP inhibition [] Subsequent studies however attempted to anteriorize deï¬nitive endoderm to foregut endoderm through TGFβ inhibition alone SOX2 a combination of endogenous WNT TGFβ and BMP inhibition not quantiï¬ed [] and via Sonic Hedgehog SHH and FGF2signaling FOXA2 EPCAM [] A comparison of the lattertwo strategies demonstrated that SHH and FGF2 are insufï¬cient inproducing reliable NKX21 lung progenitors [] possibly becauseFGF2 is involved in promoting thyroid lineages [] In general TGFβand BMP inhibition [] is the basis for currently applied endodermanteriorization strategies []Factors involved in early versions of ventralization in directed differentiation protocols included WNT3a FGF7 FGF10 BMP4 epidermalgrowth factor EGF and RA have now been reduced based on elimination studies [] As such CHIR99021 CHIR WNT agonist BMP4and RA are necessary and sufï¬cient for producing lung progenitorsfrom anterior foregut endoderm derived from both mouse and humanPSCs [] Despite ï¬nding that FGF7 and FGF10 are nonessential for inducing NKX21 expression they continue to be used forventralization in some protocols [] Although each protocol differsin terms of the duration of each phase NKX21 lung progenitors aregenerally achieved by days with the exception of a study by deCarvalho in which they maintained their cultures for an additional days in FGF7 FGF10 and CHIR99021 to attain NKX21FOXA2 lung progenitors In all cases these lung progenitors are theneither sorted or directly guided towards proximal or distal progeny in2D or 3D culture systems Ideally products of directed differentiationprotocols should mimic the cell proportions present in human airwaysand lungs Table however current protocols have not progressedthat far While these protocols continue to be reï¬ned the percentageof select cell populations generated from these protocols have beensummarized in Table Creation of human proximal lung epitheliaProtocols to create proximal lung epithelia have focused on the production of the four major cells types present ciliated goblet club andbasal cells see Table for a summary of markers for each cell type Motivation for creating proximal epithelia in the ï¬eld has primarily been todevelop patientspeciï¬c cystic ï¬brosis CF models [] andor toproduce epithelia with multiciliated cell populations for protocol validation [] A shift towards human PSCderived CF models hasbeen critical as mouse models do not accurately represent CF diseaseprogression and phenotypes seen in humans [] As such theï¬rst evidence of human PSC proximalization using CF patientderivedPSCs was shown by Mou who exposed anterior foregut endodermto BMP4 GSK3iXV WNT agonist FGF2 and RAsupplemented B27 togenerate NKX21 cells by Day Although contaminatingneuroectodermal and distal lung NKX21SOX9 cells were presentday populations included proximal NKX21SOX2 progenitorsSubcutaneous implantation of this population in immunodeï¬cientmice for days resulted in emergence of NKX21P63 cells howeverno mature epithelial markers for ciliated goblet and club cells werefoundWong employed a longer 2D differentiation approach to produce mature proximal airway epithelia in vitro Through a processthey called proximal speciï¬cation they generated day lung progenitors via low levels of BMP4 mimicking signaling gradients in theairway FGF7 and FGF10 which began expressing proximal genes Further culture with FGF7 FGF10 and FGF18 resulted in upregulated geneexpression of KRT5 P63 FOXJ1 SOX17 cystic ï¬brosis transmembraneconductance regulator CFTR and SCGB1A1 to a lesser extent alongwith low levels of distal SOX9 and SPC by day Protein expressionamounted to NKX21 panKRT P63 FOXJ1 cells These cells were subsequently matured in airliquid interface ALI culture for weeks week of submerged culture withFGF18 followed by weeks of ALI culture to generate CFTRpanKRT FOXJ1 with coexpressing CFTR and CFTRLHS28 cells The resulting epithelium ranged from being squamous to cuboidal with sparse pseudostratiï¬ed regions implying thatthis protocol lacked speciï¬c maturation cues Contaminating thyroidthyroglobulin and PAX9 liver HNF4 and AFP and pancreaticPDX1 lineages were detected through quantitative PCR while percentages of goblet club and basal cell populations barring gene expression analysis were not evaluatedA similar 2D culture approach was employed by Firth to generate proximal lung progenitors which were subsequently matured intomulticiliated epithelia They optimized lower concentrations of BMP4required during the ventralization phase day | Thyroid_Cancer |
Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A casecontrolstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction HardyWeinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 oddsratio OR confidence interval CI however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [] Graves ophthalmopathyGO is one common anspecific complication affecting of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO []Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acasecontrol study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbeccos modified Eagles medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at ¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturers protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3CMycNR3C1 pCMV3CMycFOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturers protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturers protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNAIP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM TrisHCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydIdC and glycerol] for min at ¦C in a final volume of μl FOXP3 or NR3C1nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive [] [] Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbSigniï¬cance of age were evaluated by t testP005P00010039a 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the HardyWeinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 OR CI which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG 0929bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with signiï¬canceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAG2495cGG b0154b2467bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with signiï¬canceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearsons correlation Spearmans correlation Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearsons correlation Spearmans correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3CE The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 BE Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GOatherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via Drug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE HardyWeinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves Disease N Engl J Med 101056NEJMra1510030 Brent GA Clinical practice Graves disease N Engl J Med 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves disease CMAJ Canadian Med Assoc J J de lAssoc Med Canadienne The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves disease Endocrinol Metab Clin North Am 101016S0889852905702991 Bednarczuk T Gopinath B Ploski R and Wall JR Susceptibility genes in Graves ophthalmopathy searching for a needle in a haystackClin Endocrinol Oxf 101111j13652265200702854x Anvari M Khalilzadeh O Esteghamati A Esfahani SA Rashidi A Etemadi A et al Genetic susceptibility to Graves ophthalmopathy therole of polymorphisms in proinï¬ammatory cytokine genes Eye Lond 101038eye2009244 Bahn RS Understanding the immunology of Graves ophthalmopathy Is it an autoimmune disease Endocrinol Metab Clin North Am vi Manji N CarrSmith JD Boelaert K Allahabadia A Armitage M Chatterjee VK et al Inï¬uences of age gender smoking and familyhistory on autoimmune thyroid disease phenotype J Clin Endocrinol Metab 101210jc20061402 Stan MN and Bahn RS Risk factors for development or deterioration of Graves ophthalmopathy Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy20101634 Bahn RS and Heufelder AE Pathogenesis of Graves ophthalmopathy N Engl J Med Tomer Y Barbesino G Greenberg DA Concepcion E and Davies TF Mapping the major susceptibility loci for familial Graves andHashimotos diseases evidence for genetic heterogeneity and gene interactions J Clin Endocrinol Metab Tomer Y Ban Y Concepcion E Barbesino G Villanueva R Greenberg DA et al Common and unique susceptibility loci in Graves andHashimoto diseases results of wholegenome screening in a data set of multiplex families Am J Hum Genet 101086378588 Gianoukakis AG and Smith TJ Recent insights into the pathogenesis and management of thyroidassociated ophthalmopathy Curr OpinEndocrinol Diabetes Obesity 101097MED0b013e32830eb8ab Shiina T Ota M Shimizu S Katsuyama Y Hashimoto N Takasu M et al Rapid evolution of major histocompatibility complex class I genesin primates generates new disease alleles in humans via hitchhiking diversity Genetics 101534genetics106057034 Liu YH Chen YJ Wu HH Wang TY and Tsai FJ Single nucleotide polymorphisms at the PRR3 ABCF1 and GNL1 genes in the HLA class Iregion are associated with Graves ophthalmopathy in a genderdependent manner Ophthalmology 101016jophtha201404027 Wang S Sun H Chen HY Zhao ZF Yang Y Zhao YJ et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves disease in Chinese patients Endocrine 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Gravesophthalmopathy patients Ophthalmology 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves ophthalmopathy Hum Immunol 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol Siegmund T Usadel KH Donner H Braun J Walï¬sh PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves disease Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with GravesOphthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi | Thyroid_Cancer |
Breast cancer BC is the most common malignancy among women Emerging studies have demonstrated that circular RNA circRNA zinc finger RNA binding protein circZFR serves as a crucial regulator in many human cancers However the role and mechanism of circZFR in BC tumorigenesis remain unclearMethods The levels of circZFR miR578 and hypoxiainducible factor 1α HIF1A were detected by quantitative realtime polymerase chain reaction qRTPCR or western blot Cell viability colony formation apoptosis migration and invasion capacities in vitro were determined by using the Cell Counting Kit8 CCK8 standard colony formation flow cytometry and transwell assays respectively Glucose uptake lactate product and adenosine triphosphate ATP levels of cells in vitro were measured using the commercial human assay kits Targeted relationships among circZFR miR and HIF1A in BC cell lines were verified by dualluciferase reporter and RNA pulldown assays Animal studies were performed to assess the effect of circZFR on tumor growth in vivoResults Our data indicated that circZFR was overexpressed in BC tissues and cells and the increased circZFR level predicted poor prognosis of BC patients CircZFR silencing or miR578 overexpression repressed BC cell viability colony formation migration invasion and glycolysis and enhanced cell apoptosis in vitro CircZFR silencing also hampered tumor growth in vivo Mechanistically circZFR acted as a sponge of miR578 and circZFR silencing hindered BC cell malignant behaviors by miR578 HIF1A was a functional target of miR578 in regulating BC cell viability colony formation migration invasion glycolysis and apoptosis in vitro Furthermore circZFR modulated HIF1A expression through sponging miR578Conclusion Our findings first identified that the silencing of circZFR suppressed BC malignant progression in vitro via the regulation of the miR578HIF1A axis providing evidence for the crucial involvement of circZFR in BC pathogenesisKeywords Breast cancer BC circZFR miR578 HIF1A Malignant progressionCorrespondence qiuxinguang2020163com Department of Thyroid Surgery The First Affiliated Hospital of Zhengzhou University No1 Jianshe East Road Erqi District Zhengzhou Henan ChinaFull list of author information is available at the end of the BackgroundBreast cancer BC remains the most commonly diagnosed cancer and the leading cause of cancerassociated death among females in [] Although the therapeutic methods have greatly improved over the past two decades effective treatment against metastatic BC is still limited [ ] Therefore a deeper understanding of what drives this disease is the first step to design innovative interventions The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cChen a0et a0al Cancer Cell Int Page of Circular RNAs circRNAs are covalently closed endogenous RNAs that have crucial noncoding functions in human physiologic and pathologic processes [] Work in biological functions has demonstrated the roles of circRNAs as microRNA miRNA sponges [] Dysregulation of circRNAs has recently implicated in the pathogenesis of BC [] For example Yuan et a0al uncovered that hsa_circ_0068033 a downregulated circRNA exerted a repressive impact on BC malignant progression via sequestering miR659 [] Cao and colleagues demonstrated that hsa_circ_0087784 functioned as a potential promoter in BC development through sponging miR487a [] Xu et a0al reported that circTADA2As attenuated BC progression and metastasis by the regulation of miR203a3p [] Moreover hsa_circ_001783 and circABCB10 were reported as oncogenic regulators in BC through functioning as specific miRNA sponges [ ] As for circRNA zinc finger RNA binding protein circZFR hsa_circ_0072088 it has been identified as an oncogenic modulator in many human cancers such as renal carcinoma bladder cancer and nonsmall cell lung cancer [] Nevertheless the biological roles of circZFR in BC tumorigenesis remain largely unknownMiRNAs modulate gene expression but are frequently dysregulated in human cancers including BC [] Danza et a0al reported that miR578 was underexpressed in BRCABC and it regulated tumor angiogenesis [] However the precise function of miR578 in BC progression is still undefinedHere we undertook to examine the biological effect of circZFR in the malignant progression of BC in a0 vitro and in a0 vivo We identified that circZFR a prominently upregulated circRNA in BC controlled BC progression in a0vitro via targeting the miR578hypoxiainducible factor 1α HIF1A axisMaterials and a0methodsClinical tissues and a0cellsIn this study we enrolled BC patients admitted to The First Affiliated Hospital of Zhengzhou University from April to June The clinicopathological features of these patients were provided in Table a0 Seventy pairs of primary tumor tissues and matched healthy breast tissues were collected and stored at a0°C to detect the expression levels of circZFR miR578 and HIF1A Accession NM_1810543 These patients were followedup for at least a0 months and the followup information was obtained by telephone calls every a0 months The study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University and written informed consent was provided by all participantsTable Correlation and a0the a0clinicopathological features of a0BC patientsbetween a0circZFR expression CharacteristicsNumbercircZFR expressionPHighLowAge years ¥ Distant metastasis Present AbsentTumor size cm TNM stage I II III IVHER2 status Positive NegativePR status Positive NegativeER status Positive NegativeHER2 human epidermal growth factor receptor2 PR progesterone receptor ER estrogen receptorP P BC cell lines MCF7 ATCC ®HTB22 BT549 ATCC ®HTB122 MDAMB231 ATCC ®HTB26 and MDAMB453 ATCC ®HTB131 American Type Collection Culture ATCC Manassas VA USA were cultured in RPMI1640 medium supplemented with fetal calf serum FCS and antibiotic solution all from cell line ATCC ®CRL10317 ATCC was maintained in HyClone Logan UT USA The immortalized MCF10A Dulbeccos modified Eagles mediumNutrient Mixture F12 DMEMF12 with FCS a0ngmL epidermal growth factor a0 μgmL hydrocortisone and a0 μgmL insulin all from HyClone All cells were cultured in a CO2 incubator at a0°CQuantitative realtime polymerase chain reaction qRTPCRThe expression levels of circZFR HIF1A and miRNAs were gauged by qRTPCR Complementary DNA cDNA synthesis was done using total RNA a0 ng 0cChen a0et a0al Cancer Cell Int Page of isolated by Isogen Nippon Gene Tokyo Japan from tissues and cell lines The levels of circZFR and HIF1A were quantified using the TaqMan Gene Expression Assays with the indicated primers and mature miRNAs were assayed using the TaqMan MicroRNA Assays with TaqManspecific primer probes as recommended by the manufacturers Applied Biosystems Rotkreuz Switzerland qRTPCR was run in triplicate on the iCycler iQ5 device BioRad Munich Germany using the PCR conditions as previously reported [] Results were normalized to the expression of glyceraldehyde3phosphate dehydrogenase GAPDH or U6 internal control using the ÎÎCt method [] Primer sequences for circZFR forward ²ATG GTC TGC AGT CCT GTG TG3² were and reverse ²TGG TGG CAT GTT TTG TCA TT3² for HIF1A were forward ²TTC CCG ACT AGG CCC ATT C3² and reverse ²CAG GTA TTC AAG GTC CCA TTTCA3² for miR578 were forward ²GTG CAG GGT GTT AGGA3² and reverse ²GAA GAA CAC GTC TGGT3² for miR944 were forward ²GAG TAG GCT ACA TGT TAT TAAA3² and reverse ²GTG CAG GGT CCG AGGT3² for miR5323p were forward ²ATC CTC CCA CAC CCA AGG ² and reverse ²GTG CAG GGT CCG AGGT3² for GAPDH and U6 were described previously []Lentivirus transduction and a0transient transfectionCircZFR knockdown in MCF7 and BT549 cell lines was achieved by the transduction of corresponding lentiviruses expressing three different sequence shRNAs specific to circZFR shcircZFR1 shcircZFR shcircZFR2 and shcircZFR3 Fulengen Guangzhou China and nontarget shRNA lentiviruses shNC were used as the negative control Vectortransduced cells were selected by puromycin Solarbio Beijing China at a final concentration of a0μgmL at least a0h MiR578 overexpression and knockdown cell lines were generated using the synthetic miR578 mimic a0nM Ribobio Guangzhou China and inhibitor antimiR578 a0nM Ribobio respectively with a corresponding nontarget oligonucleotide miRNC mimic or antiNC Ribobio as the negative control To elevate HIF1A expression in BC cell lines a recombinant overexpressing plasmid for HIF1A HIF1A a0 ng Ribobio or negative control plasmid vector Ribobio was transiently transfected into cell lines using Lipofectamine reagent Invitrogen Breda The Netherlands as per the manufacturers protocols Each experiment was performed in triplicateCell viability colony formation and a0apoptosis assaysshcircZFRinfected or shNCtransduced cell lines were transfected with or without antiNC or antimiR578 and MCF7 and BT549 cell lines were carried out the indicated transfections followed by the incubation for a0h at a0°C Cell viability assay was done using the Cell Counting Kit8 CCK8 Abcam Cambridge UK assay as per the manufacturers guidance Cell colony formation was tested using standard colony formation protocols as previously reported [] Cell apoptosis measurement was performed by flow cytometry using doublestaining with fluorescein isothiocyanate FITClabeled Annexin V and propidium iodide PI based on the directions of manufacturers Invitrogen events were analyzed by a flow cytometer and the apoptotic cells were determined by calculating the sum of early Annexin VPI and late Annexin VPI apoptotic cells All experiments were done in triplicateTranswell migration and a0invasion assaysCell migration and invasion were detected by the transwell assay using modified Boyden chambers in 24transwell plates a0 μm pores Corning Amsterdam The Netherlands Chambers of cell invasion assays consisted of Matrigelprecoated membrane inserts Corning After transfection BC cell lines were seeded into the top chamber of a 24transwell insert and medium containing FCS was used as a chemoattractant in the lower chamber a0h later the well was stained with crystal violet Solarbio and the number of cells that had migrated or invaded to the basal side of the membrane was counted under a microscope Nikon Shinagawa Tokyo Japan at à magnification Each experiment was performed in triplicateMeasurement of a0glucose uptake lactate product and a0adenosine triphosphate ATP levelThe Colorimetric Glucose Uptake Assay Kit LLactate Assay Kit and ATP Assay Kit all from Abcam were used to determine the levels of glucose uptake lactate product and ATP according to the recommendations of manufacturers Briefly the lysates of transfected cells were prepared using the Assay buffer and incubated with standard protocols for the indicated time point followed by the measurement of the absorbance with a microplate reader Invitrogen at OD a0nm for glucose uptake a0nm for lactate product and a0nm for ATP level All assays were done in triplicate 0cChen a0et a0al Cancer Cell Int Page of Animal studiesThe xenograft models were constructed to assess the role of circZFR on tumor growth in a0vivo Animal studies were implemented in accordance with a protocol approved by the Ethics Committee on Animal Use and Care of the First Affiliated Hospital of Zhengzhou University Twelve a0weeks BALBc female mice Shanghai Animal Laboratory Center Shanghai China were used and randomly divided into two groups n per group shNC and shcircZFR Approximately à shNCinfected or shcircZFRtransduced BT549 cell line was subcutaneously injected into the dorsal flank of the mice One week later tumor size was measured every week with callipers and tumor volume was calculated using the following formula volume à length à width2 All mice were sacrificed at a0days after implantation and tumor tissues were collected for weightBioinformatics dualluciferase reporter and a0RNA pulldown assaysAnalysis for the targeted miRNAs of circZFR was performed using the online web CircInteractome https circi ntera ctome nianihgovindex html and CircBANK httpwwwcircb ankcnsearc hCirc html The putative targets of miR578 were predicted by TargetScan v7 online software httpwwwtarge tscan vert_71Targeted relationships among circZFR miR578 and HIF1A were confirmed by dualluciferase reporter and RNA pulldown assays In dualluciferase assays the fragment of circZFR containing the miR578binding sites and HIF1A ²UTR were individually cloned into pmirGLO vector Promega Madison WI USA to construct corresponding wildtype reporters circZFRWT and HIF1A3²UTRWT The TaKaRa MutanBest Kit was used to construct the corresponding mutations circZFRMUT and HIF1A3²UTRMUT as per the insturctions of manufacturers TaKaRa Dalian China Each reporter construct a0ng and a0nM of miR578 mimic or miRNC mimic were cotransfected into BC cell lines Cell line extracts were prepared with RIPA buffer TaKaRa a0h posttransfection and the ratio of Renilla to firefly luciferase was detected using the Promega Dualluciferase Assay In RNA pulldown assays cell lysates were incubated with the biotinlabeled miR578 mimic BiomiR578 Ribobio or nontarget control sequence BioNC Ribobio for a0 h at a0 °C before adding to the streptavidin beads SigmaAldrich for a0 h The beads were collected and total RNA was extracted for circZFR quantification Each experiment was performed in triplicateWestern blot for a0HIF1A expressionWestern blot was used to determine the expression of HIF1A using standard protocols The preparation of cell line extracts was done using RIPA buffer with proteinase inhibitors Roche Charente France Proteins a0 μg were resolved on a SDSpolyacrylamide gel electrophoretically blotted onto nitrocellulose membranes GE Healthcare Little Chalfont UK and probed with antibody against HIF1A ab51608 a0μgmL Abcam or GAPDH MA515738 a0 μgmL Invitrogen Following the incubation with horseradish peroxidasecoupled IgG secondary antibody ab97051 a0 μgmL Abcam the signals were visualized by Cheniluminescence GE Healthcare as recommended by the manufacturers All experiments were done in triplicateStatistical analysisData were shown as the mean ± standard deviation from at least three independent assays Pairwise comparisons were done using a twotailed Students t test MannWhitney U test or analysis of variance ANOVA with SPSS version software SPSS Chicago IL USA For survival analysis the KaplanMeier survival curve and logrank test were used Correlations among circZFR miR578 and HIF1A expression levels in BC tissues were determined by the Spearman correlation test All tests were considered statistically significant at pvalue ResultsOverexpression of a0circZFR predicted poor prognosis of a0BC patientsAs demonstrated by qRTPCR circZFR was significantly upregulated in BC tissues and cell lines compared with their counterparts Fig a01a b To determine its clinical relevance we preliminarily examined the link between circZFR level and the prognosis of BC patients KaplanMeier survival curves showed that the patients in low circZFR level group had a longer survival time than those in high circZFR group Fig a0 1c Additionally circZFR expression was remarkably associated with the distant metastasis and TNM stage of these patients Table a0Silencing of a0circZFR hindered BC cell viability colony formation and a0enhanced apoptosis in a0vitro and a0weakened tumor growth in a0vivoTo study the biological role of circZFR in BC progression the lossoffunction experiments were carried out using shRNAs against circZFR shcircZFR1 shcircZFR2 and shcircZFR3 In contrast to the negative control the transfection of the three shRNAs prominently reduced circZFR expression in both MCF7 and BT549 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR was overexpressed in BC and associated with poor prognosis CircZFR expression by qRTPCR in pairs of tumor tissues and adjacent normal tissues a MCF10A MCF7 BT549 MDAMB231 and MDAMB453 cell lines b c Analysis for the overall survival of BC patients in high n or low n circZFR level group divided by the median of circZFR expression in BC tissues using KaplanMeier survival analysis and logrank test P cell lines Fig a02a b Notably shcircZFR1 also named shcircZFR caused the most significant downregulation in circZFR expression so we used it for further analyses Functional experiments data revealed that the silencing of circZFR led to a striking inhibition in cell viability Fig a02c colony formation Fig a02d e as well as a strong promotion in cell apoptosis Fig a02f gTo determine whether circZFR regulated BC tumor development in a0vivo we performed the xenograft model assays When we infected BT549 cell line with shcircZFR tumor growth was remarkably blocked compared with the shNC control Fig a02h iSilencing of a0circZFR suppressed BC cell migration invasion and a0glycolysisWe also asked whether circZFR regulated BC cell migration invasion and glycolysis in a0 vitro Transwell assays showed that in comparison to the control group cell migration Fig a0 3a and invasion Fig a0 3b were significantly repressed by circZFR knockdown Moreover in both cell lines circZFR silencing resulted in decreased glucose uptake Fig a0 3c lactate product Fig a0 3d and ATP level Fig a0 3e demonstrating the suppression of circZFR knockdown on cell glycolysisCircZFR directly interacted with a0miR by a0binding to a0miRTo further understand the role of circZFR in BC pathogenesis we performed a detailed analysis for its targeted miRNAs The two online algorithms CircInteractome and CircBANK collectively revealed that circZFR harbored a putative complementary sequence for miR578 miR944 and miR5323p Fig a04a The data of qRTPCR showed that the silencing of circZFR led to a striking overexpression in miR578 and miR5323p levels but miR expression was not affected by circZFR knockdown in the two BC cell lines Fig a0 4b c Previous work had reported that circZFR regulated colorectal cancer progression through acting as a sponge of miR5323p [] So we aimed to identify whether miR578 was a molecular mediator of circZFR in BC progression By contrast miR was prominently upregulated in the two BC cell lines transfected with miR578 mimic Fig a04d We then cloned circZFR fragment containing the miR578binding sites into a luciferase plasmid and mutated the miR578binding sites Fig a0 4e The elevated miR578 expression significantly reduced the activity of circZFR wildtype reporter circZFRWT Fig a04f However the mutation of the target sites circZFRMUT completely abrogated the effect of miR578 on reporter gene expression Fig a04f indicating the validity of the target sequence for interaction Additionally RNA pulldown assays revealed that the enrichment level of circZFR was remarkably elevated by BiomiR578 in both cell lines Fig a04gOverexpression of a0miR restrained BC cell viability colony formation migration invasion and a0glycolysis and a0promoted apoptosis in a0vitroIn BC tissues miR578 expression was significantly decreased compared to the normal control Fig a05a and it was inversely correlated with circZFR level Fig a0 5b Moreover miR578 level was lower in BC cell lines than that of control Fig a05c Subsequently we analyzed the biological effect of miR578 on BC progression In contrast to the control group the increased expression of miR578 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR silencing suppressed BC progression in vitro and in vivo a b qRTPCR for circZFR expression in MCF7 and BT549 cell lines transduced with shNC shcircZFR1 shcircZFR2 or shcircZFR3 CCK8 assay for cell viability c colony formation assay for cell colony formation d e flow cytometry for cell apoptosis f g in shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines h i shNCinfected or shcircZFRtransduced BT549 cell line was subcutaneously injected into the nude mice n per group followed by the measurement of tumor volume and weight and the capture of representative pictures P 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR silencing suppressed BC cell migration invasion and glycolysis Transwell assay for cell migration and invasion a b corresponding assay kits for glucose uptake lactate product and ATP level ce in shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines P induced a distinct repression in cell viability Fig a05d and colony formation Fig a0 5e and a strong promotion in cell apoptosis Fig a0 5f as well as a striking reduction in cell migration Fig a05G invasion Fig a05H and glycolysis Fig a05ikSilencing of a0circZFR regulated BC cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitro by a0upregulating miRA crucial question was whether circZFR regulated BC progression by miR578 To address this we reduced miR578 expression in shcircZFRtransduced MCF7 and BT549 cell lines As expected in comparison to the negative control the reduced level of miR578 significantly reversed circZFR knockdowninduced antiviability Fig a0 6a anticolony formation Fig a0 6b proapoptosis Fig a0 6c antimigration Fig a0 6d antiinvasion Fig a0 6e and antiglycolysis Fig a06fhACA AGA A was CircZFR modulated HIF1A expression via a0acting as a0a a0sponge of a0miRUsing the software TargetScan a predicted miR578binding sequence identified within the ²UTR of HIF1A Fig a0 6a When we cloned the ²UTR fragment containing the putative miR578binding sites downstream of a luciferase coding sequence the cotransfection of the luciferase reporter HIF1A3²UTRWT and miR578 mimic into the two BC cell lines produced lower luciferase activity than in cell lines cotransfected with the miRNC control Fig a07b c However the mutation of the target sequence HIF1A²UTRMUT prominently abolished the suppression of miR578 Fig a07b c By contrast HIF1A mRNA and protein levels were significantly reduced by miR578 overexpression in the two BC cell lines Fig a07d e These data together pointed that HIF1A in BC cell lines was directly targeted and inhibited by miR578 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR directly interacted with miR578 by binding to miR578 a Venn diagrams representing the putative miRNAs that bind to circZFR identified by CircInteractome and CircBANK online algorithms b c The levels of miR578 miR944 and miR5323p by qRTPCR in shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines d qRTPCR for miR578 expression in the two BC cell lines transfected with miRNC mimic or miR578 mimic e Schematic of the miR578binding sites within circZFR and the mutation of the seed sequence f Relative luciferase activity in MCF7 and BT549 cell lines cotransfected with circZFRWT or circZFRMUT and miRNC mimic or miR578 mimic g qRTPCR for circZFR level in cell lysates incubated with BioNC or BiomiR578 and streptavidin beads P We then examine whether circZFR influenced HIF1A expression in BC cell lines As expected in comparison to their counterparts HIF1A expression was remarkably downregulated by circZFR silencing at both mRNA and protein levels in the two BC cell lines and this effect was significantly abrogated by antimiR578 introduction Fig a0 7f g Additionally qRTPCR data showed a striking upregulation of HIF1A mRNA level in BC tissues Fig a0 7h Furthermore in BC tissues HIF1A mRNA expression was positively correlated with circZFR expression and negatively correlated with miR578 level Fig a07iHIF1A was a0a a0functional target of a0miR in a0regulating BC cell viability colony formation migration invasion glycolysis and a0apoptosis in a0vitroTo provide further insight into the link between miR and HIF1A in BC progression we elevated HIF1A expression using a recombinant overexpressing plasmid in miR578 mimictransfected BC cell lines As a result HIF1A protein level was prominently increased by the overexpressing plasmid in the two cell lines Fig a0 8a Functional experiments results revealed that the upregulation of HIF1A dramatically abolished miR578 overexpressionmediated antiviability Fig a0 8b proapoptosis 0cChen a0et a0al Cancer Cell Int Page of Fig MiR578 overexpression hindered BC progression in vitro a qRTPCR for miR578 expression in pairs of tumor tissues and adjacent normal tissues b Correlation between miR578 expression and circZFR level in BC tissues using the Spearman test c MiR578 expression by qRTPCR in MCF10A MCF7 BT549 MDAMB231 and MDAMB453 cell lines MCF7 and BT549 cell lines were transfected with miRNC mimic or miR578 mimic followed by the determination of cell viability by CCK8 assay d colony formation using a standard colony formation assay e cell apoptosis by flow cytometry f cell migration g and invasion h by transwell assay glucose uptake lactate product and ATP level using assay kits ik P Fig a08c antimigration Fig a08d antiinvasion Fig a08e and antiglycolysis Fig a08fhDiscussionTo date the emerging links between circRNAs and BC progression have opened up a novel field for cancer diagnosis and treatment [ ] In the meantime understanding the molecular basis underlying the actions of circRNAs has been still challenging In this study we identified the biological role of circZFR in BC progression in a0 vitro and in a0 vivo and investigated the mechanisms governing itHere we firstly demonstrated that circZFR was overexpressed in BC and the elevated expression of circZFR was associated with the poor prognosis of these patients By using lossoffunction in a0vitro and in a0vivo analyses we were first to uncover that the silencing of circZFR performed a suppressive effect in BC progression Previous reports had highlighted the potential oncogenic role of circZFR in several other malignancies such as hepatocellular carcinoma papillary thyroid cancer and nonsmall cell lung cancer [ ] Conversely circZFR was reported as a tumor suppressor in colorectal cancer and gastric cancer [ ] These contradictory findings may attribute to different type tumor or complex tumor microenvironmentUsing the online algorithms we first identified that circZFR sequestered miR578 through sponging miR in BC cell lines Ji et a0al showed that the increased miR578 level weakened osteosarcoma progression via directly interacting with circ_001621 [] Farhana et a0al unraveled that in pancreatic cancer miR578 was 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR knockdown repressed BC malignant progression by miR578 in vitro shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines were transiently transfected with antiNC or antimiR578 a CCK8 assay for cell viability b A standard colony formation assay for cell colony formation c Flow cytometry for cell apoptosis d e Transwell assay for cell migration and invasion fh Glucose uptake lactate product and ATP level using assay kits P associated with the tumor cell apoptosis [] As it has been reported [] our data validated the downregulation of miR578 expression in BC tissues and cells Moreover we first identified that miR578 overexpression restrained BC cell malignant behaviors in a0 vitro More importantly for the first time we substantiated that circZFR knockdown hampered BC progression in a0vitro by miR578Using TargetScan software we identified that miR in BC cell lines directly targeted and inhibited HIF1A HIF1A a transcriptional regulator in response to intratumoral hypoxia [ ] contributes to BC metastasis and malignant progression [] We also uncovered that the upregulation of miR578 hampered BC malignant progression via downregulating HIF1A in a0 vitro Previous studies had reported that several 0cChen a0et a0al Cancer Cell Int Page of Fig CircZFR modulated HIF1A expression via sponging miR578 a Schematic of the putative miR578binding sequence and mutated the target sequence b c Relative luciferase activity in MCF7 and BT549 cell lines cotransfected with HIF1A3²UTRWT or HIF1A3²UTRMUT and miR578 mimic or miRNC mimic HIF1A mRNA and protein levels by qRTPCR and western blot in MCF7 and BT549 cell lines transfected with miR578 mimic or miRNC mimic d e shNCinfected or shcircZFRtransduced MCF7 and BT549 cell lines transfected with antiNC or antimiR578 f g h Relative HIF1A mRNA expression by qRTPCR in pairs of tumor tissues and adjacent normal tissues i Correlation between HIF1A expression with circZFR or miR578 level in BC tissues using Spearman test P other miRNAs such as miR18a and miR497 exerted an antitumor activity in BC through targeting HIF1A [ ] Furthermore our data first illuminated the role of circZFR as a sponge of miR578 to mediate HIF1A expression in BC cell lines The findings by Liang et a0al underscored that circRNA circDENND4C contributed to BC cell proliferation under hypoxia via regulating HIF1A []In conclusion our present study demonstrated that circZFR was overexpressed in BC and the silencing of 0cChen a0et a0al Cancer Cell Int Page of Fig The repression of miR578 upregulation on BC progression in vitro was mediated by HIF1A a HIF1A protein level by western blot in MCF7 and BT549 cell lines transfected with vector or HIF1A MCF7 and BT549 cell lines were transfected with miRNC mimic vector miR578 mimic vector or miR578 mimic HIF1A followed by the determination of cell viability by CCK8 assay b cell apoptosis by flow cytometry c cell migration and invasion by transwell assay d e glucose uptake lactate product and ATP level using assay kits fh vector negative control plasmid HIF1A recombinant HIF1A overexpressing plasmid P circZFR suppressed BC malignant progression via the regulation of the miR578HIF1A axis This is the first report of circZFR in BC pathogenesis providing evidence for the crucial involvement of circZFR in BC progressionSupplementary informationSupplementary information accompanies this paper at https doi101186s1293 Additional file a0 Supplement material The STR authentication of MCF7 A and BT549 B cellsAdditional file a0 Supplement material The detailed quantification of the western blot analysisAbbreviati | Thyroid_Cancer |
Ofï¬cial Case Reports Journal of the Asian Paciï¬c Society of RespirologyRespirology Case ReportsEndobronchial metastases from a primary embryonalcarcinomaChiKang Teng1ChihYen Tu121Division of Pulmonary and Critical Care Medicine Department of Internal Medicine China Medical University Hospital Taichung Taiwan2School of Medicine China Medical University Taichung Taiwan3Division of Pathology China Medical University Hospital Taichung Taiwan WenChien Cheng12 ChiehLung Chen1 TingHan Chen1 YunShan Lin3 AbstractWe report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung Bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus Cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma The patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin BEP chemotherapy regimenKeywordsCryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourCorrespondenceWenChien Cheng Division of Pulmonary and Critical Care Medicine Department of Internal MedicineChina Medical University Hospital No YudeRoad North Dis Taichung City TaiwanEmail wcchengdrgmailcomReceived July Revised July Accepted July Associate Editor James HoRespirology Case Reports e00644 101002rcr2644IntroductionLung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases EBMs from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] Primary lung cancer is the most common cause of endobronchialtumours Extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] Although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma EBMs from embryonal carcinomas are extremely rare [] In this report we present acase of EBM from a primary embryonal carcinomaCase ReportA 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days Upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days He reported no fever chills coldsweats weight loss or decreased appetite A chest radiograph at admission revealed complete collapse of the leftlung Fig 1A Computed tomography CT was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumBronchoscopytumourobstructing theFig 1B Theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung Immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 TTF1negative sallike protein SALL4positive and cluster of differentiation CD30positive These ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomaFig We checked the levels of tumour markers in thepatient including those of alphafetoprotein AFP betahuman chorionic gonadotropin BhCG and lactate dehydrogenase LDH Each tumour marker was found to be The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of RespirologyThis is an access under the terms of the Creative Commons AttributionNonCommercialNoDerivs License which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made Vol Iss e00644Page 0cEBM from embryonal carcinomaCK Teng et alFigure Chestradiograph and bronchoscopic view of the endobronchial metastasesEBM A Complete collapse of the left lungon chest radiograph B Bronchoscopic viewof the endobronchial tumour within the leftmain bronchusFigure Tumour pathology of metastatic embryonal carcinoma A Embryonal carcinoma with a complex glandular growth pattern The characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation B Immunohistochemical staining with antithyroid transcription factor1 TTF1 highlighting cells in the alveolar space original magniï¬cation C Immunohistochemical staining with antisallikeprotein SALL4 revealed diffuse nuclear staining original magniï¬cation D Immunohistochemical staining with anticluster of differentiation CD30 highlighting diffuse membranous staining original magniï¬cation presentin high levels AFP ngmL BhCG mIUmL and LDH IUL The patientunderwent a right orchiectomy followed by a BEPbleomycin etoposide cisplatin chemotherapy regimenDiscussionWe report here the case of a young man with an EBMfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumoursLikewise manykindsLung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverEBMs from extrapulmonary malignancies are rare [] Primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith EBM primarily breast colon and renal carcinomas[] EBMs from testicular seminomas are also extremelyrare The majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0cCK Teng et alEBM from embryonal carcinomaTable Reports of previous cases of EBMsLocationDiagnostic methodPathologyzt¼rk []MoreiraMeyer []Case Case The oriï¬ce of right upper lobeRight main bronchusLeft main bronchus main carina andright main bronchus Fibreoptic bronchoscopy Mixed GCTFibreoptic bronchoscopy Mixed GCTVideobronchoscopyEmbryonal carcinomazsu []The oriï¬ce of the right upper lobeFibreoptic bronchoscopyTesticular seminomaTuran []Varkey []Our caseand right intermediary lobeRight intermediate bronchusLeft main bronchusLeft main bronchusEBM endobronchial metastases GCT germ cell tumourembryonic carcinomas or seminomas There are only a few published reports of primary testicularembryonic carcinomas resulting in EBMs []The mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently Howeversome patients are asymptomatic [] In our patient symptoms on presentation included haemoptysis and shortnessof breathResults from chest radiography in patients with EBMcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation [] Our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyHowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone Toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue The ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy The former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure [] The patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomaWe had evaluated the presence of AFP BhCG andLDH tumour markers Elevated AFP levels can be secretedby germ cell tumours GCTs including embryonal carcinoma yolk sac tumour or teratoma In GCTs detectableRigid bronchoscopyBronchoscopyFibreoptic bronchoscopyand cryosurgerySomatictype GCTEmbryonal carcinomaEmbryonal carcinomaBhCG elevation is observed in both seminomas and nonseminomas The serum level of LDH was directly correlated with tumour burden in nonseminomatous GCTswhich is also useful for the surveillance of patients withadvanced seminoma [] The tumour markers in ourpatient showed elevated levels of AFP BhCG and LDHThis was compatible with the diagnosis of embryonal carcinoma MoreiraMeyer also evaluated the patienttumour markers and found elevated levels of AFP ngmL and BhCG mIUmL The elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma [] zsu onlyevaluated the patients BhCG level which was found to beelevated mIUmL and the ï¬nal diagnosis wasmetastatic testicular seminoma []On comparison with previous case reports Table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery Other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation APC to control bleedingCryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining Cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree samplesHaemorrhage was observed to be similar during both procedures [] Further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesIn conclusion EBMs from primary GCTs notably thoseassociated with total or partial collapse are extremely rareWe have presented this case to emphasize the importanceof distinguishing EBM from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0cEBM from embryonal carcinomaDisclosure StatementAppropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisReferences zt¼rk A Aktas¸ Z and Yılmaz A Endobronchialmetastasis of mixed germ cell tumors two cases TuberkToraks Lee SH Jung JY Kim DH Endobronchialmetastases from extrathoracic malignancy Yonsei Med J Ikemura K Lin DM Martyn CP Endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing Lung MoreiraMeyer A BautistaHerrera D Hern¡ndezembryonal EndobronchialGonz¡lez MCK Teng et alcarcinomaJ BronchologyInterv Pulmonol zsu S Erol MM Oztuna F Endobronchial metastasis from testicular seminoma Med Princ Pract tumoraltesticular germ cell Turan D Akif zg¼l M Kirkil GEndobronchial metastasis ofEurasian J Pulmonol et Varkey B and Heckman MG Diagnosis of a case ofembryonal carcinoma by bronchial biopsy Chest Paradis TJ Dixon J and Tieu BH The role of bronchoscopy in the diagnosis of airway disease J Thorac Dis Aktas Z Gunay E Hoca NT Endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisAnn Thorac Med Barlow LJ Badalato GM and McKiernan JM Serumtumor markers in the evaluation of male germ cell tumorsNat Rev Urol The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0c' | Thyroid_Cancer |
incidence and death rate of nonsmall cell lung cancer NSCLC in China ranks the first among the malignant tumors Circular RNA circRNA was reported to be involved in the progression of NSCLC Our study aimed to investigate the underlying mechanism of circ_0020123 in NSCLC progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was used to detect the expression of circ_0020123 miR5905p and Thrombospondin THBS2 in NSCLC tissues and cells Cell proliferation and migration were examined by Cell Counting Kit8 CCK8 assay and Transwell assay respectively Flow cytometry assay was used to detect the apoptosis of NSCLC cells The protein levels of Ki67 matrix metalloprotein9 MMP9 Cleavedcaspase9 Cleavedcasp9 and THBS2 were detected by Western blot The targets of circ_0020123 and miR5905p were predicted by starBase and TargetScan and then confirmed by dualluciferase reporter assay and RNA immunoprecipitation RIP assay The animal experiment showed the effect of circ_0020123 on tumor growth in vivoResults The expression of circ_0020123 was upregulated in NSCLC tissues and cells Functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells Interestingly circ_0020123 directly targeted miR5905p and inhibition of miR5905p reversed the knockdown effects of circ_0020123 on NSCLC cells More importantly THBS2 was a target of miR5905p and THBS2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in NSCLC cells Finally suppression of circ_0020123 inhibited tumor growth in vivo through miR5905pTHBS2 axisConclusion Circular RNA circ_0020123 regulated THBS2 by sponging miR5905p to promote cell proliferation and migration and inhibit cell apoptosis in NSCLC cellsKeywords NSCLC Circ_0020123 miR5905p THBS2Highlights Circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in NSCLC cellsCorrespondence bskrju163comDepartment of Thoracic Surgery Lianyungang Second Peoples Hospital No Hailian East Road Haizhou District Lianyungang Jiangsu China Circ_0020123 directly targeted miR5905p and miR5905p downregulation reversed the knockdown effects of circ_0020123 on NSCLC progression THBS2 acted as a target of miR5905p and overthe effects of expression of THBS2 reversed circ_0020123 knockdown on NSCLC progression Downregulation of circ_0020123 suppressed tumor growth in vivo through miR5905pTHBS2 axis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of BackgroundLung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide [] Lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis Small cell lung cancer SCLC accounts for about of all lung cancer cases [] However nonsmall cell lung cancer NSCLC accounts for of lung cancer and the a0years overall survival rate OS is only about [] Therefore it is important to find the effective treatment and potential molecular targets of NSCLC progressionCircular RNA circRNA is a single stranded RNA molecule with a closed circular structure Recently amounts of circular DNA have been discovered and most of which were thought to be the byproducts of typical splicing [ ] Previous reports indicated that the expression of circRNA was tissuespecific and the change of its expression intensity was associated with some diseases [] Furthermore circRNA was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] For example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging miR34a5p to regulate CC Chemokine ligand CCL22 [] Meanwhile hsa_circ_0043256 participated in the progression of NSCLC cells by mediating the cinnamaldehyde treatment [] A previous report suggested that circ_0020123 acted as an oncogene in NSCLC and circ_0020123 regulated zincfingerenhancer binding protein ZEB1 and enhancer of zeste homolog EZH2 by competitively binding with miR144 to induce cell progression and migration [] These reports suggested that circ_0020123 was a vital factor in the pathogenesis of NSCLC and its function and molecular mechanism need to be further studiedAs a small endogenous RNA microRNA miRNA is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers [] Recently some aggregated miRNAs have been found in prostate cancer such as miR221222 miR143145 miR23b27b241 and miR1133a which were downregulated and had tumor inhibiting functions [] A previous study found that circulating miR5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy Besides overexpression of miR5905p reduced the development of NSCLC cells and regulated the expression of epithelialmesenchymal transformation EMTrelated proteins by targeting the signal transducers and activators of transcription STAT3 [] However the precise mechanism by which miR5905p affects NSCLC needs further investigationThrombospondin THBS2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer [] colorectal cancer [] and NSCLC [] A previous report suggested that THBS2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by miR20a [] Tian et a0al found the expression and clinicopathological features of THBS2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis [] However the molecular function of THBS2 in NSCLC remains poorly definedIn this study the targeting relationship between circ_0020123 and miR5905p was firstly detected The effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesMaterials and a0methodsPatients and a0specimensNSCLC tissues and the adjacent healthy lung tissues were taken from NSCLC patients in the Lianyungang Second Peoples Hospital All volunteers signed written informed consents NSCLC tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at a0 °C for further experiments This research was approved by the Ethics Committee of Lianyungang Second Peoples HospitalCell culture and a0cell transfectionTwo NSCLC cell lines A549 and H1299 and one normal lung cell line IMR90 were obtained from the Beijing Concorde Cell Library Beijing China A549 H1299 and IMR90 cells were cultivated in Dulbeccos modified eagle medium DMEM HyClone Logan UT USA supplementing with fetal bovine serum FBS HyClone and cultured in an incubator at a0 with CO2Small interfering RNA siRNA targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin RNA shRNA targeting circ_0020123 shcirc_0020123 miR5905pinhibitors siRNA negative control siNC shNC and NCinhibitors were all obtained from Biomics Biotech Jiangsu China Full length of THBS2 cDNA Sangon Biotech Shanghai China was subcloned into pcDNA31 plasmid EKBioscience Shanghai China Then cell transfection was performed by Lipofectamine Thermo Fisher Scientific Waltham MA USA 0cWang a0et a0al Cancer Cell Int Page of RNA isolation and a0quantitative realtime polymerase chain reaction qRTPCRThe TRIzol reagent Invitrogen Carlsbad CA USA was used for extracting the total RNAs Next the reversed transcription was carried out by RTPCR kit Invitrogen The qRTPCR was performed using the ABI SYBR Green Master Mix Invitrogen The primers in our study were as follows F5²TTC GGA CGA CCG TCA AAC AT3² and R5²AGG ATC CCT GCA CCA CAA TG3² for circ_0020123 F5²TGA AAG ACG TGA TGG CAC AC3² and R5²CTT CCA TTT TGG for miR5905p F5²AGA AGG GGT TTT TGG3 ² CTG GGG CTC ATT TG3² R5²AGG GGC CAT CCA CAG TCT TC3² for glyceraldehyde3phosphate dehydrogenase GAPDH [] F5²GCG GCT GGG TCT ATT TGT C3² and R5²GCA GGA GGT GAA GAA CCA TC3² for THBS2 [] F5²ATT GGA ACG ATA CAG AGA AGATT3² and R5²GGA ACG CTT CAC GAA TTT G3² for U6 [] GAPDH and U6 were the internal parametersCell Counting Kit CCK assayThe proliferation viability of A549 and H1299 cells were detected by the CellCounting Kit8 MSK Wuhan China A549 and H1299 cells were cultivated into a 96well plate with a density of à a0cellswell and incubated in a0°C for or a0h Then a0μL fresh medium and CCK8 solution was added After incubation at a0°C for a0h the OD values were detected by the Multiskan Ascent microplate reader Abcam Cambridge MA USATranswell assayTranswell chamber Corning Life Sciences Corning NY USA was used to detect cell migration Firstly the serumfree DMEM Thermo Fisher Scientific was fixed with cell suspension cells and seeded into the upper chamber and the DMEM containing serum was put into the lower chamber After incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed Finally the migrated cells were stained with crystal violet Corning Life Sciences and observed by using a microscopeFlow cytometryFirstly A549 and H1299 cells were cultured and PBS was used for washing cells Then the binding buffer was used to resuspend cells and the Annexin Vfluorescein isothiocyanate VFITCpropidium iodide PI Apoptosis Detection Kit Thermo Fisher Scientific was used to stain cells Finally cell apoptosis was detected by flow cytometry Thermo Fisher ScientificWestern blot analysisThe total proteins of NSCLC tumors or cells were collected by RIPA lysis buffer Sangon Biotech Then the proteins were separated by Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDSPAGE and transferred to polyvinylidene fluoride PVDF membranes Thermo Fisher Scientific The skimmed milk was added and incubated with primary antiGAPDH antibody Invitrogen Carlsbad CA USA antiβactin antibody Invitrogen antiKi67 antibody Invitrogen antimatrix metalloprotein9 MMP9 antibody Invitrogen antiCleavedcaspase9 Cleavedcasp9 antibody Invitrogen or antiTHBS2 antibody Invitrogen at a0°C overnight Finally the membranes were incubated with the secondary antibody for a0 h at room temperature The results were viewed using Kodak film developer Fujifilm JapanDualluciferase reporter assaysThe wild type circ_0020123 sequences circ_0020123WT mutant circ_0020123 sequences circ_0020123MUT wild type THBS2 ²UTR sequences THBS2WT mutant THBS2 ²UTR sequences THBS2MUT were cloned into pGL3 luciferase reporter plasmid Promega Madison WI USA Then the plasmid and miR5905p or miRNC were cotransfected into A549 and H1299 cells by Lipofectamine Thermo Fisher Scientific After transfection for a0h the DualLuciferase Reporter Assay System Promega was performed to detect the luciferase activityRNA immunoprecipitation RIPFirstly the Magna RIP RNAbinding Protein Immunoprecipitation Kit gzscbio Guangzhou China was performed to verify the relationship between circ_0020123 and miR5905p In brief the magnetic beads and antiAgo2 antibody Abcam were added into cells and incubated for a0h Then the proteinase K and the phenolchloroformisoamyl alcohol reagent were added for purifying RNAs Finally qRTPCR was used to measure circ_0020123 enrichmentAnimal experimentsThe 4weekold BALBc male nude mice Vitalriver Beijing China were raised in a sterile environment for 0cWang a0et a0al Cancer Cell Int Page of experiments Then PBS was used to suspend A549 cells à transfected with shcirc_0020123 or shNC Next the nude mice were divided into two groups n A549 cells transfected with shcirc_0020123 or shNC were shcirc_0020123 or shNC inoculated into the nude mice The tumor volume was detected every a0 days After a0days the nude mice were euthanatized and the tumor weight was detected Besides the tumor tissues from each group were collected to detect the expression of circ_0020123 miR5905p and THBS2 The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second Peoples HospitalStatistical analysisThe software GraphPad Prism was performed for statistical analysis The data was displayed as mean ± standard deviation SD The significant difference was calculated by Students t test and oneway analysis of variance ANOVA P was considered as statistically significantResultsCirc_0020123 was a0upregulated in a0NSCLC tissues and a0cellsTo begin with qRTPCR was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in NSCLC tissues compared with the adjacent healthy tissues Fig a0 1a Similarly the expression of circ_0020123 in NSCLC cells A549 and H1299 was markedly higher than that in normal cells IMR90 Fig a01b From these data it is speculated that circ_0020123 might be acted as an oncogene in NSCLCFig Circ_0020123 was upregulated in NSCLC tissues and cells a QRTPCR was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b The expression of circ_0020123 in normal cell line IMR90 and NSCLC cell lines A549 and H1299 was detected by qRTPCR P Downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0NSCLC cellsTo investigate the functional effects of circ_0020123 on NSCLC cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into A549 and H1299 cells Firstly the transfection efficiency was detected by qRTPCR Fig a02a Next CCK8 was used to detect the proliferation and the results showed that the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced Fig a0 2b Moreover the migration of A549 and H1299 cells was significantly downregulated by circ_0020123 knockdown Fig a02c In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with siNC Fig a02d Finally the protein levels of cell proliferationrelated protein Ki67 and cell migrationrelated protein MMP9 were inhibited while cell apoptosisrelated protein Cleavedcasp9 was upregulated in NSCLC cells transfected with sicirc_00201231 or sicirc_00201232 Fig a02e These data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in NSCLC cellsCirc_0020123 directly targeted miR5pBy searching in the online software starBase the potential binding sites between circ_0020123 and miR5905p were detected Fig a0 3a To confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123WT reporter plasmid was reduced by miR5905p mimic while the circ_0020123MUT reporter plasmid activity was not changed in A549 and H1299 cells Fig a03b Furthermore the expression of miR5905p was lower in A549 and H1299 cells compared with that in IMR90 cells Fig a0 3c In contrast miR5905p expression was elevated in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a0 3d Finally the RIP assay was also used to confirm the targeting relationship between circ_0020123 and miR5905p and the results showed that circ_0020123 and miR5905p were enriched in antiAgo2 group Fig a03eMiR5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0NSCLC cellsTo further explore the functional effects between circ_0020123 and miR5905p miR5905pinhibitor was established QRTPCR was used to detect the transfection efficiency Fig a0 4a Interestingly miR5905p was upregulated in A549 and H1299 cells transfected with sicirc_00201231 while the expression of miR5905p was recovered in cells transfected with 0cWang a0et a0al Cancer Cell Int Page of Fig Downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of NSCLC cells a The transfection efficiency of sicirc_00201231 and sicirc_00201232 in A549 and H1299 cells was detected by qRTPCR b CCK8 assay was used to detect the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 c The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by Transwell assay d Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 e The protein levels of cell proliferation related protein Ki67 cell migration related protein MMP9 and cell apoptosis related protein Cleavedcasp9 were detected by Western blot P Fig a0sicirc_00201231 miR5905pinhibitors 4b Moreover circ_00201231 knockdown inhibited cell proliferation and migration while the miR5905p inhibitor reversed these effects Fig a0 4c d In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 was increased which was abolished by miR5905pinhibitor Fig a0 4e Similarly miR5905p inhibitors reversed the effects on the protein levels of Ki67 MMP9 and Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 Fig a0 4f These results that miR5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of NSCLC cellsindicated MiR5p targeted THBS2 in a0NSCLC cellsThe THBS2 ²UTR was predicted to contain the binding sites of miR5905p through the online software TargetScan Fig a05a Then the dualluciferase reporter assay was used to confirm the targeting relationship The results showed that cotransfection of miR5905p and THBS2WT significantly limited the luciferase activity in both A549 and H1299 cells the luciferase activity was not altered in cells cotransfected with miR5905p and THBS2MUT Fig a05b Importantly the mRNA and protein level of THBS2 was enahnced in NSCLC cells Fig a05c d We further explored whether circ_0020123 affected the functions of THBS2 in NSCLC cells The mRNA and protein expression of THBS2 were repressed in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a05e f 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0020123 directly targeted miR5905p a The binding site between circ_0020123 and miR5905p was detected by the online software starBase b The luciferase activity of circ_0020123WT or circ_0020123MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p was detected by dualluciferase reporter assay c QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells d The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR e RIP assay was used to confirm the relationship between circ_0020123 and miR5905p P THBS2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0NSCLC cellsBased on the work ahead of us the pcDNA31THBS2 was constructed Then the qRTPCR and Western blot were used to detect the transfection efficiency and the THBS2 expression was increased in A549 and H1299 cells transfected with pcDNA31THBS2 Fig a0 6a b In addition the proliferation and migration rates of A549 and H1299 cells transfected with sicirc_00201231 pcDNA31THBS2 were higher than that transfected with sicirc_00201231 Fig a0 6c d Meanwhile a similarly phenomenon was also observed in cell apoptosis the pcDNA31THBS2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis Fig a0 6e Furthermore the effects of circ_0020123 deletion on Ki67 MMP9 and Cleavedcasp9 protein levels were also reversed by THBS2 overexpression Fig a0 6f These data suggested that overexpression of THBS2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisReduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123miR5pTHBS2 axisTo further explore the function of circ_0020123 in NSCLC cells the shcirc_0020123 was constructed and the xenograft tumor was established Then A549 cells transfected with shcirc_0020123 or shNC were injected into the nude mice The xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shNC group Fig a07a Moreover tumor weight was inhibited by circ_0020123 knockdown Fig a0 7b Furthermore the expression circ_0020123 and THBS2 was decreased while the miR5905p was increased in xenograft tumor transfected with shcirc_0020123 Fig a0 7c Western blot assay also revealed that the protein level of THBS2 was repressed by circ_0020123 knockdown Fig a07d Finally the digital tomosynthesis DTS was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group Fig a07e The results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivoDiscussionClinically only a few NSCLC patients were diagnosed at an early stage and treated by surgical resection More than of NSCLC patients were diagnosed with the advanced stage or metastatic tumors [] Thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of NSCLCRecently circRNA was no longer considered as a random product in the RNA shearing process and its biological significance and function in malignant tumors 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p downregulation reversed circ_0020123 knockdown effects in NSCLC cells a QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells transfected with miR5905pinhibitors b The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was detected by qRTPCR c The proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was tested by CCK8 assay d Transwell assay was used to measure the migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors e Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors were detected by Western blot P had received more and more attention Previous reports revealed that circ_0020123 was involved in the development of NSCLC [] Moreover the level of circ_0020123 was elevated in NSCLC cells [] Consistently we found that the expression of circ_0020123 was markedly higher in NSCLC tissues and cells Moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of NSCLC cells in a0 vitro Besides circ_0020123 promoted tumor growth in a0vivoEndogenous circRNAs could act as microRNA sponges to inhibit their function and some studies linked miRNA sponges to human diseases including cancer [] A previous study indicated that circRNA ctransferrin receptor cTFRC regulated TFRC by sponging miR107 to facilitate bladder carcinoma development [] MiR5905p was studied in different cells such as airway smooth muscle cells [] colon epithelial cells [] and NSCLC cells [] However the potential relationship between miR5905p and circRNA has not been researched In this study circ_0020123 directly targeted miR5905p and miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC progression These data provided a clue to the therapeutic strategy for NSCLC 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p targeted THBS2 in NSCLC cells a The potential binding site between THBS2 ²UTR and miR5905p was predicted by the online software TargetScan b Dualluciferase reporter assay was used to measure the luciferase activity of THBS2WT or THBS2MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p c QRTPCR was used to detect the mRNA expression of THBS2 in NSCLC cells d The protein level of THBS2 in NSCLC cells was tested by Western blot e The mRNA expression of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR f Western blot was used to measure the protein level of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 P Our study also confirmed that miR5905p could target THBS2 directly in NSCLC cells THBS2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase MMP genes involved in tissue formation and repair [ ] A previous document suggested that THBS2 acted as a target of miR2213p and participated in lymph node metastasis in cervical cancer [] The data in this research showed that the expression of THBS2 in NSCLC cells was markedly higher than normal healthy cells Furthermore overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells suggesting that circ_0020123 promoted the progression of NSCLC cells through miR5905pTHBS2 axisConclusionIn conclusion our research showed that the expression of circ_0020123 was higher in NSCLC tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of NSCLC cells and also suppressed tumor growth in a0 vivo Moreover circ_0020123 directly targeted miR5905p while miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC cells More importantly circ_0020123 regulated the expression of THBS2 by sponging miR5905p and upregulation of THBS2 reversed the effects of circ_0020123 knockdown on NSCLC cells Therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cWang a0et a0al Cancer Cell Int Page of Fig Overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells a b The mRNA and protein expression of THBS2 in A549 and H1299 cells transfected with pcDNA31THBS2 was detected by qRTPCR and Western blot c CCK8 assay indicated the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 d The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was measured by Transwell assay e The apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was detected by Flow cytolysis assay f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 were detected by Western blot P apoptosis of NSCLC cells by sponging miR5905p to regulate THBS2results and develop the manuscript All authors read and approved the final manuscriptAbbreviationsNSCLC Nonsmall cell lung cancer circRNA Circular RNA qRTPCR Quantitative realtime polymerase chain reaction CCK8 Cell Counting Kit8 MMP9 Matrix metalloprotein9 Cleavedcasp9 Cleavedcaspase9 Cleavedcasp9 Cleavedcaspase9 RIP RNA immunoprecipitation ZEB1 Zincfingerenhancer binding protein EZH2 Zeste homolog STAT3 Signal transducers and activators of transcription THBS2 Thrombospondin AcknowledgementsNot applicableAuthors contributionsLW collaborated to design the study LZ were responsible for experiments analyzed the data YW wrote the paper All authors collaborated to interpret FundingNoneAvailability of data and materialsPlease contact corresponding author for data requestsEthics approval and consent to participateThis research was approved by the Ethics Committee of Lianyungang Second Peoples Hospital The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second Peoples HospitalConsent for publicationAll listed authors have actively participated in the study and have read and approved the submitted manuscript 0cWang a0et a0al Cancer Cell Int Page of Fig Reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123miR5905pTHBS2 axis a A total of à A549 cells transfected with shcirc_0020123 or shNC were injected into nude mice to establish the xenograft tumor Tumor volume was measured every d after injection b Tumor weight was measured on d c The expression of circ_0020123 miR5905p and THBS2 in xenograft tumor was measured by qRTPCR d The protein level of THBS2 in xenograft tumor was evaluated by Western blot e The number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis DTS P Competing interestsThe authors declare that they have no competing interestsReceived April Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin Abe H Takase Y Sadashima E Fukumitsu C Murata K Ito T Kawahara A Naito Y Akiba J Insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value Cancer Cytopathol Li C Zhang L Meng G Wang Q Lv X Zhang J Li J Circular RNAs pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer J Cancer Res Clin Oncol Belousova EA Filipenko ML Kushlinskii NE Circular RNA new regulatory molecules Bull Exp Biol Med Zhang Z Xie Q He D Ling Y Li Y Li J Zhang H Circular RNA new star new hope in cancer BMC Cancer Li L Chen Y Nie L Ding X Zhang X Zhao W Xu X Kyei B Dai D Zhan S Guo J Zhong T Wang L Zhang H MyoDinduced circular RNA CDR1as promotes myogenic differentiation of skeletal muscle satellite cells Biochim Biophys Acta Gene Regul Mech Greco S Cardinali B Falcone G Martelli F Circular RNAs in muscle function and disease Int J Mol Sc | Thyroid_Cancer |
peripheral serum metabolomic profiles inform central cognitive impairmentJingye Wang1 Runmin Wei12 Guoxiang Xie1 Matthias Arnold Alexandra KueiderPaisley Gregory Louie Siamak Mahmoudian Dehkordi3 colette Blach5 Rebecca Baillie Xianlin Han7 Philip L De Jager David A Bennett9 Rima KaddurahDaouk Wei Jia The incidence of Alzheimers disease AD increases with age and is becoming a significant cause of worldwide morbidity and mortality However the metabolic perturbation behind the onset of AD remains unclear In this study we performed metabolite profiling in both brain n and matching serum samples n to identify differentially expressed metabolites and metabolic pathways associated with neuropathology and cognitive performance and to identify individuals at high risk of developing cognitive impairment The abundances of metabolites glycolithocholate GLCA petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and the deoxycholatecholate DCACA ratio along with the dysregulation scores of metabolic pathways primary bile acid biosynthesis fatty acid biosynthesis and biosynthesis of unsaturated fatty acids showed significant differences across both brain and serum diagnostic groups Pvalue Significant associations were observed between the levels of differential metabolitespathways and cognitive performance neurofibrillary tangles and neuritic plaque burden Metabolites abundances and personalized metabolic pathways scores were used to derive machine learning models respectively that could be used to differentiate cognitively impaired persons from those without cognitive impairment median area under the receiver operating characteristic curve AUC for the metabolite level model median AUC for the pathway level model Utilizing these two models on the entire baseline control group we identified those who experienced cognitive decline in the later years AUC sensitivity specificity for the metabolite level model AUC sensitivity specificity for the pathway level model and demonstrated their preAD onset prediction potentials Our study provides a proofofconcept that it is possible to discriminate antecedent cognitive impairment in older adults before the onset of overt clinical symptoms using metabolomics Our findings if validated in future studies could enable the earlier detection and intervention of cognitive impairment that may halt its progressionAlzheimers disease AD one of the top leading causes of death in the United States is an increasing challenge for health care systems and will result in increased economic burden as increasing numbers of new cases are diagnosed annually12 Currently there is no therapy to prevent or slow AD progression which may be due to the inability to detect AD before its progression into evident cognitive decline Identification of early 1University of Hawaii Cancer Center Ilalo Street Honolulu HI USA 2Department of Molecular Biosciences and Bioengineering University of Hawaii at Manoa Honolulu HI USA 3Department of Psychiatry and Behavioral Sciences Duke University Durham NC USA 4Institute of Computational Biology Helmholtz Zentrum M¼nchen German Research Center for Environmental Health Neuherberg Germany 5Duke Molecular Physiology Institute Duke University Durham NC USA 6Rosa Co LLC San Carlos CA USA 7University of Texas Health Science Center at San Antonio San Antonio TX USA 8Center for Translational Computational Neuroimmunology Columbia University College of Physicians and Surgeons Department of Neurology New York NY USA 9Rush Alzheimers Disease Center Rush University Medical Center Chicago IL USA 10Institute of Brain Sciences Duke University Durham NC USA 11Department of Medicine Duke University Durham NC USA email kaddu001mcdukeedu wjiacchawaiieduScientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cbiomarkers associated with preclinical symptoms would allow early intervention or preventive strategies to be developed3 Research has identified multiple neurochemical perturbations in AD including amyloid precursor protein metabolism phosphorylation of tau protein and a wide range of metabolic perturbations4 Unfortunately current biomarkers for early disease including cerebrospinal fluid CSF betaamyloid and tau levels5 structural and functional magnetic resonance imaging6 the recent use of brain amyloid imaging7 or inflammaging8 and CSF markers to track brain atrophy and deposition of cortical betaamyloid and neurofibrillary tangles are limited because they are either invasive timeconsuming or expensiveRecent studies have focused on obtaining biomarkers to identify features that differentiate persons a0with cognitive impairment from persons without cognitive impairment Molecular markers sensitive to the underlying pathogenic factors would be highly relevant to early disease detection and facilitation of disease monitoring and treatment responses Metabolomics is an unbiased approach to study smallmolecule metabolites that offers hope for the discovery of more biomarkers for AD This profiling technology has already been used to identify differential metabolites that can distinguish mild cognitive impairment MCI subjects who will develop AD from stable MCI9 Mounting evidence suggests that AD is closely accompanied with the abnormal bile acid BA metabolism10 free fatty acid FFA metabolism14 lipid metabolism1718 and neurotransmitter metabolism19 BAs have become increasingly recognized as important metabolic signaling molecules that modulate lipid glucose and energy metabolism20 More importantly BAs in brain act as neuroactive steroids21 Different classes of BAs can either inhibit or potentiate GABAα a0and inhibit NMDA receptors while also exerting neuroprotective effects Recent crosssectional studies have shown differences in blood BAs in AD compared with noncognitively impaired individuals2425 Additionally researchers found an accumulation of FFAs in the hippocampus and cortex of AD mice compared to control mice2627 Another animal study examined the role of elevated FFA in the pathogenesis of AD and established a potential mechanism of FFA causing hyperphosphorylation of tau through astrogliamediated oxidative stress28 Alterations of FFAs have also been detected in postmortem AD brains tissues14 and serum samples16 which may indicate an alternative fuel source before the onset of clinical symptoms29 These observations have given rise to the possibility that metabolic perturbations could presage the onset of cognitive impairment and therefore aid in the identification of individuals with higher risks by providing additional information to use with standard clinical markersIn this study we performed metabolomic profiling in participants from a large longitudinal cohort with the goal of identifying metabolic changes as well as key metabolic pathways that might serve as new predictors of future cognitive impairment in older adultsMaterials and methodsParticipants The Religious Orders Study ROS which began in is a longitudinal clinicalpathologic cohort study of risk factors of cognitive decline and incident dementia run from the Rush Alzheimers Disease Center that is comprised of individuals from religious communities eg Catholic brothers nuns and priests across the USA3031 The Rush Memory and Aging Project MAP which began in includes participants from northeastern Illinois USA with a broader range of socioeconomic status and life experiences31 Participants in both studies enroll without known dementia agree to annual clinical evaluation and an donation Both studies were approved by an Intuitional Review Board of Rush University Medical Center All subjects signed an informed consent an Anatomic Gift Act and a repository consent to allow their biospecimens and data to be used for ancillary studies All research was performed in accordance with relevant guidelinesregulations set forth by the Rush University Medical Center Both studies are conducted by the same team of examiners and share a large common core of data collection at the item level to allow for efficient merging of dataCognitive performance tests Cognitive performance was measured using a battery of cognitive performance tests of which could be summarized in five cognitive domains ie episodic memory working memory semantic memory perceptual orientationvisuospatial ability and perceptual speed Table a0S1 Domains are created by averaging the zscores based on mean and standard deviation from all baseline data for tests in each domain The global cognitive function score is calculated by averaging zscores for all tests to yield a global measure of cognitive function Additionally the MiniMental State Examination was also administered to characterize the cohortClinical diagnoses Medical conditions were documented via selfreport and clinical evaluation Clinical diagnoses each year were determined blinded to previously collected data A threestep process starts with an actuarial decision tree based on the history of cognitive decline and impairment ratings in five cognitive domains based on cutoffs for cognitive tests32 a0followed by clinical judgment by a neuropsychologist for cognitive impairment and determination of dementia and its causes by a clinician ie neurologist geriatrician second neuropsychologist geriatric nurse practitioner33 The diagnosis of AD follows the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimers Disease and Related Disorders Association NINCDSADRDA34 Participants were categorized as a AD b MCI if diagnosed cognitive impairment by the neuropsychologist but not diagnosed dementia by the clinician32 and c no cognitive impairment NCI if diagnosed without AD or MCI35 At the time of death brain autopsies and histopathological exams were performed by clinicians to confirm the diagnosis After an autopsy was completed a spectrum of neuropathologic diagnoses was obtained such as a pathologic diagnosis of AD as defined using the modified NIA Reagan criteria However many other pathologies were present in the brains of older individuals the mean age of death is a0years old in ROSMAP and they were catalogued for each participantScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cNCI converters were those participants who were cognitively normal at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followupNeuropathology Upon death a postmortem neuropathological evaluation was implemented and the procedures follow those outlined by the pathologic dataset recommended by the National Alzheimers Disease Coordinating Center Brains of deceased subjects were removed weighed cut into one cmthick coronal slabs and stored Each brain was examined for the neuropathological indices of common pathologies that contribute to cognitive impairment The location age and volume of all macroscopic infarcts were recorded and tissue was obtained for histological confirmation in addition to the identification of microscopic infarctions as previously described3637 AD pathology was identified using the modified Bielschowsky silver stain technique and by use of the Consortium to Establish a Registry for Alzheimers Disease CERAD criteria38 and NIAReagan criteria39 while the assessment of neurofibrillary tangles was based on Braak criteria40 as described previously41 The CERAD score a semiquantitative measure of neuritic plaque burden is made of levels no AD possible AD probable AD and definite AD As recommended CERAD scores were reclassified to a binary level score score Seven categories of Braak stages were based on the region and severity of neurofibrillary tangles pathologyMetabolites quantification Using targeted metabolomics protocols42 and profiling protocols43 established in previous studies BAs were quantified by ultraperformance liquid chromatography triple quadrupole mass spectrometry UPLCTQMS Waters XEVO TQS Milford USA and other metabolites were quantified by gas chromatography timeofflight mass spectrometry GCTOFMS Leco Corporation St Joseph USA Details are described in the Supporting InformationStatistical analysis Stratifying by clinical diagnosis continuous demographic variables were expressed as mean [standard deviation SD] and tested by Wilcoxon ranksum test while categorical demographic variables were expressed as n percentage and tested by Chisquare test Missing values in quantitative metabolites due to limits of quantification were regarded as leftcensored missing and imputed by GSimp4445 Individual BA concentrations were normalized to the total BAs concentration ie the proportion of total BAs Metabolites were reported as median quantile quantile and tested by univariate analysis Wilcoxon ranksum test Due to the limited sample size of the AD group participants in serum samples we combined MCI and AD participants into an aggregate group MCIAD for the following data analysis Logtransformed abundances were used in the following data analysis We additionally generated BA ratios based on the BA metabolic pathway topologyTo identify metabolites differentially expressed in participants with cognitive decline we used ordinal logistic regression to compare metabolites across three groups NCI MCI AD for brain samples and logistic regression across two groups NCI MCIAD for serum samples To control the positive false discovery rate Qvalues were calculated based on Pvalues Additionally for serum samples we adjusted for potential confounders eg fasting status supplements diabetic and lipid lowering medications using logistic regressions The relationships between logtransformed brain metabolites levels with neurofibrillary tangle burden and neuritic plaque burden were expressed as boxplots across Braak scores KruskalWallis test and CERAD scores Wilcoxon ranksum test respectively Using Spearmans rank correlation test we further evaluated the associations between the abundances of each identified metabolite and the global cognitive function score in both brain and serum samples Linear regression models with each individual metabolite used as the predictor and each cognitive test as the response variable adjusted for age gender years of education and presence of APOE ε4 were used to test the associations between metabolite and cognitive function Similar analyses with an additional adjustment of BMI were conducted for serum samples The Wilcoxon ranksum test was carried out to explore whether identified variables were differentially expressed between NCI converters vs NCI nonconverters and between NCI converters vs MCIAD in sera Then we built a random forest RF predictive model to differentiate NCI nonconverters vs MCIAD using glycolithocholate GLCA deoxycholatecholate DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid and age as the predictorsTo differentiate MCIAD vs NCI nonconverters we randomly split the data into training set and testing set times Each time we trained an RF model on the training set to differentiate the MCIAD from NCI nonconverters and evaluated it on the testing set using the area under the receiver operating characteristic curve AUROC sensitivity SE and specificity SP A final model was built on the whole NCI nonconverters and MCIAD dataTo investigate the preclinical predictive potentials as well as to validate the classification performance of our model we utilized this model on the entire baseline NCI group to identify those NCI converters from NCI nonconverters The differences of RF scores between NCI nonconverters vs NCI converters and NCI nonconverters vs MCIAD groups were tested by the Wilcoxon ranksum test To determine whether RF scores could independently differentiate NCI converters from NCI nonconverters in the presence of potential confounders we used the logistic regression method with RF scores as the predictor adjusting for gender years of education APOE ε4 and BMI Additionally we fit linear mixed effects models to evaluate correlations between RF scores with global cognitive function and each of the five cognitive domains separately with a random effects term for education and BMI and fixed effects terms for RF score gender and APOE ε4For the personalized pathway level analyses we extracted metabolite information from the Human Metabolome Database HMDB46 and metabolic pathway information from the Kyoto Encyclopedia of Genes and Genomes KEGG database47 to map affiliated metabolites to metabolic pathways We used the pathifier Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cOverallNCIMCIADBrain samplesNAge mean SDMale n Education mean SDAPOE ε4carrier n Serum samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Serum NCI samplesNAge years mean SDMale n Education years mean SDAPOE ε4carrier n Overall Overall NCI MCIAD NCI nonconverters NCI converters Table Detailed demographic characteristics of study samples Chisquare test Pvalue comparing AD vs NCI Wilcoxon rank sum test Pvalue comparing MCIAD vs NCI Wilcoxon rank sum test Pvalue comparing NCI converters vs NCI nonconverters Chisquare test Pvalue comparing NCI converter vs NCI nonconverter NCI cognitively normal MCI mild cognitive impairment AD Alzheimers disease APOE ε4 apolipoprotein E epsilon allele SD standard deviation algorithm48 to transfer metabolic level information of each sample to pathway level by generating a pathway dysregulation score PDS For each pathway each sample was projected onto a directed principal curve49 which was yielded depending on leading principal components of the pathway to optimally pass through the cloud of samples PDS was the distance along the curve between the projection of each sample and that of NCI Thus PDS could capture the pathwaylevel extent of abnormality increments or decrements for each participant relative to those with NCI We performed similar data analysis on pathway level data to what we did on metabolomics level data We tried to identify differential pathways using ordinal logistic regression across NCI MCI and a0AD groups in brain samples and logistic regression for NCI and MCIAD in serum samples Next we explored the associations between identified pathways with neuropathology KruskalWallis test for Braak scores Wilcoxon ranksum test for CERAD scores and cognitive performance Spearmans rank correlation test for the global cognitive function linear regression with adjustments for each cognitive test Then we examined the predictive potential of identified pathways in serum samples using univariate analysis Wilcoxon ranksum test for NCI converters vs NCI nonconverters NCI converters vs MCIAD Finally we built RF models on training sets and tested them on testing sets according to times random splitting on the model construction data and applied the final model on the validation data using ROC SE SP as evaluation methods The overall workflow chart of the data and the analysis are shown in Fig a0S9Data were analyzed using R version with packages including pROC pathifier randomForest ggplot2 ggsignif and MASS The statistically significance was determined by a threshold of unadjusted Pvalues and Qvalues ResultsParticipants and characteristics For the joint analyses of the ROSMAP study we measured metabolomics of serum samples NCI MCI and AD at the blood draw and postmortem brain tissues from dorsolateral prefrontal cortex NCI MCI and AD at the time of death Among brain samples and serum samples a total of participants had both brain and blood metabolomics data NCI participants n were further categorized into NCI converters and NCI nonconverters NCI converters were those participants who were cognitively normal NCI at the time of blood draw and then experienced the cognitive decline MCI or AD at the time of death while NCI nonconverters were participants who remained cognitively normal during followup Among NCI participants the time between the blood draw and conversion ranged from to a0years with a median of a0years Fig a0S10 Detailed demographic characteristics of the serum samples and postmortem brain samples are included in Table a0 Among participants with postmortem brain samples AD patients tended to have at least one APOE ε4 allele compared to the NCI group as expected The mean age of NCI and MCIAD group at the time of blood draw among serum samples was a0years SD and SD respectively Similarly the age and the percentage of APOE ε4 Scientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0cFigure a0 Brain metabolome and serum metabolome composition and alterations a Left panel the brain metabolome composition Right panel a0log10 Pvalue across clinical groups of brain tissues NCI MCI AD b Left panel the serum metabolome composition Right panel a0log10 Pvalue across clinical groups of serum tissues NCI MCIADcarriers were higher in the NCI converters group than the NCI nonconverters group We did not observe other significant demographic characteristics differences across clinical groups Table a0Identifying metabolites differentially expressed in participants with cognitive impairment In this study metabolites and metabolites overlapping metabolites were detected in brain tissues and serum samples respectively Tables a0S7 S8 Amino acids BAs carbohydrates anic acids and fatty acids were the predominant types of annotated metabolites accounting for of all the metabolites in brain tissues and in serum samples Fig a01ab left panel A total of seven a0metabolites BA BA ratio anic acid known as a longchain fatty acid fatty acids showed significant differences across clinical groups in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a01ab right panel Tables a0S14 S15 After adjusting for confounders ie fasting status supplement use diabetic and lipid lowering medications most of serum metabolites remained statistically significant Table a0S18 In brain tissues increments of the levels of GLCA DCACA ratio petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid followed the pattern NCI MCI AD We observed increments of GLCA DCACA ratio and decrements of petroselinic acid linoleic acid myristic acid palmitic acid palmitoleic acid in sera of MCIAD compared to controls Table a0 The trend of increments of identified metabolites in brain samples increments of BAs and decrements of FFAs in serum samples were further validated within individuals with both brain and serum samples From NCI to MCI and AD groups increments of identified metabolites were observed in brain samples Table a0S11 The increasing trend of GLCA DCACA ratio and decreasing trend of FFAs among MCIAD group relative to NCI group were detected in sera Table a0S11The seven brain metabolites were all negatively correlated with global cognitive function where higher scores indicate better cognitive performance Ï a0 for GLCA Ï a0 for DCACA ratio Ï a0 for petroselinic acid Ï a0 for linoleic acid Ï a0 for myristic acid Ï a0 for palmitic acid and Ï a0 for palmitoleic acid using Spearmans rank correlation analysis Fig a02a Similarly after adjusting for age gender years of education and APOE ε4 all identified metabolites remained negatively correlated with tests in five Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cBrain samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]Serum samplesGLCA median [IQR]DCACA median [IQR]Petroselinic acid median [IQR]Linoleic acid median [IQR]Myristic acid median [IQR]Palmitic acid median [IQR]Palmitoleic acid median [IQR]NCIMCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ]NCI [ ] [ ] [ ] [ ] [ ] [ ] [ ]MCIAD [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]Table Levels of metabolites differentially expressed in participants a0by diagnostic group Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing AD vs NCI Pvalue Pvalue Pvalue by Wilcoxon rank sum test comparing MCIAD vs NCI NCI cognitively normal MCI mild cognitive impairment AD Alzheimers disease IQR interquartile rangeFigure a0 Associations between metabolites level and global cognitive function a Boxplots showing group differences and P values for identified metabolites across Braak groups for brain tissue abundances b Boxplots showing group differences and significances for identified metabolites across CERAD groups for brain tissue abundances Ï correlation coefficient of Spearmans rank correlation testScientific RepoRtS 101038s4159802070703wVol1234567890wwwnaturecomscientificreports 0ccognitive domains and the MiniMental State Exam see Table a0S2 for significant correlation pairs The serum concentration of two BAs showed negative correlations with global cognitive function Ï a0 for GLCA Ï a0 for DCACA ratio conversely fatty acids demonstrated positive correlations Ï for petroselinic acid Ï for linoleic acid Ï for myristic acid Ï for palmitic acid and Ï for palmitoleic acid Fig a02b Linear regression revealed similar consistent results in serum samples with adjustment for age gender years of education APOE ε4 and BMI see Table a0S2 Results of associations between identified metabolitesratio and each cognitive performance domains are shown in Table a0S13 Correlations with global cognitive function were further validated in individuals with both brain and serum samples and the directions were consistent with our previous findings among the a0entire cohort Seven identified metabolites were all negatively correlated with global cognitive function in brain samples while two BAs showed negative correlations and five FFAs showed positive correlations in serum samples Fig a0S6 Additionally the serumbrain ratio of identified FFAs were positively correlated with global cognitive function ie lower levels of identified FFAs in serum and higher levels of identified FFAs in brain were associated with worse cognition Fig a0S7Identified metabolites predicted antecedent cognitive impairment before the manifestation of clinical symptoms The concentrations of GLCA and DCACA were significantly lower in the NCI nonconverters group than in the NCI converters group By contrast the abundances of petroselinic acid linoleic acid myristic acid palmitic acid and palmitoleic acid were higher in the NCI nonconverters group than in the NCI converters group Fig a03a There were no significant differences in these metabolites between participants in NCI converters group vs MCIAD group Fig a03a Using the seven metabolites and age we built RF models on the training set according to 100times randomly splitting approach to differentiate MCIAD patients from NCI nonconverters group The median of times AUC on testing set was CIs with SE CIs and SP CIs using Youdens index to maximize the sum of SE and SP Fig a03b RF models showed decent classification performances in differentiating MCIAD group from NCI nonconvertersNext we were interested in studying the models early diagnostic capability for predicting NCI converters before clinical diagnosis The model was thus applied on the entire NCI group at baseline to differentiate NCI converters from NCI nonconverters We achieved an AUC of CIs SE SP at the cutoff value of Fig a03c with significant differences in RF scores between NCI converters vs NCI nonconverters between NCI nonconverters vs MCIAD group using the Wilcoxon ranksum test Pvalue Fig a03d After additional adjustment for gender years of education APOE ε4 and BMI fasting status and medications supplements diabetes lipid lowing the RF scores remained significant as an independent predictor with a coefficient of Pvalue Table a0S3 Additionally the RF scores showed significant negative correlations with global cognitive function and the five cognitive domains with the same adjustment in mixed effects models Table a0S12Personalized metabolic pathwaybased study for the association and prediction of cognitive impairment Considering altered metabolite levels were significantly associated with cognitive impairment and showed early predictive value of clinical symptoms onset we then employed the pathifier algorithm to summarize metabolite information to pathways level for further examinations All PDS scores ranged from to where larger scores represent the higher extent of the abnormality in the corresponding metabolic pathway out of metabolites detected in brain tissues and out of metabolites detected in sera were successfully mapped to the KEGG metabolic pathways This method identified metabolic pathways in brain tissues and metabolic pathways in serum samples overlapping pathways Figs a0S1ab left panel Table a0S9 Table a0S10 three of which ie primary BAs biosynthesis FFAs biosynthesis and biosynthesis of unsaturated FFAs were significantly shifted in both brain and serum samples Pvalue and Qvalue ordinal logistic regression for brain samples logistic regression for serum samples Fig a0S1ab right panel Table a0S16 Table a0S17 We noted increased PDS for all three identified pathways from NCI to MCIAD that suggested dysregulation of these metabolic pathways in MCIAD patients compare to NCI Detailed PDS of these pathways stratified by diagnostic groups are described in Table a0S4 Results also indicated that higher PDS were significantly associated with lower global cognitive function ie worse cognitive performance in both brain Ï a0 for primary BAs biosynthesis pathway Ï a0 for FFAs biosynthesis pathway Ï a0 for biosynthesis of unsaturated FFAs pathway Fig a0S4 and serum samples Ï a0 for primary BAs biosynthesis pathway Ï a0 for FFAs biosynthesis pathway Ï a0 for biosynthesis of unsaturated FFAs pathway Fig a0S5 respectively In Table a0S5 we show the significant negative associations between each cognitive test and PDS of three pathways after adjusting for age gender years of education and APOE ε4 additional adjustment for BMI in serum samples Two fatty acid pathways showed significantly different PDS between the NCI nonconverters group and the NCI converters group Pvalue and Pvalue respectively A gradually increasing trend was noted for the BAs pathway across groups ie NCI nonconverters NCI converters and MCIAD Fig a04aWe then constructed a discriminant RF model in training data and tested on testing data based on three identified metabolic pathways along with age to differentiate MCIAD from NCI nonconverters in model construction data using 100times randomly splitting approach The median AUC on the a0testing set was CIs with SE CIs and SP CIs Fig a04b Applying the RF model to the whole NCI data at baseline could successfully discriminate NCI converters from NCI nonconverters with an AUC of CIs SE SP cutoff value Fig a04c Similarly predictive RF scores were significantly different between NCI converters vs NCI nonconverters and NCI nonconverters vs MCIAD group Pvalue Fig a04d After adjusting for gender Scientific RepoRtS 101038s4159802070703wVol0123456789wwwnaturecomscientificreports 0cFigure a0 The identified panel of metabolites and its predictive performance a Boxplots showing group differences and P values for identified metabolites across NCI nonconverters NCI converters and MCIAD for serum abundances b ROC curves of metabolite models trained on the training data and tested on the testing data according to 100times randomly trainingtesting splitting c The ROC curve of the final metabolite model on the validation data d RF scores of the final metabolite model across NCI nonconverters NCI converters and MCIAD Pvalue Pvalue Pvalue Wilcoxon rank sum test The optimal cutoff was determined by the Youden index AD Alzheimers disease AUC area under | Thyroid_Cancer |
pulmonary disease COPD is due to structural changes and narrowing of small airways and parenchymaldestruction loss of the alveolar attachment as a result of pulmonary emphysema which all lead to airï¬ow limitation Extracorporeal shock waves ESW increase cell proliferation and diï¬erentiation of connective tissue ï¬broblasts To date no studiesare available on ESW treatment of human bronchial ï¬broblasts and epithelial cells from COPD and control subjects We obtainedprimary bronchial ï¬broblasts from bronchial biopsies of patients with mildmoderate COPD and control smokers with normallung function 16HBE cells were also studied Cells were treated with a piezoelectric shock wave generator at low energy mJmm2 pulses After treatment viability was evaluated and cells were recultured and followed up for and h Cellgrowth WST1 test was assessed and proliferation markers were analyzed by qRTPCR in cell lysates and by ELISA tests in cellsupernatants and cell lysates After ESW treatment we observed a significant increase of cell proliferation in all cell types CKitCD117 mRNA was significantly increased in 16HBE cells at h Protein levels were significantly increased for cKit CD117 at h in 16HBE p and at h in COPDï¬broblasts p � for PCNA at h in 16HBE p � for y1 CD90 at and h in CSï¬broblasts p � and p � for TGF1 at h in CSï¬broblasts p � for procollagen1 at h inCOPDï¬broblasts p � and for NFκBp65 at and h in 16HBE p � and p � In the peripheral lung tissueof a representative COPD patient alveolar type II epithelial cells TTF1 coexpressing cKit CD117 and PCNA were occasionally observed ese data show an increase of cell proliferation induced by a low dosage of extracorporeal shock waves in16HBE cells and primary bronchial ï¬broblasts of COPD and control smoking subjects Backgrounde progressive chronic airï¬ow limitation in chronic obstructive pulmonary disease COPD is due to two majorpathological processes i remodeling and narrowing ofsmall airways and ii destruction of the lung parenchymawith loss of the alveolar attachments as a result of pulmonaryemphysema [] Chronic ammation in the lung plays a 0cCanadian Respiratory Journaltherapycentral role in both the small airway remodeling and thepulmonary emphysema [] Lung volume reductionsurgery and lung transplantation while possible in endstageCOPD are restricted to just a few selected patients []httpwwwgoldcopdcom RegenerativeforCOPD includes mesenchymal stromal cell MSC or tissueengineering therapies But while bone marrow MSC oradipose tissue MSC treatments showed promising results inmice with induced emphysema [] clinical trials performedin COPD patients have been discouraging [ ] ere are alarge number of animal studies in which lung regenerationhas been successfully stimulated For instance in a rat modelof elastaseinduced emphysema administration of alltransRA ATRA stimulated alveolar regeneration [] keratinocyte growth factor KGF FGF7 administered afterpneumonectomy augmented alveolarization [] administration of HGF stimulated alveolar regeneration enhancedlung vascularization and improved exercise tolerance andgas exchange [] intratracheal administration of bFGF torats and dogs with elastaseinduced emphysema improvedalveolar dimensions and lung microvessel density [] andVEGF administration enhanced postpneumonectomy alveolar growth in mice [] But again the attempts tostimulate lung regeneration in COPD patients with emphysema with orally administered ATRA yielded no differences in computed tomography CT lung function orquality of life scores between treatment groups [ ] andRARc selective agonist administration also showed nodiï¬erences in CT scores or lung function in treated vsnontreated COPD patients [ ] However the therapeutic potential of regenerative pharmacology is still at thebeginning of its development And many authors haveshown that the human lung also in adulthood retains asignificant regenerative potential from the large to the smallairways and in terminal and respiratory bronchioles [] andthat tissue regeneration is achieved in two ways by proliferation of common diï¬erentiated cells andor by deployment of specialized stemprogenitor cells [ ]Extracorporeal shock wave therapy ESWT is applied inmany musculoskeletal diseases and in regenerative medicinebased on its capability to induce neoangiogenesis osteogenesis regeneration and remodeling through stem cellstimulation [] ESW in combination with tenogenicmedium improved the diï¬erentiation of human adiposederived stem cells hASCs into tenoblastlike cells []ESW combined with osteogenic medium increased the osteogenic diï¬erentiation of treated hASCs [] while stemcell diï¬erentiation into myoï¬broblasts was partially reducedby ESW treatment [] But to our knowledge no data areavailable on ESW treatment of primary bronchial ï¬broblastsof patients with COPD and control healthy smokers orbronchial epithelial cells 16HBEMarkers of cell proliferation include CD117 cKit orSCFR a receptor tyrosine kinase protein that binds to stemcell factor SCF expressed on hematopoietic stem cells Itcan also be expressed by mast cells melanocytes in the skininterstitial cells of Cajal in the digestive and urogenital tract[] cardiac pericytes [] amniotic ï¬uid stem cells []stemprogenitor cells in conducting airway epithelium ofporcine lung [] and dendritic cells in the lung []Another marker of cell proliferation is proliferating cellnuclear antigen PCNA It is expressed in the nuclei of cellsand is involved in DNA replication DNA repair andchromatin remodeling [ ] In the lung of COPD patients alveolar type II epithelial cells and endothelial cells[] and small airway bronchiolar epithelium [] expressdecreased PCNA levels compared with related nonCOPDcontrol groups A third marker of cell proliferation is CD90y1thymocyte diï¬erentiation antigen1 a glycophosphatidylinositol cell surface protein expressed by thymocytes CD34 cells mesenchymal stem cells endothelialcells and cardiac ï¬broblasts It is also considered a marker ofmultipotent mesenchymal stem cells when expressed inassociation with other markers CD29 CD44 CD73CD105 [ ]We aimed in this study to analyze the proliferative eï¬ectof shock waves when applied as an external challenge toprimary bronchial ï¬broblasts of COPD patients and controlsmokers and to immortalized bronchial epithelial cells16HBE To this end we investigated cell markers expression related to this proliferative stimulus Methods Ethics Statement Collection and processing of bronchialbiopsies at the Institute of Veruno NO and collection andprocessing of the peripheral lung tissues at the UniversityHospital of Orbassano during lung resection for a solitaryperipheral neoplasm were approved by the ethics andtechnical committees ofthe Istituti Clinici Scientiï¬ciMaugeri CTS p102 and San Luigi Hospital OrbassanoTO CE N Italy the study complied withthe Declaration of Helsinki and written informed consentwas obtained from each participant Cell Culture and Treatments We used the SV40 large Tantigentransformed 16HBE cellline which retains thediï¬erentiated morphology and function of normal humanbronchial epithelial cells NHBE [] and primary humanbronchial ï¬broblasts obtained from bronchial biopsies ofpatients with COPD n � and control smoking subjectsn � with normal lung function Primary bronchial ï¬broblasts were obtained from bronchial biopsies obtainedfor diï¬erent protocol studies [] Bronchial biopsies weretreated with type II collagenase min at °C InvitrogenGIBCA and cultured in DMEM until conï¬uentprimary ï¬broblasts were obtained 16HBE cells and primarybronchial ï¬broblasts were maintained in Dulbeccos modiï¬ed minimum essential medium DMEM supplementedwith vv fetal bovine serum FBS IUmL penicillin μgmL streptomycin 1x nonessential amino acids mMsodium pyruvate and mM glutamine °C CO2 []When cells were conï¬uent the complete mediumwas replaced with DMEM with FBS for starvation time h e shockwave generator utilized for the in vitroexperiments was a piezoelectric device Piezoson Richard Wolf Knittlingen Germany designed for clinical 0cCanadian Respiratory Journaluse in orthopedics and traumatology Aliquots of mL ofcell suspension adjusted to à cellsmL were placed in mm polypropylene tubes completely ï¬lled with culturemedium e shock wave unit was kept in contact with thecellcontaining tube by means of a waterï¬lled cushionCommon ultrasound gel was used as a contact mediumbetween the cushion and tube ESW treatment was as follows energy ï¬ux density EFD � mJmm2 pulsesfrequency � shockss is EFD is a mediumhigh energywe already used for previous in vitro diï¬erentiation studiesin tendons [] After treatment cell viability was evaluatedby trypan blue exclusion and primary ï¬broblasts werepassaged in DMEM complete for hours 16HBEcells were cultured for and h because of their lowerresistance to starvation T0 corresponds to hours post ESWtreatment for all experiments reported Nontreated ï¬broblasts or 16HBE cells were used as controls Cell growth wasevaluated by the colorimetric test WST1 All experimentswere performed in quadruplicate ie four independentexperiments for each type of treatment ESW or noESWand each time exposure Extraction and Quantiï¬cation of RNA and qRTPCRfrom Primary Bronchial Fibroblasts and 16HBE Total RNAfrom treated and nontreated cells was puriï¬ed and isolatedusing an RNAspin Mini RNA Isolation kit GE HealthcareLife Sciences Pittsburgh USA following the manufacturersinstructions Total RNA was resuspended in μL nucleasefree water RNA concentration was determined using aUVvisible spectrophotometer λ260280 nm EppendorfBioPhotometer plus and stored at °CQiagenQT00000728e expression of genes of interest was measured usingSYBR Green Qiagen UK for qPCR in a Corbett RotorGene Corbett Cambridge UK system Onestep realtime PCR was carried out by amplifying mRNA using theQuantiFast¢ SYBR Green RTPCR kitITaccording to the manufacturers instructions and the genespeciï¬c primers Qiagen IT We detected the expression ofcKit or SCFR CD117 Cat QT01844549 Qiagen PCNACat QT00024633 y1 CD90Cat QT00023569TGF1CatProcollagenIQT01005725 and NFκBp65 Cat QT01007370 Weperformed independent experiments and quantitative PCRmeasurements in quadruplicate for each type of treatmentESW or noESW and each time exposure Brieï¬y the PCRreaction mix prepared in a total volume of μL was run onthe Rotor Gene Q Qiagen IT and the following PCR runprotocol was used °C for min reverse transcription°C for min PCR initial activation step ampliï¬cationcycles of °C for s denaturation and °C for scombined annealingextension followed by melting curveanalysis to ensure the speciï¬city of PCR ampliï¬cationGlyceraldehyde phosphate dehydrogenase GAPDHQT01192646 Qiagen was used as the reference gene forevery target gene per sample and the data were normalizedagainst the respective GAPDH signaling Cycle thresholdCT values were determined using the Rotor Gene Qsoftware RotorGene Q Series Software eCatexpression levels of all genes studied were normalized toGAPDH levels in each sample to determine the expressionbetween treated and nontreated cells using the ÎCt method[] for primary bronchial ï¬broblasts and the ÎÎCt for16HBE cells [] ELISA Tests in the Supernatants or Cell Lysates of ESWTreated and Nontreated Cells Protein extraction andquantiï¬cation in the supernatants or cell lysates of ESWtreated and nontreated cells were performed as reported inTable Suppliers Cat Numbers dilution conditions anddetection limits of the ELISA kits used are also reported eELISA kits WST1 cell proliferation kit and MPERmammalian protein extraction kit were used according tothe manufacturers instructions Table CKit CD117PCNA and NFκBp65 were quantiï¬ed in cell lysates CD90TGF1 and procollagen1 were quantiï¬ed in the cellsupernatants Immunohistochemistry of the Lung Parenchyma of Patients with COPD Samples were frozen in liquid nitrogenprecooled is tane after embedding in OCT and used forcryostat sectioning and immunostaining of some cellproliferationrelated molecules Single immunostainings offrozen sections were performed with mouse antithyroidtranscription factor1 TTF1 sc53136 Santa Cruz rabbitanticKit CD117 ARG51826 ARGBIO and rabbit antiPCNA PAS27214 ermo Fisher primary antibodiesAntibody binding was demonstrated with secondary antibodies antimouse Vector BA and antirabbitVector BA followed by ABC kit AP AK5000VECTASTAIN and FastRed Substrate red color Doublestainings were performed using also ABC kit Elite PK6100VECTASTAIN and diaminobenzidine substrate browncolor for identiï¬cation of TTF1 positive alveolar type IIepithelial cells [] coexpressing cKit CD117 or PCNAantigens Slides were included in each staining run usinghuman tonsil nasal polyp or breast cancer as positivecontrols For the negative control slides normal nonspeciï¬cmouse or rabbit immunoglobulins Santa Cruz Biotechnology Santa Cruz CA USA were used at the same proteinconcentration as the primary antibodiesmean± standardthe unpaired ttest Probability values of p were Statistical Analysis Group data were expressed asorinterquartile range IQR for morphologichistologic dataDiï¬erences between treatment groups were analyzed usingor mediandeviationrangeconsidered significant Data analysis was performed usingthe Stat View SE Graphics program Abacus Concepts IncBerkeley CA USA Results ESW Eï¬ects on Cell Proliferation ESW treatment at adosage of mJmm2 pulses frequency � shockssof primary bronchial ï¬broblasts from COPD patients n � 0cCanadian Respiratory JournalPackyearsExsmokercurrent smokerTable Clinical characteristics of chronic obstructive pulmonary disease COPD patients and control smokers who provided bronchialï¬broblasts for in vitro experimentsSubjectsCOPD1COPD2COPD3± Mean± SD± Mean± SDIndividual and mean± standard deviation SD data M male F female FEV1 forced expiratory volume in s BD bronchodilator FVC forced vitalAge years MFMMMMMM± ± ± ± ± ± ± CurrentCurrentCurrentFEV1 postBDFEV1 preBDCurrentCurrentNDNDNDFEV1FVCCS1CS2CS3Excapacity ND not determined Patients were classiï¬ed according to the Global Initiative for Chronic Obstructive Lung disease httpgoldcopdorg levels ofseverity for COPD For COPD patients FEV1FVC are postbronchodilator values ANOVA test FEV1 p � FEV1FVC p � No significantdiï¬erences were observed for age p � and packyears p � smokedTable List of ELISA tests cell proliferation and protein extraction kits used For ELISA tests dilution of the supernatants or cell lysatesamples used and detection limits are also reportedTargetcKit or SCFR CD117PCNAy1 CD90TGF1Procollagen1NFκBp65WST1 cell proliferationMPER mammalian protein extraction ermo Scientiï¬c ngmL ngmL ngmL ngmL pgmL pgmL pgmL pgmL pgmL pgmL17pgmL pgmLCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpCloudClone CorpSEA121 HuSEA591MiSEB404 HuSEA124 HuSEA957 HuKHO0371KA1384Dilution PBS PBSDetection limit range diluent buï¬erInvitrogenAbnovaNo dilNo dilNo dilSupplierCat ashowed a significantly increased proliferation index at and h after treatment compared with nontreated bronchial ï¬broblasts Figure 1a ESWtreated primary bronchial ï¬broblasts from control smokers with normal lungfunction n � also showed a significant increase of theproliferation index at and h aftertreatmentFigure 1b Treated bronchial epithelial cells 16HBEshowed significantly increased proliferation index values at and h after treatment when compared with nontreated16HBE cells Figure 1c ESW Eï¬ects on mRNA and Protein Levels of Cell Proliferation and Cell Remodeling Markers Primary bronchialï¬broblasts from COPD patients n � control smokersn � and human bronchial epithelial cells 16HBE werestimulated with extracorporeal shock waves at a dosage of mJmm2 pulses and compared with paired nonstimulated primary bronchial ï¬broblasts and 16HBE cellsCKit mRNA was significantly increased in ESWtreated16HBE cells at h p � and decreased in CSï¬broblasts at h p � compared with nontreated cellsFigures 2b and 2c Furthermore a tendency to increased cKit mRNA levels was observed after ESW treatment for COPDï¬broblasts Figure 2a CKit protein wassignificantly increased in the cell lysates at h after ESWtreatment in primary bronchial ï¬broblasts of COPD patientsp � Figure 2d and in 16HBE cells p at h after ESW treatment Figure 2f No significantchanges were observed for cKit protein in ESWtreatedprimary bronchial ï¬broblasts from control smokers CSbronchialï¬broblastswith normal lung function Figure 2e PCNA mRNAlevels were not significantly changed in ESWtreated ï¬broblasts and 16HBE cells when compared with nontreatedcells Figures 3a3c PCNA protein in the cell lysatesshowed a tendency to be increased in primary bronchialï¬broblasts of CS p � at h after ESW treatmentFigure 3e and a significant increase was observed at hT0 in 16HBE cells p � after ESW treatmentFigure 3f No significant changes were observed inprimaryof COPD patientsFigure 3d y1 CD90 mRNA levels were not significantly diï¬erent in ESWtreated ï¬broblasts and 16HBE cellscompared with nontreated cells Figures 4a4c y1CD90 protein in the cell supernatants was significantlyincreased in primary bronchial ï¬broblasts of CS at hp � after ESW treatment Figure 4e No significant changes were observed in primary bronchial ï¬broblastsof COPD patients or in 16HBE cells Figures 4d and 4fTGF1 mRNA levels were not significantly changed in ESWtreated ï¬broblasts and 16HBE cells when compared withnontreated cells Figures 5a5c TGF1 protein in thecell supernatants was significantly increased in primarybronchial ï¬broblasts of CS at h p � after ESWtreatment Figure 5e No significant changes were observed in primary bronchial ï¬broblasts of COPD patients orin 16HBE cells Figures 5d and 5f Procollagen1 mRNAlevels were not significantly diï¬erent in ESWtreated ï¬broblasts and 16HBE cells compared with nontreated cellsFigures 6a6c Procollagen1 protein in the cellsupernatants wasincreased in primarysignificantly 0cCanadian Respiratory JournalabIncreased cell proliferation was observed in all cellular types studied after challenge with ESW Ttestp andp Figure WST1 test for evaluation of cell proliferation after extracorporeal shock wave ESW stimulation of primary bronchial ï¬broblastsof COPD patients n � a primary bronchial ï¬broblasts of control smokers n � b and bronchial epithelial cells 16HBE ccbronchial ï¬broblasts of COPD patients at h p � after ESW treatment Figure 6d No significant changeswere observed in primary bronchial ï¬broblasts of CS or in16HBE cells Figures 6e and 6f NFκBp65 mRNAlevels were not significantly changed in ESWtreated ï¬broblasts and 16HBE cells when compared with nontreatedcells Figures 7a7c NFκBp65 protein in the cell lysates was decreased in primary bronchial ï¬broblasts ofCOPD patients at h p � after ESW treatmentFigure 7d and increased in 16HBE cells at h p � and h p � after ESW treatment Figure 7f Nosignificant changes were observed in primary bronchial ï¬broblasts of CS Figure 7e Immunohistochemistry in the Lung Parenchyma of COPDPatients of Alveolar Type II Epithelial Cells Expressing cKitand PCNA In the lung parenchyma of COPD patientsalveolar type II epithelial cells were identiï¬ed by the use ofantithyroid transcription factor1 TTF1antibodyImmunopositivity for cKit CD117 and PCNA was alsooccasionally observed in alveolar septa Figure Doublestaining used for identiï¬cation of TTF1 cells coexpressingcKitFigures 9a and 9b and PCNAFigures 9c and 9d showed that alveolar type II epithelial cells coexpressing cKit and PCNA were present eventhough rarely observedCD117 Discussionis study shows that extracorporeal shock waves induce cellproliferation of bronchial epithelial cells 16HBE and primary bronchial ï¬broblasts of COPD patients and controlsmokers As far as markers of cell proliferation are concerned cKit CD117 was increased in bronchial epitheliumat both mRNA and protein levels h after ESW treatmentand it was also increased in primary bronchial ï¬broblasts at h after ESW challenge Other markers indicative of cellproliferation were also increased PCNA protein increased inCOPDWST1NO ESWESWT24T48T72T0012345CST0T24T48T72NO ESWESWWST101234516HBET0T24T48NO ESWESWWST1012345 0cCanadian Respiratory JournaladbecfFigure CKit CD117 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPDpatients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBEcKit increased at mRNA c and protein f levels In primary bronchial ï¬broblasts of COPD patients cKit increased at protein level d Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Proliferating cell nuclear antigen PCNA mRNA a b c and protein d e f expression after ESW treatment in primary bronchialï¬broblasts of COPD patients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In bronchialepithelium 16HBE PCNA increased at protein f level Ttest was used for comparative purposes and p values are reported in the graphsT0T24T48T7201234102030COPD CNCOPD ESWcKit CD117 Ct0180021103960767CS CNCS ESWcKit CD117 CtT0T24T48T7202461020304050002016HBE CN16HBE ESWcKit CD117 CtT0T24T480246204060800435041800320010020030006839038680037305624COPD CNCOPD ESWcKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESW000500100015002000250006727038680877507636cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW16HBE CN16HBE ESW020406080P 000010791501364cKit CD117 ngmLT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT0T24T48T72PCNA Ct0005101520COPD CNCOPD ESWT0T24T48T7205101520PCNA CtCS CNCS ESWT0T24T48000510152025PCNA Ct16HBE CN16HBE ESW01450027520139305288COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00102030PCNA ngmL00002004006008010000512084270367304489PCNA ngmLCS CNCS ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW0123004620190109820PCNA ngmL16HBE CN16HBE ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SW 0cCanadian Respiratory JournalacebdfFigure y1 CD90 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPDpatients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial ï¬broblasts ofcontrol smokers y1 increased at protein level at and h e Ttest was used for comparative purposes and p values are reported in thegraphsbronchial epithelial cells at h after ESW challenge y1CD90 protein increased in CSprimary bronchial ï¬broblasts at and h after ESW treatment molecules morerelated to remodeling such as TGF1 protein were increased in CSprimary bronchial ï¬broblasts at h afterESW treatment and procollagen1 protein increased at hfollowed by a decrease at h in COPDprimary bronchialï¬broblasts after ESW treatment A marker of ammationtranscription factor NFκBp65 protein was decreased inCOPDprimary bronchial ï¬broblasts at h after ESWtreatment but it was increased in CSprimary bronchialï¬broblasts and in bronchial epithelial cells after ESWtreatment Markers of cell proliferation such as cKit andPCNA were observed in the peripherallung of COPDT0T24T48T72COPD CNCOPD ESWThy1 CD90 Ct0005101520CS CNCS ESWThy1 CD90 CtT0T24T48T72012316HBE CN16HBE ESWThy1 CD90 CtT0T24T4801020304009523087570853209221COPD CNCOPD ESWT0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW00200040006000800010000Thy1 CD90 pgmLCS CNCS ESW01500003150239300410T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000200004000060000Thy1 CD90 pgmL16HBE CN16HBE ESW035960811001447T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW010203040Thy1 CD90 pgmL 0cCanadian Respiratory JournaladbecfFigure TGF1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPD patients ad primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial ï¬broblasts of controlsmokers TGF1 increased at protein level at h e Ttest was used for comparative purposes and p values are reported in the graphsadbecfFigure Procollagen1 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPDpatients a d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In primary bronchial ï¬broblasts ofCOPD patients procollagen1 increased at protein level d at h T0 followed by a decrease at h panel d Ttest was used forcomparative purposes and p values are reported in the graphsT0T24T48T72TGF 1 Ct0005101520COPD CNCOPD ESWT0T24T48T72TGF 1 Ct00051015CS CNCS ESWT0T24T48TGF 1 Ct0005101516HBE CN16HBE ESW07196046450373903445T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESWTGF 1 pgmL005010015004487044930863500385T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCS CNCS ESWTGF 1 pgmL0005001000150004757089490102101199T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW16HBE CN16HBE ESWTGF 1 pgmL010203040T0T24T48T72Procollanen1 Ct000510152025COPD CNCOPD ESWT0T24T48T72Procollagen1 Ct000510152025CS CNCS ESWT0T24T48Procollagen1 Ct00051015202516HBE CN16HBE ESW00220057350024202359T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SWCOPD CNCOPD ESW00100020003000Procollagen1 pgmL00541053750944605958T0 CNT0 SWT24 CNT24 SWT48 CNT48 SWT72 CNT72 SW000100002000030000Procollagen1 pgmLCS CNCS ESW010340898407490T0 CNT0 SWT24 CNT24 SWT48 CNT48 SW050100150200Procollagen1 pgmL16HBE CN16HBE ESW 0cCanadian Respiratory JournaladbecfFigure NFκBp65 mRNA a b c and protein d e f expression after ESW treatment in primary bronchial ï¬broblasts of COPD patientsa d primary bronchial ï¬broblasts of control smokers b e and bronchial epithelial cells c f In bronchial epithelium 16HBE NFκBp65 increased at protein panel f level at and h of exposure In primary bronchial ï¬broblasts of COPD patients NFκBp65 decreased atprotein level d at h In primary bronchial ï¬broblasts of control smokers NFκBp65 increased at protein level e at h Ttest wasused for comparative purposes and p values are reported in the graphspatients and both these markers were occasionally coexpressed by alveolar epithelial type II cells TTF1 in thesepatientsExtracorporeal shock wave therapy is applied in regenerative medicine since it is capable of inducing neoangiogenesis osteogenesis and remodeling through stemcell stimulation [] On the other hand while regenerativetherapy applied to mice with induced emphysema has shownpromising results [] clinical trials performed in COPDpatients were discouraging [ ] Since the human lung alsoin adulthood maintains a significant regenerative potential[] due to proliferation of diï¬erentiated of stemprogenitor cells andor by their stimulation [ ] we hereinvestigated the proliferative action of ESW at low dosage inbronchial epithelial cells and in primary bronchial ï¬broblasts of control smokers CS and patients with COPD Ourdata show that all the cell types studied significantly increased their proliferation index WST1 test after ESWtreatment in agreement with data previously obtained formuscle cells or tendon ï¬broblasts [] Interestingly thecKit CD117 receptor tyrosine kinase protein and mRNAwere increased in 16HBE cells and cKit protein also increased in primary bronchial ï¬broblasts of COPD patientsafter ESW stimulation It is not clear however if this cellresponse represents an intermediate dediï¬erentiation step ora simple proproliferative stimulus for stimulated 16HBEcells and COPDprimary bronchial ï¬broblasts Since weexposed welldiï¬erentiated cells we believe that this transitory increment may be interpreted as a proproliferativestimulus induced by ESW exposureIn bronchial epithelial cells 16HBE proliferating cellnuclear antigen PCNA considered a marker of cellproliferation was increased after ESW stimulation conï¬rming again the proproliferative role of ESW exposurefor these lung structure cells is ï¬nding in view of thedecreased PCNA levels reported in the lung of COPDpatients [ ] compared with control subjects is particularly relevant since ESW stimulation may contrastthese lower PCNA levels characterizing the damaged lungof these patientse increased y1 CD90 protein level shown afterESW exposure in CSprimary bronchial ï¬broblasts was notobserved in ESWtreated COPDprimary bronchial ï¬broblasts or in 16HBE treated cells PCNA protein alsotended to be higher in CSprimary bronchial ï¬broblastsafter ESW treatment but not in COPDprimary bronchialï¬broblasts ese diï¬erences in the response to ESWchallenge of COPD and CSprimary bronchial ï¬broblastsmay in part be due to the reduced proliferation capacity ofthese cells derived from COPD lungs as previously reported [ ] In our welldiï¬erentiated ESWexposedï¬broblasts we interpret the increment of y1 protein NFκBp65 Ct012345T24T48T72T0COPD CNCOPD ESW NFκBp65 Ct01234T24T48T72T0CS CNCS ESW NFκBp65 Ct000510152025T24T48T016HBE CN16HBE ESW00805026820020907045NFκBp65 pgmL0050000100000150000T72 CNT72 SWT24 SWT48 CNT48 SWT0 SWT24 CNT0 CNCOPD CNCOPD ESWNFκBp65 pgmL036510427702233003830020000400006000080000100000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CNT72 SWT72 CNCS CNCS ESWNFκBp65 pgmL0015500002062865000100001500002004006008001000T0 SWT24 CNT24 SWT48 CNT48 SWT0 CN16HBE CN16HBE ESW 0cCanadian Respiratory JournalFigure Photomicrographs showing thyroid transcription factor1 TTF1 expression panels a b cKit CD117 c d and proliferatingcell nuclear antigen PCNA e f in the peripheral lung tissue of a representative patient with chronic obstructive pulmonary diseaseCOPD Arrows indicate positively stained cells mainly located in the alveolar septa Bars � micronsafter ESW treatmentlike that of cKitas a proproliferative stimulus induced by the treatmentWe found increased levels of secreted TGF1 inCSprimary bronchial ï¬broblasts h after ESW stimulation TGF signaling pathways are involved in the regulationof many cell functions and in the maintenance of cellularhomeostasis [] We recently reported a decrease of TGF1and TGF3 in bronchiolar epithelium and alveolar macrophages of COPD patients compared with CS [] and thisdecrease may favor the increase of autoimmunity responsesin these patients [] We speculate that the inductionthrough ESW challenge of an increase of TGF in bronchialï¬broblasts may play a role in the TGF repositioning andgain in homeostatic function of this important protein in thelungs of COPD patientsTGF induced extracellular matrix and procollagen1production has been reported in pulmonary ï¬broblasts[] even though it was also reported that the increase ofproï¬brotic markersincluding procollagen1 in humanlung ï¬broblasts may be NLRP3 ammasome dependentand TGF independent [] and associated with increasedammation ofthe lung [] We here observed aTTF a0Lung COPDCD a0Lung COPDPCNA a0Lung COPDabcdef 0cCanadian Respiratory JournalFigure Photomicrographs showing alveolar type II epithelial cells TTF1 cells red color coexpressing cKit CD117 brown color ab and PCNA brown color c d in the peripheral lung tissue of a representative patient with COPD Positive doublestained cells can berecognized in the alveolar septa even though their presence was only rarely observed Arrows indicate positively stained cells located in thealveolar septa Bars � micronstransitory increase of procollagen1 protein in COPDprimary bronchial ï¬broblasts at h after ESW | Thyroid_Cancer |
"Older patients with cancer require specific and individualized management The 3groupMultidimensional Prognostic Index MPI based on the Comprehensive Geriatric Assessment CGA has shown apredictive interest in terms of mortality The objective of our study was to assess the prognostic value of MPI for year mortality in an external prospective French cohort of elderly patients with cancerMethods From March to March a prospective singlecenter cohort study enrolled all patients withcancer aged years and older referred to the geriatric oncology clinic We used a proportional hazard model for1year mortality adjusted for age sex tumor sites and metastatic status Cstatistics were used to assess theincremental predictive value of MPI index to these risk factorsResults overall patients underwent CGA with MPI women mean age ± years The most commontumor sites were prostate skin colorectum and breast of patients had a metastaticdisease patients belonged to the MPI1 group to the MPI2 group and patients wereclassified in the MPI3 group Oneyear mortality rate was in MPI1 in MPI2 and in MPI3 p All domains of MPI except cognition and living status were significantly associated with mortality at oneyear aswell as tumor sites and metastatic status Higher MPI was associated with a higher mortality risk adjusted HR [95CI ] and [] for MPI groups and compared to p Conclusions In addition to established risk factors MPI improves risk prediction of 1year mortality This practicalprognostic tool may help to optimize management of these vulnerable patientsKeywords Aged Neoplasms Mortality Comprehensive geriatric assessment Correspondence Evelyneliuuchupoitiersfr1Department of Geriatrics Poitiers University Hospital Poitiers France2Clinical Investigation Centre CIC1402 CHU Poitiers University of PoitiersINSERM Poitiers FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiuu BMC Geriatrics Page of BackgroundIndividuals over years old are the fastest growing segment of the population and by will represent about of Americans and of Europeans [] The incidence of cancer continues to increase worldwide it is estimated at millionyear by representing anincrease of in cases compared with [] Theincidence of cancer is times higher in people over years old and people aged and older have a higherrisk of developing invasive cancer []The older population is characterized by a very heterogeneous profile especially in terms of frailty geriatriccharacteristics and comorbidities which explains theneed for specific and adapted care [ ] Neverthelessscientific data are scarce because older subjects are oftenunderrepresented in oncological clinical trials that setthe standards of antineoplastic treatment [ ]Over the last three decades the fiveyear survival ratefor all types of cancer has increased particularly in individuals aged to [ ] Still older patients are atmore risk of toxicity in anticancer therapies such aschemotherapy and require a benefitrisk assessmentprior to treatment [] A comprehensive geriatric assessment CGA is consequently recommended in these patients to diagnose comorbidities and optimize geriatricinterventions and to improve the functional state andpossibly the survival rate by ensuring better tolerance totreatment [ ] CGA has also shown predictive valuein identifying elderly patients with cancer who are exposed to a poor prognosisincluding a higher risk ofdeath during hospitalization [] Among the CGAbased assessment tools the Multidimensional PrognosticIndex MPI has shown a predictive interest in mortalityat months and months in Italian patients aged years and older with advanced cancers []The main objective of our study was to validate theprognostic value of the MPI for 1year mortality in an external French cohort of older patients with cancer Thesecondary objective was to assess the major risk factors associated with 12month mortality in these patientsMethodsStudy population and data collectionThis prospective singlecenter cohort study enrolledfrom March to March all patients with cancer aged years and older who were referred to thegeriatric oncology clinic of Poitiers University HospitalpriorSociodemographic data and cancerrelated information werecollected during the consultationincluding age sexmarital status social environment type of cancer metastasis status and cancerspecific treatment Tumor siteswere classified as follows colorectal breast prostateupper gastrointestinaltract stomach and esophagusanticancertreatmenttoplannedand liver urinary system bladder upper urinary tractand kidney hematologic malignancies and other tumors including ovary uterus lung head and neck skinthyroid and unknown primary The CGA was performed by a senior geriatrician specialized in oncologyand provided data necessary to calculate MPI All eligible patients who had signed the consent form were included in the study The study protocol was validated bythe Poitiers University Hospital ethics committee Poitiers France All the clinical and biological data werecollected and recorded in a cohort databaseliving with familyMultidimensional prognostic indexThe MPI based on a CGA was calculated after administration of standardized and validated tests exploringeight domains Table [] Living status was categoinstitutionalized orrized asalone and functional status was evaluated by Activitiesof Daily Living ADL ranging from total dependenceto independence and Instrumental ADL IADL [] Nutrition was assessed by the Mini NutritionalAssessmentShort Form MNASF questionnaire cognitive status was evaluated by the Short Portable MentalStatus Questionnaire SPMSQ[ ] The ExtonSmith Scale ESS estimated the risk of pressure ulcer[] Comorbidities were evaluated by the CumulativeIllness Rating Scale CIRS which scores the severity of anic systems ranging from absent to mostsevere [] Based on this scale a comorbidity indexCIRSCI records the number of moderate to severean pathologies CIRS scores from to [] Thenumber of medications is classified in three groups drugs a day to drugs or ¥ drugsThe MPI was scored by matching the results of thesetests A value of or was assigned accordingto the conventional cutoff points considering as noproblem minor problem and major problemTable The sum was then divided by to obtain thefinal MPI score which was categorized into groupsthe MPI1 group final score defining patientswith low mortality risk at year the MPI2 group moderate risk and the MPI3 groupgroup higher riskDefinition of outcomesThe primary outcome in the longitudinal analyses was1year mortality Systematic followup was performedafter discharge through clinical visits every months bythe same clinical research assistant When patients werenot present at visit phone calls were made to the general practitioners to assess vital status and to obtain thedate of death if applicable 0cLiuu BMC Geriatrics Page of No problem value ¥¥Table Multidimensional Prognostic Index score assigned to each domain according to the severity of problemAssessment tests rangeADL IADL SPMSQ 10aCIRSCI 14bMNASF ESS Number of medicationsMinor problem value Institutionalized¥ ¥Living with familySevere problem value ¥¥ ¥ Living statusAbbreviations ADL Activities of Daily Living IADL Instrumental Activities of Daily Living SPMSQ Short Portable Mental Status Questionnaire CIRSCI CumulativeIllness Rating Scale Comorbidity Index MNASF Mini Nutritional Assessment Short Form ESS Exton Smith Scalea Number of errorsb Number of pathologiesLiving aloneStatistical analysisDescriptive statistics were reported as mean ± standarddeviation SD or median 25th75th percentiles forcontinuous variables or absolute number and percentagefor categorical variables The time to event was plottedas KaplanMeiersurvival curves according to MPIgroups and comparison was made using the logranktest The hazard ratio HR of 1year mortality for eachparameter was determined by Cox proportional hazardsregression Two models were used univariate modeland models adjusted for age sex metastatic statustumor sites Interactions between sex tumor site andmetastatic status for the association between MPI and year mortality were evaluated by the addition of interaction terms into the corresponding regression modelThe Akaikes information criterion AIC was used tocompare globalfit among models with and withoutMPI and the model with the smallest AIC was considered as the best modelGeneralized cstatistics were calculated to assess improvement in 1year mortality risk prediction of MPI inaddition to traditional risk factors age sex metastaticstatus tumor sites [] The CIs for the changes inthe cstatistic were computed based on bootstrapsamples P values were considered statistically significant Statistical analyses were performed with SASversion SAS Institute Cary NCResultsBaseline characteristics of study populationDuring the recruitment period eligible patients aged years and older were included mostly males n with a mean age of ± years Table Themost common tumor sites were prostate skin and breast of patients had a metastaticdisease Anticancer treatment included chemotherapyin patients surgery in and radiotherapy in Patients had comorbid conditionsregarding the CIRSscale and medication and were frequently malnourished Table In this cohort patients were classified in the MPI1 group patients in MPI2 and patients in MPI Except for metastatic status and antineoplastic treatments all variables of interest differed between the threeMPI groups P MPI and 1year mortalityAmong the patients were lost to followup Mean followup was ± months Overall mortalityat months was in MPI1 in MPI2 and in MPI3 P Fig Since we observed significant statistical interaction between sex and tumor site P we presented resultsfor the multivariate model with the inclusion in themodel of an interaction term We found no significantinteraction between tumor site and metastatic statusP The risk of 1year mortality across MPI groups isshown in Fig All functional scoring but SPMSQ and living statusnumber of daily drugs metastatic status and tumor sitewere significantly associated with mortality Table Compared to colorectal cancer reference categorybreast cancer was associated with significantly lower year mortality and upper gastrointestinal tractliver cancer and other malignancies with significantly higher year mortalityMPI groups were associated with 1year mortality inthe univariate model and remained significantly associated even after adjustment for age sex metastatic statusand tumor site Compared to the MPI1 group patientsof the MPI2 and MPI3 groups had gradual increasedrisk of 1year mortality adjusted hazard ratio [95CI] [] and [] respectively P Table 0cLiuu BMC Geriatrics Page of Table Patients baseline characteristics and evaluation by multidimensional prognostic index MPI n Sociodemographic characteristicsAgeFemale n Oncological characteristicsMost frequent tumor sitesProstateSkinColorectumBreastHematological malignanciesBladderMetastatic status n Type of antineoplastic treatment aChemotherapySurgeryRadiotherapyTotal cohortN ± MPI1N ± Comprehensive geriatric assessment and multidimensional prognostic index bHormone therapy ADL scoreADL categoryIADL scoreIADL categoryESS scoreESS categoryMNASF scoreMNASF categorySPMSQ scoreSPMSQ categoryCIRS scoreCIRSCI scoreCIRSCI categoryNumber of medicationsNumber of medications categoryLiving status familyinstitutionalone ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± MPI2N ± ± ± ± ± ± ± ± ± MPI3N ± ± ± ± ± ± ± ± ± MPI score ± ± ± ± P Numbers are mean ± SD or n Abbreviations MPI multidimensional prognostic index SD standard deviation ADL activities of daily livings IADL instrumental activities of daily livings ESS ExtonSmith Scale MNASF mini nutritional assessment short form SPSMQ Short Portable Mental Status Questionnaire CIRS Cumulative Illness Rating Scale CIcomorbidity indexa Antineoplastic treatment may combine one or several types of treatmentb Categories are reported as number of patients with nominorsevere problem to calculate MPI scoreDiscriminationWe assessed improvement in risk discrimination for theMPI group compared with the model with traditionalrisk factors age sex metastatic status and tumor siteWe observed a small but significant improvement in year mortality risk prediction difference in Cstatistic P when including the MPI group in themodel Table 0cLiuu BMC Geriatrics Page of Fig KaplanMeier curves of overall mortality in patients according to MPI groups Dotted line MPI dashed line MPI and solid line MPI Logrank test P Discussion and implicationsOur study confirmed the predictive value of the multidimensional prognostic index for 1year mortality in olderpatients with cancer MPI group had a significantlytwo to fivefold higher rate of 1year mortality We alsoshowed that the MPI improved prediction of 1yearmortality going beyond the traditional risk factors reported in the literature []Estimation of patient survival at time of the therapeutic decision is required to assess the balance of benefits and risks of performing or not performing specificoncologic interventions taking cancerspecific mortalityinto consideration Clinicians may need to know if thepatient will die of cancer or with cancer in cases wherecomorbidities or geriatric syndromes are challengingSeveral scales have been created and validated in largeepidemiologic cohorts to estimate overall survival notably at months with the Carey and Walter indexes [ ] These two scores consider dependency comorbidities with cancer and malnutrition Walter and collaborators reported independent associations betweenoneyear mortality in multivariable analysis and risk factors including male gender two medical diagnoses congestive heart failure aOR 95CI andcancer aOR for localized cancer and aOR for metastatic cancerfunctional dependency in any ADL at discharge aOR for dependencies from to ADLs and aOR for dependencies in all ADLs and laboratoryvalues creatinine level mgdL [ μmolL] aOR and albumin level gdL aOR from to gdL and aOR forvalues below gdL [] Carey confirmed thesefindings and furthered the elaboration of a prognosticindex for mortality in communityliving frail older individuals considering eight independent risk factors ofmortality weighted using Cox regression male sex dependence in toileting malignant neoplasm and renal insufficiency [] None of these tests were specificallydeveloped in cohorts with individuals with cancer andthey may consequently not be informative enough to reflect clinical and functional variability in daily care andto provide personalized corrective interventions Recentevidence reported a positive impact of geriatric interventions and monitoring in survival increase improvementof quality of life and completion of chemotherapy [] The MPI differs from other mortality indexes because it is based on a CGA with each of the eight tests 0cLiuu BMC Geriatrics Table Univariate and multivariate analyses for oneyear mortality model for MPIgroups n VariableADL score pointPvalueAdjusted HR CIIADL score pointSPMSQ score pointCIRS score pointMNA score pointESS score pointNumber of drugs drugLiving statusliving with familyliving aloneInstitutionalizedAge yearSex male vs femaleMetastatic statusTumor sitesColorectalbreastprostateUpper gastrointestinal tractliverurinary systemhematologic malignanciesother tumorsMultidimensional Prognostic Indexgroup group group HR CI reference reference reference reference reference Page of P Abbreviations HR hazard ratio CI confidence interval MPI Multidimensional Prognostic IndexMultivariate model adjusted for age sex tumor site metastatic status and MPI groupsassessing one geriatric domain Giantin and collaboratorsconfirmed the good discriminatory power for 12monthmortality in a cohort of cancer patients older than and validated higher mortality prediction compared to astandard CGA [] Use of the MPI in clinical practicemay provide rapid and comprehensive evaluation of patients and help to adapt decisionmaking in oncologyThe MPI has been developed and validated in large cohorts of in and outpatients for many causes to predict notonly mortality but also length of hospital stay P care intensity institutionalization rehospitalization andaccess to homecare services [ ] In an internationalmulticenter cohort of hospitalized older patients patients in group MPI2 OR P and the MPI3 group OR P were at higher risk of overall mortality compared to thoseof the lower risk group at admission [] This index maybe used as a decisionmaking tree for cancer managementso as to select older patients with lower mortality risk forthe same standard treatment as younger counterpartsthose who could benefit from adapted care or an exclusively supportive strategy in patients with limited life expectancy This classification in three groups is comparableto the geriatric oncology algorithm of Balducci [] ThisTable Predictive performance of MPI during 12month followupBiomarkerclinical modelAkaike criterioncindexclinical model MPIClinical model age sex metastatic status tumor site CIdifference in CstatisticsP value 0cLiuu BMC Geriatrics Page of algorithm defines three groups of patients robust vulnerable and frail according to seven criteria age dependencemeasured by ADL and IADL comorbidities with CIRSCIcognition evaluated with MMSE minimental state examination or delirium depressive mood urinary and fecalincontinence and falls in the last months Risk of deathincreased steadily from the lowest to the highest categorycompared to the fit group the patients with a vulnerableprofile had a twofold mortality risk HR and a threefold risk in the frail group HR P [] More recent classifications were suggested to improve global management for such individualsincluding nutrition data and cognitive assessment[ ]Indeed malnutrition is highly prevalent in geriatriconcology settings [] This geriatric syndrome is a wellknown risk factor for early mortality Our findings confirmed that oneyear mortality is strongly associated withnutritional status and altered MNA in its short formSome questions in this test were selected for the elaboration of the Geriatric8 G8 index to screen for vulnerability in older patients with cancer as recommended bythe International Society for Geriatric Oncology SIOG[ ]The findings ofthis study should be interpretedwith caution Firstits design as an observationalsinglecenter study may limit the extrapolation of ourresults to a more general older population with cancer Recruited patients in this cohort may not be representative as cancer specialists may not refer alltheir patients to the geriatric oncology clinic notablythose screened as notvulnerable in geriatric termsas recommended by the SIOG and National Instituteof Cancer in a twostep approach [] Cancer management of these patients may follow standard strategy without geriatric expertise After accounting fortraditional risk factors the magnitude of the improvement in risk prediction by the addition is small butsignificant Moreover our results are consistent withexisting findings in geriatric oncology settingsthisstrategyOur research on the predictive value of MPI foroneyear mortality of older patients with cancershould serve as a foundation for future studies aiming to improve therapeutic strategies for these patients A major part ofinvolvespersonalized geriatric interventions such as specificcare monitoring by nurse and physical rehabilitationIt has shown benefits for elderly cancer patients butso far no study has demonstrated any impact onsurvival [] MPI appears to be a rapid assessmenttool helping to optimize cancer care guidepatienttailored interventions and predict early mortality These findings should pave the way for prospective interventionaltaking account ofstudiesMPI groups for decisionmaking about cancer treatments and followupConclusionsIn addition to established risk factors MPI improves riskprediction of 1year mortality in older cancer patientsThis practical prognostic tool may help to optimizemanagement of these vulnerable individualsAbbreviationsADL Activities of Daily Living AIC Akaikes information criterionCGA Comprehensive Geriatric Assessment CI confidence intervalCIRS Cumulative Illness Rating Scale CIRSCI Cumulative Illness Rating Scale comorbidity index ESS ExtonSmith Scale G8 Geriatric8 HR Hazard ratioIADL Instrumental Activities of Daily Living MNASF Mini NutritionalAssessmentShort Form MPI Multidimensional Prognostic IndexSD Standard deviation SIOG International society of geriatric oncologySPMSQ Short Portable Mental Status QuestionnaireAcknowledgmentsAuthors thank Emilie Favard for her assistance in the collection of followupdata and Jeffrey Arsham who edited the English of the manuscriptAuthors contributionsEL CH and MP designed the study EL SV and AJ were responsible for theacquisition of data EL and PJS performed the statistical analysis andinterpretation EL PJS and MP wrote the manuscript EL PJS MP TB MLBand AP substantively revised the work All authors EL CH SV TB AJ MLBAP PJS and MP read and approved the final manuscriptFundingThe authors declare no fundingAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateAll eligible patients who had signed the consent form were included in thestudy The study protocol was validated by the Poitiers University Hospitalethics committee Poitiers FranceConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Geriatrics Poitiers University Hospital Poitiers France2Clinical Investigation Centre CIC1402 CHU Poitiers University of PoitiersINSERM Poitiers France 3Department Geriatric Care Orthogeriatrics andRehabilitation Frailty Area EO Galliera Hospital Genova Italy 4Departmentof interdisciplinary Medicine Aldo Moro University of Bari Bari ItalyReceived December Accepted August ReferencesExtermann M Aapro M Bernabei R Cohen HJ Droz JP Lichtman S et alUse of comprehensive geriatric assessment in older cancer patientsrecommendations from the task force on CGA of the International Societyof Geriatric Oncology SIOG Crit Rev Oncol Hematol Cancer Research UK Worldwide cancer incidence statistics Availablefrom httpwwwcancerresearchukhealthprofessionalcancerstatisticsworld widecancer incidence Accessed Apr Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin 0cLiuu BMC Geriatrics Page of Pamoukdjian F Liuu E Caillet P Herbaud S Gisselbrecht M Poisson J Howto optimize Cancer treatment in older patients an overview of availablegeriatric tools Am J Clin Oncol Kalsi T BabicIllman G Ross PJ Maisey NR Hughes S Fields P The impact ofcomprehensive geriatric assessment interventions on tolerance tochemotherapy in older people Br J Cancer Rao AV Hsieh F Feussner JR Cohen HJ Geriatric evaluation andmanagement units in the care of the frail elderly cancer patient J GerontolA Biol Sci Med Sci Meyer AM Becker I Siri G Brinkkötter PT Benzing T Pilotto A Polidori MCNew associations of the Multidimensional Prognostic Index Z GerontolGeriatr Pilotto A Veronese N Daragjati J CruzJentoft AJ Polidori MC MattaceRasoF Using the multidimensional prognostic index to predict clinicaloutcomes of hospitalized older persons a prospective multicentreinternational study J Gerontol A Biol Sci Med Sci Ferrat E Paillaud E Caillet P Laurent M Tournigand C Lagrange JL et alPerformance of four frailty classifications in older patients with cancerprospective elderly cancer patients cohort study J Clin Oncol Caillet P Liuu E Raynaud Simon A Bonnefoy M Guerin O Berrut GAssociation between cachexia chemotherapy and outcomes in oldercancer patients a systematic review Clin Nutr Decoster L Van Puyvelde K Mohile S Wedding U Basso U Colloca G et alScreening tools for multidimensional health problems warranting a geriatricassessment in older cancer patients an update on SIOG recommendationsAnn Oncol Soubeyran P Bellera C Goyard J Heitz D Curé H Rousselot H et alScreening for vulnerability in older cancer patients the ONCODAGEprospective multicenter cohort study PLoS One 20149e115060 Caillet P CanouiPoitrine F Vouriot J Berle M Reinald N Krypciak S et alComprehensive geriatric assessment in the decisionmaking process inelderly patients with cancer ELCAPA study J Clin Oncol Galvão DA Taaffe DR Spry N Joseph D Newton RU Combined resistanceand aerobic exercise program reverses muscle loss in men undergoingandrogen suppression therapy for prostate cancer without bonemetastases a randomized controlled trial J Clin Oncol Goodwin JS Satish S Anderson ET Nattinger AB Freeman JL Effect ofnurse case management on the treatment of older women with breastcancer J Am Geriatr Soc Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsBalducci L Extermann M Management of Cancer in the older person apractical approach Oncologist Walter LC Brand RJ Counsell SR Palmer RM Landefeld CS Fortinsky RH Development and validation of a prognostic index for 1year mortalityin older adults after hospitalization JAMA Gouverneur A Salvo F Berdaï D Moore N FourrierRéglat A Noize PInclusion of elderly or frail patients in randomized controlled trials oftargeted therapies for the treatment of metastatic colorectal cancer asystematic review J Geriatr Oncol Talarico L Chen G Pazdur R Enrollment of elderly patients in clinical trialsfor cancer drug registration a 7year experience by the US Food and DrugAdministration J Clin Oncol Zeng C Wen W Mans AK Pao W Shu XO Zheng W Disparities by raceage and sex in the improvement of survival for major cancers results from theNational Cancer Institute surveillance epidemiology and end results SEERprogram in the United States to JAMA Oncol Ellis G Gardner M Tsiachristas A Langhorne P Burke O Harwood RH et alComprehensive geriatric assessment for older adults admitted to hospitalCochrane Database Syst Rev 20179CD006211 Wildiers H Heeren P Puts M Topinkova E JanssenHeijnen ML ExtermannM International Society of Geriatric Oncology consensus on geriatricassessment in older patients with cancer J Clin Oncol AvelinoSilva TJ Farfel JM Curiati JA Amaral JR Campora F JacobFilho WComprehensive geriatric assessment predicts mortality and adverseoutcomes in hospitalized older adults BMC Geriatr Angleman SB Santoni G Pilotto A Fratiglioni L Welmer AK MPI_AGEProject Investigators Multidimensional Prognostic Index in Association withFuture Mortality and Number of Hospital Days in a PopulationBasedSample of Older Adults Results of the EU Funded MPI_AGE Project PLoSOne 201510e0133789 Giantin V Falci C De Luca E Valentini E Iasevoli M Siviero P Maggi S et alPerformance of the multidimensional geriatric assessment andmultidimensional prognostic index in predicting negative outcomes inolder adults with cancer Eur J Cancer Care httpsdoi101111ecc12585 Pilotto A Ferrucci L Franceschi M D'Ambrosio LP Scarcelli C Cascavilla L Development and validation of a multidimensional prognostic indexfor oneyear mortality from comprehensive geriatric assessment inhospitalized older patients Rejuvenation Res Pilotto A Rengo F Marchionni N Sancarlo D Fontana A Panza F et alComparing the prognostic accuracy for allcause mortality of frailtyinstruments a multicentre 1year followup in hospitalized older patientsPLoS One 20127e29090Katz S Downs TD Cash HR Grotz RC Progress in development of the indexof ADL Gerontologist Lawton MP Brody EM Assessment of older people selfmaintaining andinstrumental activities of daily living Gerontologist Pfeiffer E A short portable mental status questionnaire for the assessmentof anic brain deficit in elderly patients J Am Geriatr Soc Rubenstein LZ Harker JO Salva A Guigoz Y Vellas B Screening forundernutrition in geriatric practice developing the shortform mininutritional assessment MNASF J Gerontol A Biol Sci Med Sci M366 Bliss MR McLaren R ExtonSmith AN Mattresses for preventing pressuresores in geriatric patients Mon Bull Minist Health Public Health Lab ServLinn BS Linn MW Gurel L Cumulative illness rating scale J Am Geriatr Soc Conwell Y Forbes NT Cox C Caine ED Validation of a measure of physicalillness burden at autopsy the cumulative illness rating scale J Am GeriatrSoc Pencina MJ D'Agostino RB Overall C as a measure of discrimination insurvival analysis model specific population value and confidence intervalestimation Stat Med Giantin V Valentini E Iasevoli M Falci C Siviero P De Luca E Does themultidimensional prognostic index MPI based on a comprehensivegeriatric assessment CGA predict mortality in cancer patients Results of aprospective observational trial J Geriatr Oncol Carey EC Covinsky KE Lui LY Eng C Sands LP Walter LC Prediction ofmortality in communityliving frail elderly people with longterm careneeds J Am Geriatr Soc Epub Nov 0c" | Thyroid_Cancer |
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14 cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classiï¬ed into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in speciï¬c chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22 ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and ï¬broblastgrowth factor receptor FGFR gene fusions30 Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients beneï¬t from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classiï¬cation andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aï¬atoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver ï¬ukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difï¬culty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespeciï¬c survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Paciï¬c Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11 Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14 Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is aï¬rm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was ï¬rst described in cHCCICC was not systematicallydescribed until when it was classiï¬ed into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly deï¬ned areas type C mixedtype presents as intimate association without clear boundaries18 In another classiï¬cation system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identiï¬able intermediatetransition between HCC and ICC type III ï¬brolamellar tumors resemblesthe ï¬brolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classiï¬cationis commonly used in which cHCCICC is classiï¬ed into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a uniï¬ed classiï¬cation system greatly adds to the difï¬culty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdeï¬ned cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identiï¬ed stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA ampliï¬cations are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in of cases Recently some oncogenic genes were identiï¬ed in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identiï¬ed as a driver ofHCC45 VEGFA and CCND1FGF19 have also identiï¬ed in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identiï¬ed For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22 and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately of cases22 Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30 Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in of ICC cases32 Fusions ampliï¬cations translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC and ICC Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate ï¬lament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difï¬cult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166 Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efï¬cacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L ï¬rstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5ï¬uorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71 This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current ï¬rstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onï¬rstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand ï¬uoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the ï¬rstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe beneï¬t of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] conï¬dence intervalPFS months versus months p This effectiveness wasconï¬rmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the ï¬rstlinetreatment for advanced cholangiocarcinoma[CI] and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe ï¬rstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efï¬cacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efï¬cacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased beneï¬t for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095 Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI months CI months CI and months CI respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as ï¬rst line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir ï¬rstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the ï¬rst US Food and Drug Administration FDAapproved ï¬rstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPaciï¬c have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the ï¬rst generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have beneï¬tedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1 FGFR1 PDGFR RET and KIT In August theFDA approved lenvatinib for ï¬rstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the ï¬rstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modiï¬cations Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can speciï¬cally inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPaciï¬cREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further conï¬rmed the efï¬cacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival beneï¬t observed across all age subgroups and atolerable safety proï¬le supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety proï¬le apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic proï¬le and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a ï¬rstinhuman phase I study NCT02508467 of ï¬sogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123 Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identiï¬ed in recent years is the inhibitor of the ï¬broblast growth factorFGF signaling pathway which consists of members labeled FGF1FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1 FGF signals regulate cell proliferation in which FGFR2fusions occurred in of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the ï¬rst and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on ï¬ndings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may beneï¬t from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPï¬zeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial Identiï¬erTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi | Thyroid_Cancer |
"production process errors may bediscovered which could affect the content and all legal disclaimers that apply to the journalpertain Published by Elsevier 0cSARSCoV2 another kind of liver aggressor how does it do that Sonia A LozanoSepulveda1 Kame GalanHuerta Natalia MartnezAcu±a Daniel ArellanosSoto and Ana Mara RivasEstilla1 1Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario Dr Jose E Gonzalez Autonomous University of Nuevo Len Monterrey Ana Mara RivasEstilla Department of Biochemistry and Molecular Medicine School of Medicine and Hospital Universitario Dr Jose E Gonzalez Autonomous University of NL Mxico Corresponding author Nuevo Leon Ave Francisco I Madero y Ave Gonzalitos sn Col Mitras Centro Journal Preproofbut complications such as pneumonia respiratory distress syndrome and multian Monterrey Nuevo Len Mxico Telfax Email amrivas1yahooca Abstract Clinical manifestations of SARSCoV2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities Liver dysfunction is one of the most striking affections among patients suggesting that SARSCoV2 may represent a new king of liver aggressor However the molecular process underlying this phenomenon is 0cstill unclear In this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients Abbreviations AaDO2 ACE2 AIH ALT AST COVID19 GGT GI GTEx Alveolararterial Oxygen Gradient Angiotensinconverting enzyme Autoimmune Hepatitis Alanine transaminase Aspartate aminotransferase Coronavirus infectious disease Gamma glutamyl transpeptidase Gastrointestinal GenotypeTissue Expression Metabolicassociated fatty liver disease Nonstructural proteins Reading Frame Journal Preproofcomplex disease in many severely ill patients In other infected subjects an infection is Keywords SARSCoV2 liver liver impairment COVID19 ACE2 MAFLD nsp ORF Introduction SARSCoV2 is the etiological agent of the disease known as COVID19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients liver alterations COVID19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm All of these factors make COVID19 highly unpredictable it is what specialists call a multisystem disease 0cAround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as AST aspartate aminotransferase and ALT alanine transaminase have been documented among patients infected with SARSCoV2 There is still no certainty whether the COVID19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor In this paper we describe a brief overview of the implications for researchers in the field of It is important to understand how liver function can be altered by direct infection with this predisposed to develop COVID19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the respiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this Journal Preproofliver disease of the most recent findings between the molecular biology of the virus This emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure [] The angiotensinconverting enzyme ACE2 the functional receptor of the spike glycoprotein of SARSCoV2 is widely distributed in the anism Historically Hamming and colleagues reported ACE2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients Clinical characteristics and liver injury in patients with COVID19 0ccells and arterial smooth muscle cells [] Posterior transcriptomic and proteomic analyses confirmed their findings and added high ACE2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid [] ACE2 is also expressed in liver but in lesser extent One of the most worrisome severe cases of COVID19 [] Regarding the gastrointestinal GI tract and liver over COVID19associated liver injury is defined as any liver damage that occurs during disease progression andor COVID19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with COVID19 even those who were receiving anticoagulants Researchers at Mount Sinai in New York published studies suggesting that clots in the lungs play an important role in the most of COVID19 patients develop GI symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests [] Journal PreproofDecreased albumin levels are associated with severe infection and poor prognosis Still AST elevation is the most common abnormality in patients presenting with COVID19 observed more frequently in men and is mainly documented in more severe cases [] liver disease In general the incidence of increased liver biochemical markers in hospitalized patients with COVID19 mainly AST and ALT and slightly elevated bilirubin varies between to of cases [] The increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with COVID19 The largest cohort study that included cases of COVID19 from China showed that had preexisting chronic liver disease Lei and colleagues reported that impaired liver function was related to mortality in COVID19 patients [] Elevated AST was more frequent and significant than the increase of ALT in severe 0chospitalized patients Moreover elevated AST was shown to be associated with highest mortality risk [] In the study reported by Yijin Wang [] they found that of COVID patients had elevated AST activity The median levels of ALT were UL vs UL respectively AST were UL vs UL respectively in abnormal and normal aminotransferase groups Liver enzymes abnormality were associated with disease severity protein levels In addition they found by ultrastructural examination of coronavirus ps in hepatocytes in COVID19 cases SARSCoV2 infected hepatocytes displayed as well as a series of laboratory tests including higher Alveolararterial Oxygen Gradient AaDO2 higher gamma glutamyl transpeptidase GGT lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased Histological findings showed apoptosis and binuclear hepatocytes Journal Preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis AIH developing COVID19 Taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection [] All these findings by different reports demonstrates that SARSCoV2 infection in liver is a crucial cause of hepatic impairment in COVID19 patients However alteration of cellular metabolism that give rise to systematic alterations and metabolic Report from Alessio Gerussi et al[] demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cMolecular biology of SARSCov2 Coronaviruses are enveloped viruses that contain a positively polarized unsegmented RNA genome belonging to the Coronaviridae family and the order of Nidovirales they are distributed in humans and other mammals[] The size of the SARSCoV2 virions is approximately to nm in diameter [] SARSCoV2 has a genome that consists polymerase RdRp which is nsp12 and is responsible of the replication and transcription of the virus[] which are encoded by the various genetic loci on the genome [] At the center of the virion lies a nucleocapsid composed of the genomic RNA and the nucleocapsid protein[] The virus glycoprotein S consists of two subunits S1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two reading frames ORF 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 []The virions have a structural Sspike protein outer spiky glycoprotein Mmembrane protein a type III transmembrane glycoprotein Nof nucleotides [] encoding amino acids and it is composed of a region Journal Preproofvirus [] as well as protein M which is a type III transmembrane glycoprotein[] and participates in the cellular membrane rearrangements for the replication and transcription complexes[] Among the encoded proteins is an RNAdependent RNA provides the receptor binding site and S2 which is at the carboxyl terminus responsible for membrane fusion [] The envelope protein E has a role in the assembly and release of the Nonstructural proteins have several functions during de viral cycle For example nsp 0cThe virus enters the cell by endocytosis through the interaction between envelope glycoprotein S with the cell receptor ACE2 and with the participation of the type II transmembrane serine protease TMPRSS2 [] Once it enters the cell the N protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free Next the polyprotein containing the viral proteins that are How does the virus select which cells to infect Viruses can infect only certain species of hosts and only permissive cells within that host Permissive cells make all the necessary proteins and viral factors to allow virus to replicate Once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the viruss genetic material []Viral replication may cause exocytosis[] will translate into viral proteins this entire process will occur in the cell cytoplasm The processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic RNA is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome[] Furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic RNAs these are the ones that Journal PreproofBoth SARSCoV and the new SARSCoV2 are very similar in structure and pathogenicity but the major structural protein S protein is slightly different between them[] Compared to other beta coronaviruses the presence of a furinlike cleavage site in SARSCoV2 enables the S protein priming and facilitates an increase on the efficiency of the spread of SARSCoV2 as is reported wide world [] 0cbiochemical changes producing cell damage called cytopathic effects Like other coronaviruses SARSCoV2 requires cellular receptors to initiate its internalization to the cells that carry these factors []Li SARSCoV2 uses the angiotensinconverting enzyme ACE2 as a host cell receptor SARSCoV2 spike S protein binds ACE2 with significantly high affinity[] In addition the main host protease that suggested to promote the pathogenesis of this coronavirus [] program httpsportalgdccancergov They compared ACE2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues Furthermore other reports have analyzed the correlations between ACE2 In order to provide insights into the mechanism of SARSCoV2 infection Li [] analyzed the expression of ACE2 in various normal human tissues using the datasets from the GenotypeTissue Expression GTEx project and The Cancer Genome Atlas TCGA transmembrane serine protease[] Other host proteases such as furin have also been mediates Sprotein activation on primary target cells and initial viral entry is the type II Journal Preproofreported by Li ACE2 expression levels showed no significant difference between have no significant association with sex age or race Is the liver a direct target for SARSCoV2 males and females between younger and older persons or between Asian and nonAsian races This finding suggests that the infection risk of SARSCoV2 and SARSCoV may expression levels and immune signature enrichment levels in individual tissues As As we expected because the systemic manifestations of COVID19 it has been reported that SARSCoV2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of Covid19 disease and aggravates preexisting conditions The ACE2 protein is found at high levels in the GI tract as the colon biliary system and liver [] On the other hand it is well documented a SARSCoV2 RNA shedding in the GI tract [] supporting its tropism for architecture express ACE TMPRSS1 receptors [] The presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs Also in SARS infection the presence of viral RNA in liver tissue was documented but not as extensively as the new coronavirus Data published by Gordon [] suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated AST the GI tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury Indeed a large part of the cells distributed in the liver Journal Preproofeffect the exacerbated inflammatory response in COVID19 may play a central role in profiles are detected in these patients [] Furthermore in addition to this intracellular More recently [] identified the clinical and laboratory characteristics of COVID19 patients with abnormal liver transaminases and they reported that SARSCoV2 is able to which high levels of IL6 have been reported [] which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease Mechanisms of liver pathogenicity 0cIf SARSCoV2 replication has direct adverse effects on liver function it is still unknown Findings in liver biopsy of patients killed by COVID19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity[] This seems to indicate that a direct injury occurred while the infection that could have been directly caused by SARSCoV2 Another possibility is that a druginduced liver injury occurred To date there are the following possible mechanisms Figure infection The massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal COVID19 cases [] In these cases uncontrolled inflammation induces multian damage leading including liver failure Biomarkers of inflammation such as Creactive protein PCR serum ferritin LDH Ddimer IL6 IL2 are have been found to be significantly elevated in Immune damage from exacerbated inflammation in response to SARSCoV2 Journal Preproofpathogenesis of SARS CoV related liver disease more studies should be liver is a potential target for direct infection with this virus To understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum Golgi apparatus and lipidrafts CoV2 enters cells through the ACE2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells [] Based on this expression the Direct cytopathic effect due to active viral replication in various liver cells SARScritically ill patients with COVID19 [] into hepatocytes and liver histology characterization It is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions There are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis Anoxia respiratory failure is one of the main characteristics of COVID19 Anoxic hypoxic hepatitis is common in patients with severe symptoms [] Reactivation of preexisting liver disease Patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional Chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients [] Druginduced liver damage DILI Initial clinical guidelines recommended antiviral agents for COVID19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from SARSCoV2 Biological Journal Preproof Genetic factors Genetics may well be one of the determining factors in some reaction may also cause HBV reactivation [] and induce eventual impairment of liver function in those patients with HBV On the other hand it is still unknown whether SARSCoV2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with COVID19 but until now we cannot be sure It is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus It will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of COVID19 Ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease MAFLD have a higher risk of COVID19 severity disease and abnormal liver blood tests than patients without MAFLD [] In contrast Louise Biquard [] demonstrated that MAFLD is not associated with changes in liver expression blood test abnormalities reported by Ji and colleagues is thus likely not explained by Concluding remarks The scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease Certainly the application of new technological platforms such as singlecell increased hepatic SARSCoV2 uptake Still several contradictory reports will help of genes implicated in 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assembly and morphology J Struct Biol httpsdoi101016jjsb201011021 expression of viral envelope protein genes EMBO J al Nucleocapsidindependent assembly of coronaviruslike ps by coJournal PreproofBiogenesis and architecture of arterivirus replication anelles Virus Res httpsdoi101016jvirusres201604001 RNA Synthesis and Processing vol 1st ed Elsevier Inc httpsdoi101016bsaivir201608008 [] Rabi F Al Zoubi M Kasasbeh G Salameh D AlNasser A SARSCoV2 and Coronavirus Disease what we know so far Pathogens [] Masters P The molecular biology of coronaviruses Adv Virus Res 0c [] Shang J Ye G Shi K Wan Y Luo C Aihara H Structural basis of receptor recognition by SARSCoV2 Nature httpsdoi101038s415860202179y [] Rabaan AA AlAhmed SH Haque S Sah R Tiwari R Malik YS SARSCoV [] Li W Moore MJ Vasilieva N Sui J Wong SK Berne MA Angiotensin httpsdoi101038nature02145 [] Hoffmann M KleineWeber H P¶hlmann S A Multibasic Cleavage Site in the [] Cohen FS How Viruses Invade Cells Biophys J httpsdoi101016jbpj201602006 SARSCoV and MERSCOV A comparative overview Le Infez Med converting enzyme is a functional receptor for the SARS coronavirus Nature Journal PreproofEM structure of the 2019nCoV spike in the prefusion conformation Science httpsdoi101126scienceabb2507 Spike Protein of SARSCoV2 Is Essential for Infection of Human Lung Cells Mol Cell 202078779784e5 httpsdoi101016jmolcel202004022 [] Ziegler CGK Allon SJ Nyquist SK Mbano IM Miao VN Tzouanas CN SARSCoV2 Receptor ACE2 Is an InterferonStimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues Cell httpsdoi101016jcell202004035 [] Wrapp D Wang N Corbett KS Goldsmith JA Hsieh CL Abiona O Cryo 0c[] Follis K York J Nunberg J Furin cleavage of the SARS coronavirus spike glycoprotein enhances cellcell fusion but does not affect virion entry Virology httpsdoi101016jvirol200602003 [] Millet JK Whittaker GR Host cell proteases Critical determinants of coronavirus [] Li MYY Li L Zhang Y Wang XSS Expression of the SARSCoV2 cell receptor httpsdoi101186s4024902000662x [] Xu H Zhong L Deng J Peng J Dan H Zeng X High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci httpsdoi101038s413680200074x tropism and pathogenesis Virus Res httpsdoi101016jvirusres201411021 gene ACE2 in a wide variety of human tissues Infect Dis Poverty 20209NA Journal PreproofCoV2 protein interaction map reveals targets for drug repurposing Nature Infection of SARSCoV2 Gastroenterology 202015818311833e3 httpsdoi101053jgastro202002055 httpsdoi101038s4158602022869 [] Xu L Liu J Lu M Yang D Zheng X Liver injury during highly pathogenic human coronavirus infections Liver Int Off J Int Assoc Study Liver httpsdoi101111liv14435 [] Coomes EA Haghbayan H Interleukin6 in COVID19 A Systematic Review and [] Xiao F Tang M Zheng X Liu Y Li X Shan H Evidence for Gastrointestinal [] Gordon DE Jang GM Bouhaddou M Xu J Obernier K White KM A SARS 0cMetaAnalysis MedRxiv httpsdoi1011012020033020048058 [] Xu Z Shi L Wang Y Zhang J Huang L Zhang C Pathological findings of COVID19 associated with acute respiratory distress syndrome Lancet Respir Med httpsdoi101016S221326002030076X [] Zhang B Zhou X Qiu Y Feng F Feng J Jia Y Clinical characteristics of [] Chen G Wu D Guo W Cao Y Huang D Wang H Clinical and [] Chai X Hu L Zhang Y Han W Lu Z Ke A Specific ACE2 Expression in Invest httpsdoi101172JCI137244 death cases with COVID19 MedRxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease J Clin Journal PreproofCholangiocytes May Cause Liver Damage After 2019nCoV Infection BioRxiv patients MedRxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 [] Herold T Jurinovic V Arnreich C Hellmuth JC von BergweltBaildon M Klein M Level of IL6 predicts respiratory failure in hospitalized symptomatic COVID[] Grein J Ohmagari N Shin D Diaz G Asperges E Castagna A Compassionate Use of Remdesivir for Patients with Severe Covid19 N Engl J Med httpsdoi101056nejmoa2007016 [] U S Food and Drug Administration Fact sheet for health care providers emergency 0cuse authorization EUA of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of COVID19 in certain hospitalized patients [] Varona Prez J Rodriguez Chinesta JM Riesgo de reactivacin de la hepatitis B asociado al tratamiento con corticoides frente a SARSCoV2 COVID19 Rev Clnica Espa±ola httpsdoi101016jrce202004012 [] Ji D Qin E Xu J Zhang D Cheng G Wang Y Nonalcoholic fatty liver httpsdoi101016jjhep202003044 SARSCoV2 in metabolicassociated fatty liver disease J Hepatol diseases in patients with COVID19 A retrospective study J Hepatol Journal Preproof[] Biquard L Valla D Rautou PE No evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cFigure legends Journal PreproofFig1 Proposed mechanisms of liver pathogenicity of SARSCoV2 in infected cells SARS CoV2 Infection2 Cytokinestorm3 Drugeffects4 Hypoxia5 PreviousliverdamageBiochemicallabmarkersWhite bloodcellsGenomereleaseReplicationTranslationVirionassemblyViral proteinsMaturevirus release\uf0e9AaDO2MitochondrialproteinsHypoxicisquemicliverinjuryLIVER DAMAGELopinavirRitonavirRemdesivirChloroquineTocilizumabOxidativeimbalanceSteatosisACE2S proteinCytopathiceffect\uf0e9GMCSF\uf0e9IL6\uf0e9IL1β\uf0e9IL2\uf0e9IL8\uf0e9CCL2\uf0e9CCL3\uf0e9CCL5\uf0e9CXCL \uf0e9ALT\uf0e9AST\uf0eaAlbumin\uf0e9PCR\uf0e9LDH\uf0e9Ddimer\uf0e9Ferritin\uf0e9Bilirubin 0c" | Thyroid_Cancer |
"increasing number of studies have focused on the extragastrointestinal effects ofHelicobacter pylori H pylori including metabolic syndrome fatty liver and rheumatic and skin diseasesOsteoporosis is an asymptomatic disease that can eventually lead to fractures and has a significant impact on thequality of life of elderly individuals Sex is an influential factor that plays a crucial role in the development ofosteoporosis The aim of this study was to investigate the relationship between H pylori infection and osteoporosisand to identify potential influencing factorsMethods We conducted a crosssectional study of individuals older than years old who had undergone regularphysical examinations at the Beijing Shijitan Hospital Health Examination Center from July to October Weevaluated the associations of oste ia and osteoporosis with H pylori infection and related serum markers byusing multiple linear regression and logistic regression Then we analysed the correlation between sex andpotential serum biomarkersResults There were significant relationships between H pylori infection status and bone density in premenopausalfemales but not in males P according to Fishers exact test In females H pylori positivity OR P Body Mass Index BMI OR P and homocysteine HCY OR P wereassociated with osteoporosis Calcium had a trend but no statistically significant OR P relationshipwith osteoporosis Furthermore the waisttohip ratio OR P BMI OR P andtriglyceride levels OR P were significantly different by sex after adjusting for age as a confounderConclusion H pylori positivity BMI and HCY are associated with osteoporosis in premenopausal females Chronicinflammation may be involved in the relationship between H pylori and osteoporosisKeywords Female H Pylori infection Osteoporosis Premenopausal Chronic inflammationBackgroundHelicobacter pylori H pylori a gramnegative spiralshaped microaerophilic bacterium has been shown to bean important pathogen in gastrointestinal diseases []Approximately of the world population has been Correspondence maggie19950426163com3Department of Gastroenterology Beijing Shijitan Hospital Capital MedicalUniversity Beijing Beijing ChinaFull list of author information is available at the end of the affected by H pylori and approximately millionChinese individuals are affected by this disease It maycause chronic inflammation of the gastric mucosa whichmay lead to chronic atrophic gastritis peptic ulcer diseases and gastric cancer [ ] Moreover the latest reports have described the investigation ofthe extragastrointestinal effects of H pylori including metabolicsyndrome [] fatty liver [] and rheumatic and skin diseases [] These parenteral diseases associated with H The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cWang BMC Musculoskeletal Disorders Page of pylori infection seriously affect the patients general condition and cause a series of complicationsOsteoporosis is an asymptomatic disease characterizedby a decreased density of normally mineralized bone thatusually occurs in elderly persons It has been reportedthat approximately million men and million womenover the age of in the United States have been diagnosed with osteoporosis with an estimated millionsuffering from oste ia [] In the development ofosteoporosis there is often a long latent period beforethe appearance of the main clinical manifestation pathologic fractures Moreover the most prevalent sequelae ofosteoporosis are compression fractures of the vertebralbodies and fractures of the ribs proximal femurs humeri and distal radiuses which could have a significantimpact on the quality oflife of elderly individualsTherefore the prevention and early detection of osteoporosiselderlypopulationis particularlytheimportantforA majority of studies support the idea that at anygiven age women have a higher risk of fracture thanmen [] However men tend to have worse outcomesafter fracture than women they are twice as likely to dieafter a hip fracture than women [] Moreover sexrelated factors remain unclear Therefore exploring thedifferences in factors influencing osteoporosis in malesand females will be helpful to explore the pathogenesisof osteoporosis and early prevention of its occurrenceand developmentThere are some wellestablished risk factors for theemergence of osteoporosis such as age sex body massindex and alcohol consumption [] Recent shave focused on H pylori and osteoporosis Different researchers have proposed different theories including butnot limited to inflammation induced by H pylori infection [] and malabsorption of nutrients [ ] However there is still a lack of systematic data analyses onthe relationship between H pylori and osteoporosis Theassociation between osteoporosis and H pylori has beenstudied by many Japanese scientists and remains controversial In addition there are still some deficiencies inexisting research Few studies have focused on the correlation between H pylori and osteoporosis in Chinesepremenopausal females The sample sizes have been insufficient there is a lack of investigations on the influence of sex and there is a lack of sufficient serummarkers The aim of this study was to investigate the relationship between H pylori and osteoporosis and toidentify potential influencing factorsMethodsStudy populationBriefly men and women older than years old ienot including those aged years old who had regularphysical examinations at the Beijing Shijitan HospitalHealth Examination Center from July to October were included Our research used the following exclusion criteria for the data collection periods patientsusing the following drugs and having comorbidities thatmay cause secondary osteosis glucocorticoids thyroidparathyroid drugs psychotropic drugs anticonvulsantsselective estrogen receptor modulators SERMs vitaminD calcium and bisphosphonate patients who had ahistory of gastrectomy inflammatory bowel disease malignant diseases chronic kidney disease diabetes mellitushypohyperparathyroiddisorder acromegaly and rheumatoid arthritis includingcollagen disease Study participants who had been diagnosed with H pylori infection before or had potentiallyantiH pylori drugs in the past month were recruitedas wellhypohyperthyroidismWe also excluded postmenopausal women in thepresent study The criteria for determining menopausebased on the latest guidelines included any of the following prior bilateral oophorectomy age ¥ years old age years and amenorrhoeic for ormore months in the absence of chemotherapy tamoxifen or toremifene [] The female study participantswere not pregnant or lactatingData collectionAmong all the eligible individuals eligible study participants gave informed verbal consent and providedtheir basic informationincluding demographics agesex race smoking status and medicine use All ethicsapprovals were given by the Ethics Committee of BeijingShijitan Hospital affiliated with Capital Medical University and the study was performed in accordance withthe Declaration of Helsinki Blood measurements wereperformed with fresh serum obtained after a 12h fast tominimize the confounding effects of diurnal variation onhormone concentrations and included tests for glucosemetabolism liver function renal function lipid metabolism ions calcium iron tumour markers pepsinogenPG and progastrinreleasing peptide proGRP Anthropometric measurements including waist circumference cm blood pressure mmHg body weight kgand height cm were measured by trained nurses usinga standardized protocol Diastolic and systolic bloodpressure were measured in the morning Body massindex BMI was calculated by taking a persons weightin kilograms divided by their height in metres squaredThe waisttohip ratio WHR is measured as waist circumference divided by hip circumference H pylori infection status was measured by a []C breathing test onthe same day with an empty stomach Tumour markerswere measured using enzymelinked immunosorbentassay methods 0cWang BMC Musculoskeletal Disorders Page of Diagnosis of osteoporosisThe bone mineral density BMD of lumbar vertebrae L2 was measured by DXA using a DiscoveryDXA system Hologic Bedford Massachusetts The results provided BMD gcm2 and young adult meanbone mineral density The diagnosis of osteoporosis wasperformed in accordance with the World Healthanization diagnostic criteria from the World Healthanization WHO Collaborating Center for Metabolic Bone Diseases [] A value for BMD within onestandard deviation SD of the average BMD of normaladults was regarded as normal Oste ia is defined asa BMD that lies between and standard deviationsbelow the young adult mean value BMD more than SD below the young adult mean value was classified asosteoporosis []Statistical analysesWe used SPSS statistical software version for dataanalyses Continuous variables were reported as means ±standard deviations whereas categorical variables werepresented as percentages Study subjects were first classified into three groups according to BMD classificationnormal oste ia and osteoporosis The KolmogorovSmirnov test was used to verify whether the data fit anormal distribution and all continuous variables thatdid not conform to a normal distribution underwenttransformation for analysis Summary and grouping datafor baseline characteristics the laboratory examinationwere compared using a t test for continuous variablesand Fishers exact test for categorical variables in the Hpylori and H pylori groups Moreover we divided thestudy population by sex and used Fishers exact test toverify whether there were sex differences in the relationship between H pylori and osteoporosisMultivariateadjusted odds ratios ORs and confidence intervals CIs were calculated using logistic regression among the three subgroups To further analysethe relationship between H pylori infection status andosteoporosis we created a model using total cholesterolTC triglycerides TG uric acid UA BMI WHRlowdensity lipoprotein cholesterol LDLC Creactiveprotein CRP homocysteine HCY H pylori infectionstatus calcium Ca vitamin B12 and the BMD groupsthat analysed the different sexes separately The studypopulations highdensity lipoprotein cholesterol HDLC and glucose levels were all normal so we did not include them in the modelMeanwhile we analysed the differences in markers between males and females We separated the study population according to sex and further analysed the patientsbasic data in the same way as we analysed the baselinecharacteristics We further analysed the relationship between sex and markers to find sex differences in therelationship between H pylori infection and osteoporosis The markers included TC LDLC UA TG glucoseGLU CA724 CEA BMI SP systolic blood pressureBP diastolic blood pressure WHR HCY CRP vitaminB12 Ca and Ghb Other serum biomarkers were entered into the model as factors using their normal valueas the grouping criterion All the models were adjustedfor age as a confounder A twotailed Pvalue wasconsidered to be statistically significantResultthe participants areThe baseline characteristics ofshown in Table The mean age was ± years were male and were femaleAmong the study population had osteoporosis had oste ia and hadnormal BMDs The mean ages ofthe osteoporosisoste ia and normal groups were ± ± and ± respectively There wereno significant differences in age among the participantsin the osteoporosis oste ia and normal BMD groupsFifty percent of the males had normal BMDs ofthe males had oste ia and of the males hadosteoporosis In addition of the females had normal BMDs of the females had oste ia and of the females had osteoporosis There was no significant difference in sex distribution among these threegroupsAmong all the study participants had H pyloriinfections and did not have H pylori infectionsTable shows that CA724 t P was significantly different between the study participants withH pylori infection and those without H pylori infectionand there were no significant differences between the Hpylori infection status and the BMD status groupsIn Table we separate the study population accordingto sex We found that there was a significant relationshipbetween H pylori infection status and bone density infemales P but not in males P As shown in Table there was a significant association of H pylori positivity OR CI P BMIOR CI P and HCY OR CI P with osteoporosis inpremenopausalOR CI P and TC OR CI P had a trend but neither wassignificantly associated with osteoporosisfemales CaMeanwhile we analysed the differences in markers between males and females which are shown in Table We found that age P SP P BP P P WHR P Hb P BMI platelets P UA P TC P TG P HDLC P GLU P 0cWang BMC Musculoskeletal Disorders Page of Table Baseline Characteristics of the patients according to the H pylori infection statueFemaleMalenormaloste iaosteoporosisAgeSexBMDSP mmHgBP mmHgBMI kgm2WHRHbgLPlatelet109LCa mmolLUA umolLTC mmolLTG mmolLHDLC mmolLLDLC mmolLGLU mmolLCEA ngmlCA724UmlGhbIron umolLCRP mgLHCY umolLTotal ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± H pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± H pylori ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PvalueB12pmolLWHR Waisttohip ratio TC Total cholesterol Ghb Glycosylated hemoglobin TG Triglyceride UA Uric acid AST Aspartate aminotransferase ALT Alanineaminotransferase Ca Calcium Cre Creatinine DP Diastolic blood pressure Hb Hemoglobin HDLC Highdensity lipoprotein cholesterol LDLC Lowdensitylipoprotein cholesterol BMD Bone mineral density OR Odds ratio proGRP Progastrinreleasing peptide WHR WaisttoHip Ratio SP Systolic blood pressure PGPepsinogen GLU Glucose CRP Creactive protein HCY HOMOCYSTEINEBold indicates statistically significant values ± ± ± Table the relationship between the H pylori infection andthe BMD in different genderFemaleH pylori infection H pylori infection Normal BMDoste iaosteoporosisMaleH pylori infection H pylori infection Normal BMDoste iaosteoporosisBMD Bone mineral densityBold indicates statistically significant valuesPvaluePvalue CEA P Ghb P iron P HCY P and B12 P demonstrated significant differences between males and femalesThe relationship between sex and markers is shown inTable We found that WHR OR CI P TG OR CI P and BMI OR CI P were significantly different bysex adjusting for age as a confounderDiscussionOsteoporosis is an important health and societal burdenin elderly people not only in females but also in malesThere are numerous osteoporosisrelated fracture riskfactors including age sex race lifestyle and concomitant medical conditions [] In men osteoporosis isunderrecognized and undertreated Only a few men are 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and BMDOR95CIPvalueTC mmolLQ1365Q2521BMI kgm2Q1179Q2240UA umolLQ1187Q2358TG mmolLQ10Q2171LDLC mmolLQ10Q2313C13Q1without H pylorQ2with H pylorWHRQ1079Q2085HCYQ10 μmolLQ2 μmolLCRPQ1 mgLQ2 mgLCaQ1 mgLQ1 mgLB12Q10516pmolLQ2 pmolLAbbreviations as in Table OR Odds ratio C13 13C breathing test positiveBold indicates statistically significant valuesscreened for osteoporosis even after a fracture [] Thetreatment rate is much lower in males than in females[] Meanwhile more men than women die every yeardue to hip fractures [] Hence we also included menin the study population to determine the risk factors forosteoporosisSome studies about the influence of sex on osteoporosis remain controversial In our study there was a significant relationship between H pylori and osteoporosisin premenopausal females but not in males The reasonsfor the difference between males and females are asfollows First differences in clinical outcomes of osteoporosis in men and women may be rooted in the biologic properties of bone BarrettConnor E holds theview that there are sexspecific differences in the number of osteoprogenitor cells and in hormone responsesand regulation [ ] Second men have a greater bonesize trabecular BMD and bone area at the radius andtibia than women even after adjusting for weight andheight which may lead to a decrease in osteoporosis andfracture [] Third men undergo a slow decrease inBMD with increasing age while women experience aprofound period of rapid bone resorption especiallyafter entering menopause [] Last but not least somestudies support the idea that men are more likely to suffer from secondary diseasefor example rheumatoidarthritis alcoholism excessive smoking gonadal deficiencies and others [] which may lead to sustainablebone lossUnfortunately the relationship between osteoporosisand H pylori infection is still controversial Some studieshold the view that there is no difference between menand women in the relationship between H pylori andosteoporosis [ ] Some studies hold the view that Hpylori is related to osteoporosis only in women ShihChun Lin conducted a retrospective study including women and showed that H pylori is related to osteoporosis in females [] while others think that there is nocorrelation between them in females Daisuke Chindaconducted a study of healthy women and found thatH pylori is not a significant risk factor for oste ia[] In our study we analysed the relationship betweenH pylori infection and osteoporosis We found a significant relationship between H pylori infection status andbone density in premenopausal females but not in malesWe suspect this may be due to the difference in the aetiology of osteoporosis between males and females However we did not find any other studies on this and itrequires more systematic research for analysisAfter analysing the differences between males and females we found that there were significant differencesin BMI WHR and TG in the study population Thisstudy provides evidence for followup research on sexdifferences in the relationship between H pylori andosteoporosisMost studies hold the view that obesity is related toosteoporosis [] It is generally believed that obesitymay be a protective factor against bone loss and osteoporosis [] However the effect of obesity remains unclear On the one hand obesity has traditionally beenconsidered positive for bone because of the beneficial effect of mechanicalloading [] On the other handpeople hold the view that BMI may harm BMD Osteoblasts and adipocytes both stem from marrow mesenchymal stromal cells Osteoblasts and adipocytes are in a 0cWang BMC Musculoskeletal Disorders Page of Table Baseline Characteristics of the patients in different genderAgeSP mmHgBP mmHgBMI kgm2WHRHbgLPlatelet109LCa mmolLUA umolLTC mmolLTG mmolLHDLC mmolLLDLC mmolLGLU mmolLCEA ngmlCA724UmlGhbIron umolLCRP mgLHCY umolLTotal ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± B12pmolLAbbreviations as in Table OR Odds ratioBold indicates statistically significant values ± Female ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Male ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Pvaluecompetitive relationship and an increase in adipocyteswill inhibit osteoblasts [] In our study there was asignificant relationship between BMI and osteoporosisIncreased BMD levels in obese people may be associatedwith increased mechanical loading and strain this is acomplicated problem that cannot be generalizedIn our study we found that H pylori infection is associated with a decrease in bone density The possible reasons are as follows First H pylori infection may causesystemic inflammation and increase the production oftumour necrosis factorα interleukin1 and interleukin [] These cytokines are directly involved in the reduction of BMD We found that HCY is related toosteoporosis which supports this hypothesis Secondosteoporosis may be related to a decrease in vitamin B12levels [] Serin et als study examined patientswithout atrophy erosions or ulcers and they found thatthe histopathological scores for both antral and corpusH pylori density and inflammation were significantly inversely associated with serum vitamin B12 levels [] Inour study although we did not find a significant relationship between B12 and osteoporosis we still supportthe relevant theory The absence of our results may bedue to a lack of sufficient data and the influence of confounding factors Last but notleast most patientschronically infected with H pylori manifest pangastritiswith reduced acid secretion due to bacterial virulencefactors inflammatory cytokines and various degrees ofgastric atrophy [] Calcium is ionized in acidic conditions and absorbed in the small bowel Therefore in either hypochlorhydria or achlorhydric stomachs calciumabsorption is impaired [] Moreover the longtermuse of acid suppressants for example proton pump inhibitors may lead to osteoporosis or a decrease in BMDLimited experimental evidence indicates that PPI mayinfluence calcium absorption leading to compensatoryphysiologic responsesincluding secondary hyperparathyroidism which may cause an increase in the rate ofosteoclastic bone resorption [] The results showedthat calcium had a trend though it was not statisticallysignificant P with osteoporosis Our results donot support the theory that there is a correlation between Ca and osteoporosis but it may be that Helicobacter pyloriinfection may cause calcium absorptiondamage and affect BMD We did not analyse vitamin Dlevels which could affect both bone homeostasis and theinflammatory state [] Although H pyloriinfectioncausing a decrease in bone density is supported by mostresearchers the effect of early eradication therapy is stillinsufficient Replogle ML holds the view that early 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and genderWHRQ1079Q2085TC mmolLQ10Q2 LDLC mmolLQ10Q2313UA umolLQ1187Q2358TG mmolLQ10Q2171GLU mmolLQ10Q2611CA724UmlQ10Q2690CEA ngmlQ10Q2500BMI kgm2Q1179Q2240SP mmHgQ190Q2 BP mmHgQ160Q2 HCY μmolLQ10Q2 CRP mgLQ1 Q2 B12pmolLQ10Q2 Ca mgLQ1 OR95CI Pvalue 0cWang BMC Musculoskeletal Disorders Page of Table Multivariable analysis for different markers and gender ContinuedQ1 GhbQ10Q261OR95CIPvalueAbbreviations as in Table OR Odds ratioBold indicates statistically significant valuesMale and female have different normal value in UA and WHR UA 149416umolL in male89357umolL in female WHR in male in femaleeradication therapy may eliminate chronic inflammationfrom H pylori [] Some s have also reported animprovement in B12 levels after complete eradication[ ] which requires further investigationDespite its relevant findings our study had several limitations First because most patients cannot rememberthe time of HP infection accurately we were not able toobtain the time of HP infection so different infectiontimes may have had an impact on the results Secondwe did not collect vitamin D data the sample size of ourdata was not large enough and the study populationonly included participants from Beijing Shijitan Hospitalmeaning that there might have confounding factors because of differences in the distribution of hospital studypopulations Further largescale studies in the generalpopulation are needed to validate our results Third thestudy participants were all Chinese and the findingsmight not be generalizable to other ethnic populationsIn addition we only found some differences betweenmen and women but failed to further explore themConclusionsH pyloriis associated with osteoporosis in premenopausal females BMI and HCY are related to osteoporosis in premenopausal females Chronic inflammationmay be involved in the relationship between H pyloriand osteoporosisSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12891020035867Additional file AbbreviationsDXA Lumbar dualenergy xray absorptiometry ORs Odds ratiosCIs Confidence intervals H pylori Helicobacter pylori SERMs Selectiveestrogen receptor modulators proGRP Progastrinreleasing peptideBMI Body mass index WHR Waisttohip ratio BMD Bone mineral densityWHO World health anization SD Standard deviation TC Totalcholesterol TG Triglycerides UA Uric acid LDLC Lowdensity lipoproteincholesterol Ghb Glycosylated haemoglobin HDLC Highdensity lipoproteincholesterol GLU Glucose PG Pepsinogen CA Calcium DP Diastolic bloodpressure Hb Haemoglobin SP Systolic blood pressure C13 13C breathingtest positiveAcknowledgementsNot applicableAuthors contributionsLH has made substantial contributions to the design of the work SSJ hasprovided the data obtained the consent of participants and analyzed thedata preliminarily ZLC has made contributions to the collection of data andparticipated in the drafting of the DFX has analyzed and interpretedof the data WJW has drafted the manuscriptSH has helped to revise themanuscript All authors have read and approved the manuscriptFundingThe study was supported by a program from the Beijing City Health System High Levels of Health Technical Personnel Training Aid and the CapitalClinical Characteristic Applied Research Project NoZ181100001718120Funds are used for the data collection portionAvailability of data and materialsThe datasets analyzed during the current study are not publicly availablebecause it includes the study population personal information which isillegal to but are available from the corresponding author onreasonable requestEthics approval and consent to participateAll the ethics approval has been given by the ethics committee of Beijingshijitan hospital affiliated to capital medical university and have beenperformed in accordance with the Declaration of Helsinki We used theparticipants data by anonymous All involved study populations were fromthe previous physical examination group and part of the population are notin Beijing All the participants received the informed consent by email Weread informed consent to patients or their immediate family members bytelephone and inform them of the purpose and significance of the studyand obtain their oral consent which is approved by the ethics committeeConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests in this sectionAuthor details1Department of Gastroenterology Beijing Shijitan Hospital Capital MedicalUniversity No Tieyi Road Beijing China 2Department of Physicalexamination center Beijing Shijitan Hospital Capital Medical University No Tieyi Road Beijing China 3Department of Gastroenterology BeijingShijitan Hospital Capital Medical University Beijing Beijing ChinaReceived March Accepted August ReferencesGlaser DL Kaplan FS Osteoporosis Definition and clinical presentationSpine Phila Pa Suppl12s6s Malfertheiner P Megraud F O'Morain CA Atherton J Axon AT Bazzoli F Management of Helicobacter pylori infectionthe Maastricht IVFlorence consensus report Gut 0cWang BMC Musculoskeletal Disorders Page of Matsuhisa T Aftab H Observation of gastric mucosa in Bangladesh the Pan BL Huang CF Chuah SK Chiang JC Loke SS Relationship betweenHelicobacter pylori infection and bone mineral density a retrospectivecrosssectional study BMC Gastroenterol GÅogowskaSzelÄ
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g M Stolecki M KudÅa M Obesity andosteoporosisconnections between adipose tissue and bone Wiad Lek cz Fassio A Idolazzi L Rossini M Gatti D Adami G Giollo A The obesityparadox and osteoporosis Eat Weight Disord Tucker KL Hannan MT Qiao N Jacques PF Selhub J Cupples LA Lowplasma vitamin B12 is associated with lower BMD the Framinghamosteoporosis study J Bone Miner Res Smolka AJ Schubert ML Helicobacter pyloriinduced changes in gastric acidsecretion and upper gastrointestinal disease Curr Top Microbiol Immunol Wright MJ Proctor DD Insogna KL Kerstetter JE Proton pumpinhibitingdrugs calcium homeostasis and bone health Nutr Rev Bellavia D Costa V De Luca A Maglio M Pagani S Fini M Vitamin Dlevel between calciumphosphorus homeostasis and immune system newperspective in osteoporosis Curr Osteoporos Rep Shih HM Hsu TY Chen CY Lin CL Kao CH Chen CH et alAnalysis ofPatients with Helicobacter pylori Infection and the Subsequent Risk ofDeveloping Osteoporosis after Eradication Therapy A NationwidePopulationBased Cohort Study PLoS One 2016119e0162645 Published Sep httpsdoi101371journalpone0162645 Avcu N Avcu F Beyan C Ural AU Kaptan K Ozyurt M The relationshipbetween gastricoral helicobacter pylori and oral hygiene in patients withvitamin B12deficiency anemia Oral Surg Oral Med Oral Pathol Oral RadiolEndod Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationscountry with the lowest incidence of gastric cancer and Japan the countrywith the highest incidence Helicobacter Upala S Jaruvongvanich V Riangwiwat T Jaruvongvanich S Sanguankeo AAssociation between helicobacter pylori infection and metabolic syndromea systematic review and metaanalysis J Dig Dis Tang DM Kumar S The association between helicobacter pylori infectionand nonalcoholic fatty liver disease Curr Gastroenterol Rep Smyk DS Koutsoumpas AL Mytilinaiou MG Rigopoulou EI Sakkas LIBogdanos DP Helicobacter pylori and autoimmune disease cause orbystander World J Gastroenterol Office of the Surgeon G Reports of the Surgeon General Bone Health andOsteoporosis A Report of the Surgeon General Rockville MD Office of theSurgeon General US Nguyen ND Ahlb HG Center JR Eisman JA Nguyen TV Residual lifetimerisk of fractures in women and men J Bone Miner Res Haentjens P Magaziner J ColonEmeric CS Vanderschueren D Milisen KVelkeniers B Metaanalysis excess mortality after hip fracture amongolder women and men Ann Intern Med Yoshimura N Suzuki T Hosoi T Orimo H Epidemiology of hip fracture inJapan incidence and risk factors J Bone Miner Metab 200523Suppl78 Chung YH Gwak JS Hong SW Hyeon JH Lee CM Oh SW et alHelicobacter pylori a possible risk factor for bone health Korean J FamMed Recker RR Calcium absorption and achlorhydria N Engl J Med Serin E Gumurdulu Y Ozer B Kayaselcuk F Yilmaz U Kocak R Impact ofhelicobacter pylori on the development of vitamin B12 deficiency in theabsence of gastric atrophy Helicobacter Tyagi NK DhesyThind S Clinical practice guidelines in breast cancer CurrOncol 201825Suppl 1S151s60Kanis JA Melton LJ 3rd Christiansen C Johnston CC Khaltaev N Thediagnosis of osteoporosis J Bone Miner Res Dontas IA Yiannakopoulos CK Risk factors and prevention of osteoporosisrelated fractures J Musculoskelet Neuronal Interact Gennari L Bilezikian JP New and developing pharmacotherapy forosteoporosis in men Expert Opin Pharmacother Bougioukli S Îollia P Koromila T Varitimidis S Hantes M Karachalios T et alFailure in diagnosis and undertreatment of osteoporosis in elderly patientswith fragility fractures J Bone Miner Metab Alejandro P Constantinescu F A review of osteoporosis in the older adultan update Rheum Dis Clin N Am McMillan J FatehiSedeh S Sylvia VL Bingham V Zhong M Boyan BD et alSexspecific regulation of growth plate chondrocytes by estrogen is viamultiple MAP kinase signaling pathways Biochim Biophys Acta Lenart BA Neviaser AS Lyman S Chang CC EdoborOsula F Steele B | Thyroid_Cancer |
Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learnings groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center DiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patients life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images []Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined []Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the identityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called BONENAVI version The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed CADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing à convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the networks weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physicians scores Taking a closer lookat the results it can be concluded that networks performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physicians further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patients whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center Diagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatients cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution à pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center DiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patientscategory for example malignant_ and healthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value was assigned for malignant_ prefixes whereas the value for healthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Pythons randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po | Thyroid_Cancer |
" Despite several efforts the development of an effective vaccine for COVID19 may take a much longer timeTraditionalnatural medicine already experienced by humans could be an earlier solution Considering the researchteams experience in using nanoclays as highaffinity material for cancer metastasis melanoma treatment andbone regeneration we propose to use these nanoclays for the preventiontreatment of COVID19 Owing to highaffinity nanoclays would capture the viruses before the latter get engaged with human hACE2 In this studymolecularlevel simulations and modeling of the interaction of coronavirus spike and hACE2 proteins wereperformed with and without nanoclays The results showed a very high level of affinitycohesiveness among SARSCoV2 spike and nanoclays as compared to the one between the former and hACE2 We premise that these nanoclays since already being used as drug carriers could also be injected as claysalone medicine Recommendationshave also been provided for future in vitro and in vivo studiesBackgroundThe sudden emergence and rapid spread of novel coronavirus SARSCoV2 have significantly affected thehealth and lives of human beings in addition to criticallyaffecting the world economy SARSCoV2 spike S bindswith high affinity to human angiotensinconverting enzyme hACE2 and uses it as an entry receptor to invade target cells Fig 1a b [] The virussurface spikeprotein mediates coronavirus entry into host cellsSARSCoV2 spike protein contains a receptorbindingdomain RBD that recognizes explicitly as its receptorhACE2 [ ] The surface of hACE2 contains two virusbinding hotspots that are criticalfor SARSCoV2 Sbinding Several naturally selected mutations in SARSCoV2 RBD surround these hotspots and regulate theinfectivity pathogenesis and crossspecies and humantohuman transmissions of SARSCoV2 [ ]At present there are no clinically approved vaccinesor drugs that specifically target SARSCoV2 Followingthe real protocol of developing a vaccine it may takemuch longer time to come up with an effective vaccine Correspondence habibrehmankfupmedusa3Engineering Research International ERI Riyadh Saudi ArabiaFull list of author information is available at the end of the There is a lot of interest in the development of therapeutic antibodies against SARSCoV2 Despite many efthese antibodies have not yet beenforts howeverdiscovered [] exceptin a few trials [] One trialshowed the potent neutralization of SARSCoV2 bybinding to the RBD of its S glycoprotein [] In this trial[] antibody cocktails a mixture of different antibodiesis recommended due to the increased neutralization effect it has on SARSCoV2 However use of antibodiesin the past from convalescent patients of SARSCoV totreat SARSCoV infection has shown adverse reactionsin the patients such as AntibodyDependent Enhancement ADE causing increased viral infectivity and otherharmful immune responses [] Moreover based on theexperience with the vaccine development efforts forSARSCoV and MERS chances of the materialization ofthe efforts being made for SARSCoV2 seems quitethin Therefore naturaltraditional medicines that have ahistory of safe consumptioningestion by humans couldbe considered as one of the treatment options for SARSCoV2 Being a natural material and a history of humanuseconsumption we suggest highly charged nanoclays to be used as coronavirus blockers and inhibitorsof the spikemediated entry into the human cells The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 0cAbduljauwad Nanoscale Research Letters Page of Fig Schematics of the SARSCoV2 attack on human hACE2 and the subsequent immune system response a b RBD binding hACE2 withoutan interference c RBD complexed with the antibody at receptor attachment site hence competing with hACE2 d RBD complexed with RBD at asite other than where receptor attaches resulting in the alteration of RBD structure and interruption of lock and key binding of RBD to hACE2Nanoclays nanosized natural materials originatingfrom minerals of the sedimentary rocks have got avery high affinity to bacteria and viruses [] Due toisomorphous substitution in their molecular structurethese nanoclays exhibit charge deficiency on theirsurfaces This charge deficiency on their surfaces isneutralized by the water molecules and the dissolvedcations Fig The charged structure and large surface area of clay nanops give them an affinityfor charged entities as found on bacterial surfacesand bacterial toxins Their distinct biomedical properties include high absorption the ability to engulf microbes and no toxicity Each of the electrically activeclay minerals has its distinct morphology characteristics and interaction behavior The most studied biomedical application of nanoclays includes serving ascarriers and complexes for anticancer drugs such as5fluorouracil and trastuzumab [] They havetherefore been a potential alternate medicine for several diseases [] Clay nanops due to theiradhesive nature have also been used as carriers forsustainedrelease medicine [ ] Nanoclays havealso successfully been used to adsorb and treat bovinerotavirus and bovine coronavirus [] Researchers[] intercalated methotrexate MTX an anticanceragentinto the anionic clay to create a nanohybriddrug They used the coprecipitation and subsequenthydrothermal methodology to prepare this chemicallystructurally and morphologically welldefined twodimensional drugclay nanohybrid The researchers[] discovered that due to the biocompatibility andhigh loading capacity bentonite nanoclay could beused for the preparation of the drugdelivery vehiclesthey prepared doxorubicinbentoniteIn thisto form ananoclay complex DOXBent complexsustainedreleaseintradrugdeliverystudysystem for 0cAbduljauwad Nanoscale Research Letters Page of Fig a SEM image and b the corresponding molecular structure of Namontmorillonite showing the configuration isomorphous substitutioncharge deficiency and interlayer cations from []tumoral chemotherapy of melanoma As montmorillonite clay is recently being studied to be used as anadditive and drug carrier materialthese nanoclaycomposites appeal their use in various dosing formmainly for controlled release of the drug [] The researchers [] also discovered that nanoclays can beused into recent dual functional drug delivery systemsDDSs to have efficiency in the drug delivery and soreduce the toxicity of doxorubicin DOX that is being used for thyroid cancer treatment Using a libraryof singlesingle type photo cleavable amphiphilicJanus dendrimers researchers [] developed a selfassembling lightresponsive dendrimersomes vesicleplatform Similar to the nanoclays surface modifiedbioactive virusmimicking anic nanovesicles fromglycodendrimersomes have structural modificationsthat contribute to manifest SARSCoV2 and hostpathogenic molecular interactions that help the virusto escape from the human immune system []Through considerable previous research we developedbasic characterization and behavior modeling ofthecharged clay minerals [] and their applications in thecontrol of cancer metastasis [] in vitro and in vivo studies on melanoma treatment [] and the calcium depositionbone regeneration studies [] In a previous study bythe authors [] it was demonstrated that clay nanops had got a high affinity to the charged surfaces Thehigh attraction affinity of the nanoclays and the increasednonspecific adhesion attraction of the cancer cells makenanoclays favorable candidates to control cancer metastasis In that study we demonstrated the possible use of twocharged clay minerals to control the metastasis of thecancer cells Namontmorillonite SWy3 and palygorskitePFll Further to the findings of the authors previous research [] on the use of these nanoclays for the control ofcancer metastasis we also through in vitro and in vivostudies established that these nanoclays have inhibitory effects on melanoma cancer cells mainly on cell proliferationand viability [] In these previous studies in addition tolaboratory experiments molecularlevel simulations werealso performed on the nanoclay and cells interactionsThese simulations provided the assessment of the relativelevel of cohesivenessaffinity in the interactions with andwithout clay nanopsperceptionthroughthisestablishingBased on all the above experience of the authors onthe highaffinity potential of nanoclays we propose thatthe nanoclays could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2 This paper is a first steptowardsamolecularlevelsimulation and modeling approachBased on the results of the molecularlevel simulationsan outline of the recommendations for the next phasesof in vitro and in vivo research is also provided As thesenanoclays are also successfully being used as medicinecarriers we also premise that they can also be injectedingested as claysalone medicine and thus we haveproposed a tentative nanoclays administration methodology for this purposeMaterialsMoleculesSelection and Formulation of SARSCoV2 and hACE2Molecules of SARSCoV2 spike S and hACE2 were acquired from the protein data bank website RCSB [] 0cAbduljauwad Nanoscale Research Letters Page of The molecular models of SARSCoV2 spike S andhACE2 formulated in Materials Studio software [] arerespectively shown in Fig 3a b Before being subject tothe simulations these molecules were charged using thecharge equilibration method QEq of the softwareSelection and Formulation of Nanoclay CrystalliteNamontmorillonite one of the most active members ofthe smectite group of clay minerals was selected for thestudy Namontmorillonite is a layered phyllosilicate claysmectite Fig In the colloidal form the space between neighboring layers can contain free sodium calcium or magnesium cations that are electrostaticallyattracted to external negatively charged surfaces [] Inits dry powdered state Namontmorillonite exists asequidimensionalflakessheets with dimensions of approximately à à microns Fig 2a Thesenegative charges on their interlayer surfaces are balancedby the cations As colloids the interlayer cations get dissociated from the clay ps and associate themselveswith the other negatively charged surfaces These ps also have positively charged edges due to the presence of the broken bonds at their ends Morphology andfurther characteristics of these nanoclays are providedin Table while formulation of their crystallites in Materials Studio software are explained belowIn the software Namontmorillonite crystallites wereformulated based on fundamental properties such asCEC exchangeable cations and interlayer charges Table The size of the molecularcrystallite size was selectedbased on the results of the p size analysis usingthe dynamic light scattering DLS technique [] Thefinal form of clay crystallite created in the software istheseshown in Fig 3c Afterthe preparation ofcrystallites in the design mode of the software using theinherent properties these were charged using the chargeequilibration method QEq of the softwareMethodsMolecularLevel SimulationsThis part of the study consisted of the simulation andassessment of the interactions of the SARSCoV2 spikeS with clay crystallites and with hACE2 Although thesemodels may not be the complete replication of the actual in vitro conditions these have been incorporatedwith all the essentialinteractions and are quite wellsuited for the intended relative and comparative studyIn the software the sorption and simulations of theformulated configurations of SARSCoV2 S Namontmorillonite crystallites and hACE2 were carriedout using Monte Carlo MC and molecular mechanicsMM techniques The enhancement of affinity in all thesimulated configurations was assessed in terms of thecalculated cohesive energy density CEDCED beingconsidered as a measurement of the cohesiveness of themolecular system Due to the largesized computationsinvolved in the simulationsthese calculations werecarried out using the highperformance computing facilities HPC at KFUPM KSA The overall methodologyand the choice of particular methods and the simulationparameters were based on authors previous research[] while it is detailed in the subsequent sectionSARSCoV2 Spike S Interactions with hACE2 and ClayCrystallitesTo simulate the interaction of SARSCoV2 S with claycrystallites various numbers of the crystallites of Namontmorillonite clay were sorbed on SARSCoV2 Smodel For these sorption simulations the MetropolisFig Molecularlevel models of a SARSCoV2 spike b hACE2 and c Namontmorillonite crystallite formulated in Materials Studio software 0cAbduljauwad Nanoscale Research Letters Page of lnoitauccoFlnoitcaretnInoitcaretnInoitcaretnIninoisrepsidretawnoisrepsiDygrenelatotygreneWdvygreneBAlraopcilihpordyHaCaNretaWytiniffasγlaitnetopVmPZateZreyalretnIegrahclardeharteTlardehatcOlebaegnahcxEegrahcegrahcsnoitacqemgCECecafruSNaeragmslarenmirehtOliacmehClaumrofacilisOiSgMlAaCaNecruoSytnuoCASUYWkoorC][yWSyacletinolliromtnomaNfonoitaziretcarahcliacmehcdnalacisyhpfoyrammuSelbaT 0cAbduljauwad Nanoscale Research Letters Page of Monte Carlo method was selected in the Sorption module of the software In each sorption step clay crystallitesoccupy spaces around the spike S model to lower theoverall energy of the complex The required number ofcrystallites were sorbed in a maximum of stepsand then the energy of the system was minimized usingthe Forcite module of the software based on the MDprinciples The similar sorption process was repeated forthe interaction modeling of the SARSCoV2 spike molecule with hACE2 In this process hACE2 moleculeswere sorbed around the RBD of the spike S of SARSCoV2 After the completion of the sorption process theenergy of the formulation was minimized using MDbased module of the softwareThe Forcite module ofthe software incorporatingNPT constant number of ps pressure andtemperature ensemble was used for MD simulationswith a modified universal force field [] The simulations were run for to ps with an interval of 05fs ortill a constant volume is obtained A Berendsen thermostat with a decay constant of ps was used to controlthe temperature during the simulation During the MDsimulations the assumed temperature was kept constantat K °C with an atmospheric pressure kPaA Berendsen barostat with a decay constant of pswas used to control the pressure of the system TheBerendsen methodology was considered as the most appropriate for the single crystallites after several trials involving other thermostats and barostats available in thesoftware In the Monte Carlo method the parametersfor the ratios of exchange conformer rotate translateand regrow were selected as and respectively with the corresponding probabilities as and Amplitudes adapted for rotationand translation were ° and respectivelyCohesive Energy Density CED MeasurementIn this study the assessment of the affinitybindinglevelin the SARSCoV2clay crystallites and SARSCoV2hACE2 complexes was measured through thechanges in the CED After the sorption of clay crystallites and the subsequent performance of moleculardynamics of each of the configurations the CED wasdetermined using the cohesive energy density optionof the Forcite module of the software The authorshave experienced that the CED concept consisting ofthe total van der Waals and electrostatic CEDs canquite closely explain the various molecularlevel processes and interactions and simulate the extent of affinitybinding created among the simulated complexes[] Quantitatively CED is defined as the amountof energy needed for the transition of mol of material from the liquid to the gaseous phase It is also ameasureofaffinityattractivenessthe mutualofmolecules and is expressed both as electrostatic andvan der Waalsan NPTensembleaveraged overforcesIn the Forcite module van der Waals energies wereevaluated using atombased cutoffsIn this methodnonbond interactions are simply calculated to a cutoffdistance and interactions beyond this distance are ignored To avoid the discontinuities caused by direct cutoffs most simulations use a switching function to turnoff nonbond interactions over a range of distancessmoothly An effective potential is created by multiplyingthe actual potential by the smoothing function Thechoice of the function in the intermediate range is crucial and should be continuously differentiable in this region so that forces can be calculated In this study acubic spline smoothing function was used with a splinewidth of and a cutoff distance of Results and DiscussionsThe final configuration of the SARSCoV2 ShACE2complex is shown in Fig 4a while the complexes between SARSCoV2 spike and different numbers of clayNamontmorillonite crystallites are respectively shownin Fig 4b c For comparison purposes total CEDs ofvarious proportionsnumbers of the clay crystallites onthe SARSCoV2 spike and the interaction of the laterwith hACE2 are plotted in Fig Based on our experience we have hypothesized thatnanoclays due to their high adhesive properties couldalso act as SARSCoV2 inhibitors They can do it bystrongly associating with the spike S present on SARSCoV2 The results obtained from the molecularlevelsimulations of the interactions indicate that due to veryhigh CED between SARSCoV2 and the nanoclays ascompared to the former and hACE2 Fig they couldinhibit SARSCoV2 from getting engaged with hACE2Moreover it could also be concluded from Fig thatthe extent of inhibition due to nanoclays is increased inquantity dosagedependent wayNanoclay Interactions with SARSCoV2 Spike SAuthors in their earlier research have demonstrated therole of nanoclays in promoting adhesion among thecancer cells and their microenvironment and hence controlling metastasis [] Adhesion measurements of mix of Namontmorillonite and palygorskite showedan increase in adhesion by among cancer cells andthe extracellular matrix proteins Fig 6a A corresponding SEM of the nanoclays binding the Raji cells and thefibronectin proteins is shown in Fig 6b Sample imagingwas performed in SEM mode in an FEI ESEMFEG XL atthe Miller School of Medicine University ofMiami Florida Authors also discovered in their previousresearch that electrostatic van der Waals and ZP 0cAbduljauwad Nanoscale Research Letters Page of Fig Molecularlevel simulation results in Materials Studio Software a SARSCoV2 S and hACE2 CED Jcm3 b SARSCoV2 S modelinteracting with twelve crystallites of Namontmorillonite CED Jcm3 and c SARSCoV2 S model interacting with twentyfour crystallites ofNamontmorillonite CED Jcm3obtained using Sorption technique implemented in the softwareattractions seem to be dominating in the adhesion processes [] We conclude that the same mechanismswould have also facilitated the binding of the adhesivesurfaces of the nanoclays to the spike of SARSCOV2Fig ZP is a measure of the dispersion or flocculationtendency in the colloidal form including the interactions 0cAbduljauwad Nanoscale Research Letters Page of Fig Variation of cohesive energy density CED for SARSCoV2 ShACE2 and the complexes of the former with different numbers ofNamontmorillonite crystalliteswith the other constituents present in the suspensionmedium As a general rule a zeta potential greater than mV either positive or negative indicates dispersiontendency while a zeta potential of less than mV generally results in agglomeration Higher dispersion tendencies ZP of the clay nanops used in the study to mV lead to higher dispersion tendency andhence in the generation of higher surface area amplifyingthe interactions with the SARSCoV2 spike Althoughbased on their ZP Namontmorillonite nanopshave hydrophilic nature they in the presence of saltsalso promote secondary adhesion mechanisms betweenhydrophobic and hydrophilic surfaces [] It should alsobe noted that these clay nanops have high dispersion tendency due to their hydrophilic nature and relatively higher repulsive acidbase AB interactions Table High dispersion in turn results in the generation ofhigh surface area for increasing the attractive interactions Higher surface areas promote larger attractionsdue to the van der Waal attractions and the electrostaticforces among oppositely charged surfaces Besides although of relatively lesser degree positively chargededges of Namontmorillonite ps also get electrically attracted to the spike SThe results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites Fig also confirm the above interaction behaviors It has been observed that the sorption of the claynanops results in the formation of closely interacting strong van der Waals attraction fields These van derWaals attraction fields create higher CED of the claySARSCoV2 configuration Substantial increase in totalCED after the addition of clay crystallites Fig is alsoa testimony of a very high affinity of SARSCoV2 withthese ps as compared to the affinity of the formerwith hACE2systemagglutinationNanoclays as PseudoantibodiesBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays we premise that nanoclays could be mimicked asantibodies and can thus attract and engulf coronavirusesbefore they get engaged with human hACE2 Antibodiesare glycoproteins synthesized by plasma cells as part ofthe adaptive immune response to assist in the clearanceof infection from the body Antibodies aid in infectionclearance in multiple ways such as opsonization of pathogens to facilitate phagocytosis activation of the complementandneutralization of viruses and toxins When bound to theviral surface proteins antibodies prevent the entry of theviruses into the cell by preventing the attachment of viruses to their target receptor on the cell Antibody binding can occur at different sites on the surface proteinleading to various mechanisms that cause the same effect In the case of SARSCoV2 two viral neutralizationmechanisms by antibodies have been observed [ ]and shown in Fig 1c d One of the mechanisms involvesdirect binding of antibodies to the attachment site of theSARSCoV2RBD resulting in the antibody competingwith the target receptor hACE2 Another mechanism involves the binding of antibodies to the other sites onRBD without any competition with the target receptorThe latter is shown to be involved in neutralization byof microbes 0cAbduljauwad Nanoscale Research Letters Page of Fig a Summary of adhesion force measurements among RajiRajiFN assembly using AFM before and after treatment with various proportionsof Namontmorillonite and palygorskite clay nanops [] Error bars represent the variations in the trials b SEM image of the binding of Rajicells and Fibronectin proteins produced by nanoclaysthe most potent Monoclonal Antibody mAb discoveredin the study [ ] Analogous to the antibodies interaction with SARSCoV2 RBD inhibiting the latter toengage with hACE2 a similar molecularlevel model isprepared for nanoclays resulting in a similar inhibitionof the coronaviruses and shown in Fig Owing to theirvery high affinity nanoclays would get attracted tospikes of SARSCoV2 and thus restrict engagement ofRBDs of these spikes with hACE2Proposed Nanoclay Administration MethodologyClay use as drug carriers has been tested multiple timesyielding promising results of little to no cytotoxicity tocells of the human body Kaolinite clay mineral wastested for use in a potential drug delivery system andwas shown to have high biocompatibility and very lowas[]negligiblecytotoxicity [] Poly DLlactidecoglycolidemontmorillonite nanop cytotoxicity in vitro was alsodemonstratedPalygorskitepolyethyleneiminefluorescein isothiocyanate nanocomposites also exhibited almost no cytotoxicity in vitro[] Authors have also experienced injecting nanoclayssubcutaneously for the treatment of melanoma duringin vivo studies [] Based on the use of clay as a cancerdrug carrier and in other sustainedrelease medicine[] we propose that nanoclays may be injected asclayalone medicine subject to the verification in vivoand clinical trialsAlthough nanoclays are nonbiodegradable a comprehensive understanding of the design of the similar inanic nanops with their metabolic performancein the body carried out in the study [] could also 0cAbduljauwad Nanoscale Research Letters Page of Fig Three possible mechanisms of interactions of montmorillonite nanoclay with the SARSCoV2 spike S Electrostatic attraction amongpositively charged nanop edges and NaCa ions with negatively charged virus surfaces Van der Waals attractions ZPelectrostatic interactionscategorize these nanoclays as human body clearable inanic agentsConclusions and RecommendationsBased on all the current and past research by the authors establishing the highaffinity potential of nanoclays these could be mimicked as antibodies and canthus attract and engulf coronaviruses before they get engaged with human hACE2The results of the molecularlevel simulations for theinteraction of SARSCoV2 spike S with the clay crystallites result in the formation of closely interacting strongvan der Waals attraction fields These van der Waals attraction fields create higher CED of the claySARSCoV configuration Substantial increase in total CED afterFig Interaction mechanism of nanoclay ps with SARSCoV2 spike S inhibiting the interaction of the later with hACE2 0cAbduljauwad Nanoscale Research Letters Page of addition of clay crystallites is also a testimony of a veryhigh affinity of SARSCoV2 with these ps as compared to the affinity of the former with hACE2We propose to continue the research by carrying outin vitro interaction studies between SARSCoV2 anddifferent percentage of nanoclays Based on theoptimum dose of nanoclay developed in the in vitrophase we suggest to carry out in vivo studies on the animals The animal study should be carried out both withand without nanoclay to finalize the nanoclay dose andshould lay the foundation for the clinical trialsAcknowledgementsThe authors highly acknowledge KFUPM for providing highperformancecomputing research facilitiesAuthors ContributionsAll the authors equally participated at all the research levels The authorsread and approved the final manuscriptFundingNo fundingAvailability of Data and MaterialsAll data generated or analyzed during this study are included in thispublished Ethics Approval and Consent to ParticipateNot applicableConsent for PublicationNot applicableCompeting InterestsThe authors declare that they have no competing interestsAuthor details1Civil Environmental Engineering department King Fahd University ofPetroleum Minerals KFUPM Dhahran Saudi Arabia 2Royal College ofSurgeons in Ireland RCSI Bahrain campus Busaiteen Bahrain 3Engineering Research International ERI Riyadh Saudi ArabiaReceived July Accepted August ReferencesEwen Callaway and Nik Spencer The race for coronavirus vaccines agraphical guide eight ways in which scientists hope to provide immunityto SARSCoV2 News Feature Nature vol April Li F Li W H Farzan M Harrison S C Structure of SARS coronavirusspike receptorbinding domain complexed with receptor Science httpsdoi101126science1116480 Li WH Angiotensinconverting enzyme is a functional receptorfor the SARS coronavirus Nature httpsdoi101038nature02145Li F Structural analysis of major species barriers between humansand palm civets for severe acute respiratory syndrome coronavirusinfections J Virol httpsdoi101128jvi0044208 Wu KL Peng GQ Wilken M Geraghty RJ Li F Mechanisms of hostreceptor adaptation by severe acute respiratory syndrome coronavirus JBiol Chem httpsdoi101074jbcM111325803 Wang C Li W Drabek D Okba NMA van Haperen R Osterhaus ADME A human monoclonal antibody blocking SARSCoV2 infection NatCommun Jiang S Hillyer C Du L Neutralizing antibodies against SARSCoV2and other human coronaviruses Trends Immunol Pinto D Park YJ Beltramello M Walls AC Tortorici MA Bianchi S Crossneutralization of SARSCoV2 by a human monoclonal SARSCoV antibody Nature da Rocha Dias S Salmonson T van ZwietenBoot B Jonsson B Marchetti SSchellens JH Pignatti F The European Medicines Agency review ofvemurafenib Zelboraf® for the treatment of adult patients with BRAF V600mutationpositive unresectable or metastatic melanoma summary of thescientific assessment of the Committee for Medicinal Products for HumanUse Eur J Cancer Sahel N Abduljauwad and HabiburRehman Ahmed Enhancing cancer celladhesion with clay nanops for countering metastasis Nature ScientificReports April httpsdoi101038s4159801942498y Zhang Y Long M Huang P Yang H Chang S Hu Y Mao L Intercalated 2D nanoclay for emerging drug delivery in cancer therapyNano Res Chianelli R R Das S US Patent No Washington DC US Patent and Trademark Office Han S Liu F Wu J Zhang Y Xie Y Wu T Y Targeting of fluorescentpalygorskite polyethyleneimine nanocomposite to cancer cells Appl ClaySci Sun B Ranganathan B Feng SS Multifunctional poly D Llactidecoglycolide montmorillonite PLGAMMT nanops decorated byTrastuzumab for targeted chemotherapy of breast cancer BiomaterialsLin FH Lee YH Jian CH Wong JM Shieh MJ Wang CY A study ofpurified montmorillonite intercalated with 5fluorouracil as drug carrierBiomaterials Bothiraja C Thorat UH Pawar AP Shaikh KS Chitosan coated layeredclay montmorillonite nanocomposites modulate oral delivery of paclitaxel incolonic cancer Mater Technol 29sup3B120B126Kevadiya BD Thumbar RP Rajput MM Rajkumar S Brambhatt H Joshi GVBajaj HC Montmorillonitepolyεcaprolactone composites asversatile layered material reservoirs for anticancer drug and controlledrelease property Eur J Pharm Sci Guo MY Wang AF Muhammad F Qi WX Ren H Guo YJ Zhu GS Halloysite nanotubes a multifunctional nanovehicle for anticancer drugdelivery Chin J Chem MartÃnez C D Cationic clays upon cancer therapy Virtual MultidisciplinaryConference QUAESTI Konta J Clay and man clay raw materials in the service of man ApplClay Sci Murray HH Traditional and new applications for kaolin smectite andpalygorskite a general overview Appl Clay Sci Volzone C Retention of pollutant gases comparison between clayminerals and their modified products Appl Clay Sci Dong Y Feng SS Poly dllactidecoglycolidemontmorillonitenanops for oral delivery of anticancer drugs Biomaterials Clarka KJ Sarrb AB Grantb PG Phillipsb TD Woodea GN In vitrostudies on the use of clay clay minerals and charcoal to adsorb bovinerotavirus and bovine coronavirus Vet Microbiol Choi G Huiyan P Alothman Z Vinu A Yun C Choy J Anionic clay asthe drug delivery vehicle tumor targeting function of layered doublehydroxidemethotrexate nanohybrid in C33A orthotopic cervical cancermodel International Journal of nanomedicine DOI httpsdoi102147IJNS95611 Hosseini F Hosseini F Jafari S M and Taheri A Bentonite nanoclaybaseddrugdelivery systems for treating melanoma Clay Minerals DOI httpsdoi101180clm201842018 Inamuddin Asiri A M and Mohammad Ali Applications of nanocompositematerials in drug delivery DOI httpsdoi101016C20160050751 Avolume in Woodhead Publishing Series in Biomaterials Zhang Y Long M Huang P Yang H Chang S Hu Y Tang A and MaoL Emerging integrated nanoclayfacilitated drug delivery system forpapillary thyroid cancer therapy doi 101038srep33335 Sci Rep Li S Xia B Javed B Hasley W D MelendezDavila A Liu M Kerzner MAgarwal S Xiao Q Torre P Bermudez J G Rahimi K Kostina N YMöller M RodriguezEmmenegger C Klein M Percec V and Good M CDirect visualization of vesicle disassembly and reassembly usingphotocleavable dendrimers elucidates cargo release mechanisms ACS 0cAbduljauwad Nanoscale Resear | Thyroid_Cancer |
lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups years years years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the students t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAgeyearsDistributionno years years years� yearsMaleno Occupationno n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [][ CI]n ± n ± [][ CI] [] []Other type of exposure Health professionalOther health workersSmoking exsmokersmokernototal no Temperature at admission ËC Patients with fever �ËCnototal no Time from symptom onset tomedical visitdays¡Symptomsno ¶ ± ± ± ± ± ± NANANA [] [] [] [] ± ± ± [] [] ± [] NANANA []CoughGeneral malaiseFatigue [] [] [] [] [] []Myalgia or arthralgia [] []DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examinationnototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] NA []Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [] PAdjusted OR[ CI] []In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex Temperature distribution was ËC ËC ËC and ËC ¡ In patients data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbiditiesno Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [] [] [] [] [] [] [] [] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] []In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xraynototalno Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scannototal no ¡ [] [] [] [] [] []Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parametersnototalno [] [] [] []Leukocytes mm3 [] []Lymphocytes mm3Platelets mm3 [] [] [] []Haemoglobin gdl [] []Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [] [] [] [] [] [] [] [] [] [] [] []Creatine kinase Ulitre [] []Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [] [] [] [] [] [] [] []In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complicationsno Any complicationPneumonia NA [] [] []Adult respiratory distress [ []syndromeRenal failurePulmonary thromboembolismTreatmentsno ¡HydroxychloroquineAzithromycinLopinavirRitonavir [] [] []NA] [] [] [] [] [] []Oxygen therapy [] []Intravenous antibiotics [] []GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infectionno [] [] [] [] [] [] [] [] [] []Any cohabitant [] []Any work colleague [] []Any contact person in other [] []settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ ] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown causeChina wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh | Thyroid_Cancer |
"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing pr sity score matching In the two groups gastric neoplasms containing NEC components were dividedinto pure NEC and mixed tumors with less than GHMiNEN between and GHMiNEN andmore than GHMiNEN neuroendocrine carcinoma components OS was compared between thesegroups and the gastric adenocarcinoma groupResults The OS of gastric neoplasms containing neuroendocrine NEC components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or Cox multivariable regressionanalysis suggested that tumor category neoplasms containing NEC components or gastric adenocarcinoma wasan independent risk factor for prognosis Interestingly among patients receiving neoadjuvant therapy thedifference was not significantContinued on next page Correspondence buzhaodecjcrcn jijiafuhscpkueducn Jiahui Chen Anqiang Wang and Ke Ji contributed equally to this workDepartment of Gastrointestinal Surgery Key Laboratory of Carcinogenesisand Translational Research Ministry of Education Peking University CancerHospital Institute No Fucheng Road Haidian District Beijing China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Cancer Page of Continued from previous pageConclusions Gastric neoplasms containing any proportion of NEC components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmsKeywords Neuroendocrine carcinoma Gastric adenocarcinoma Overall survivalBackgroundGastric neoplasms containing neuroendocrine carcinomaNEC components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure NECs and mixed tumorscontaining NEC components Every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing NEC components account for approximately of thesecases [ ] Given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []According to the World Health anizationWHO digestive neuroendocrine tumor classificationneuroendocrine neoplasm NEN can be divided intothree categories based on Ki67 levels and mitotic counts HPF Grade G1 Ki67 mitoses Grade G2 Ki67 mitoses Grade G3Ki67 mitoses [] Meanwhile the AmericanJoint Committee on Cancer AJCC defines highly differentiated NEN as a neuroendocrine tumor NET and thepoorly differentiated NEN as a neuroendocrine carcinoma NEC based on the degree of tumor cell differentiation Generally G1 G2 and rare welldifferentiated G3NENs belong to the NETs while poorly differentiatedG3 NENs belong to NECs[ ] Gastric mixedneuroendocrinenonneuroendocrineneoplasm GMiNEN is a special type of gastric NEN that is definedas containing more than of both neuroendocrineand nonneuroendocrine components [] accountingfor approximately of all GNENs and of gastricneuroendocrine carcinomas GNECs [] For thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition Consideringthe heterogeneity ofMiNEN and the malignancy degree of the different components in the tumor La Rosa [ ] proposeddividing MiNEN into three categories highgradeintermediategrade and lowgrade Highgrade MiNENconsists of NEC and carcinomaadenoma intermediategrade MiMEN consists of NET and carcinoma and lowgrade MiNEN consists of NET and adenoma Thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm GHMiNEN was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandGenerally the prognosis of mixed tumors is largely determined by the most malignant component Kim et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components All of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis Therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone We sought tofind studies on the overall survival OS comparison between GHMiNEN and gastric adenocarcinoma butfailed Thus we think that a study of the comparison ofthe OS of GHMiNEN and gastric adenocarcinoma willprovide a valuable supplement to current research on GHMiNEN To overcome the bias caused by the differences between the covariates in the comparison we usedpr sity score matching PSM to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliableMethodsPatient selectionWe retrospectively collected patients diagnosed withgastric NENs and underwent radical resection at PekingUniversity Cancer Hospital Beijing from January to December The inclusion criteria were as follows pathologically confirmed pure NEC or tumorcontaining NEC components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup Patients diagnosedwith cM1 or cT4b before surgery or died from perioperative complications were excluded from the study 0cChen BMC Cancer Page of Patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for PSM analysesperformed The chisquared test and MannWhitney Utest were used to further verify the matching resultsFollowupWe followed the patients at least twice a year Serumtumor markers test gastroscope and computed tomography CT scans were used to reexamine patients aftersurgery Depending on the patients status Magneticresonance imaging MRI and Positron emission tomography computed tomography PETCT were alsoconsidered For patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationDiagnosis and classificationWe reevaluated the diagnosis and classification of GHMiNEN Mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as GHMiNEN and GHMiNENrespectively Atumor consisting of NEC is defined as pure NECAll neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the WHO classification of tumors[] Neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin Syn chromograninA CgA and neuro cell adhesion molecule CD56 orNCAM The tumor staging described in the study wasbased on the AJCC 8th Edition TNM Staging Guidelines[] All possible disagreements were discussed in ourstudy groupDefinition of variables and groupsIn this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery Patients inthe surgery group were assessed by the pTNM stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the ypTNM staging system OSrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egPr sity score matchingTo accurately compare the prognosis of GHMiNENand gastric adenocarcinoma we employed PSM to balance the differences between the two groups PSM wasperformed through the Pamatching plugin in SPSS software Logistic regression models were used toestimate pr sity scores based on gender age tumorlocation tumor size and pathological staging Given a caliper width nearest neighbor matching wasStatistical analysisAll statistical analyses were performed using SPSS statisticalsoftware IBM United States The chisquared test and MannWhitney U test were used forstatistical analysis of categorical variables and continuous variables respectively KaplanMeier method wasused for the comparison of OS The logrank test wasused to compare survival rates Multivariable Cox proportional hazards models were used to identify predictors of survival outcome P was regarded as thethreshold of significanceResultsPatient selection and PSM resultsBetween and among the patients treated atthe Gastrointestinal Cancer Center of Peking UniversityCancer Hospital a total of patients with gastric neoplasms containing NEC components met the inclusioncriteria for the study including cases of pure NECand cases of mixedtype Of these patients a total of patients received neoadjuvant therapy NEC GHMiNEN GHMiNEN GHMiNEN while the remaining patients receivedsurgery directly NEC GHMiNEN GHMiNEN GHMiNEN There were aninsufficient number of patients in group GHMiNEN group to conduct effective statistical analysisso we combined the GHMiNEN group with theNEC group for further analysis We also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery Among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly Fig Immunohistochemical specificity markers were utilizedto identify the neuroendocrine components Fig 2aSyn was expressed in almost all neoplasms containingNEC components while the positive rates ofCgA and CD56 were much lower and respectively No significant difference in the positiverate of Syn and CgA was observed between pure NEC GHMiNEN GHMiNEN and GHMiNENFig 2b c only the positive rate of CD56 was found tobe higher in the pure NEC group than that in the GHMiNEN group Fig 2dTherefore priorto OS comparison PSM wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cChen BMC Cancer Page of Fig Flow chart of patient enrolmentComparison of OS between all patients with NECcomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupBefore PSM we compared the survival curves between all patients with NEC components and patientswith gastric adenocarcinoma by the KaplanMeiermethod Fig Apparently patients with NEC components had a poorer OS than those with gastricadenocarcinoma Fig 3a p in the surgicalgroup In contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy Fig 3b p According to the proportion of NEC components patients were classifiedinto pure NEC GHMiNEN GHMiNENand GHMiNEN The OS was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison Fig 3c dFig Illustrations of immunohistochemical staining patterns in gastric neoplasms containing NEC components a An overview of the expressionof Syn CgA and CD56 in tumors containing NEC components b Syn expression in different NEC component groups c CgA expression indifferent NEC component groups d CD56 expression in different NEC component groups CD56 neuro cell adhesion molecule CgAchromogranin A NEC neuroendocrine carcinoma Syn synaptophysin Pvalue 0cChen BMC Cancer Page of Fig See legend on next page 0cChen BMC Cancer Page of See figure on previous pageFig Comparison of OS between gastric neoplasms containing NEC components and gastric adenocarcinoma a OS comparison betweengastric neoplasms containing NEC components and gastric adenocarcinoma before PSM in the surgical group b OS comparison between gastricneoplasms containing NEC components and gastric adenocarcinoma before PSM in the neoadjuvant group c OS comparison between differentNEC content groups pure NEC GHMiNEN GHMiNEN and GHMiNEN and gastric adenocarcinoma before PSM in the surgicalgroup d OS comparison between the different NEC content groups and gastric adenocarcinoma before PSM in the neoadjuvant group e OScomparison for patients in the surgical group after PSM f OS comparison for patients in the neoadjuvant group after PSM NEC neuroendocrinecarcinoma OS overall survival PSM pr sity score matchingBefore PSM significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group Table Table To balance the clinicopathological differences between the twogroups PSM was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups The detailedclinicopathological characteristics before and after PSMare shown in Table and Table As a result patients with NEC components and patients with gastric adenocarcinoma were matchedin the surgical group Table Patients with NEC components also had a poorer OS than those with gastricadenocarcinoma Fig 3e p Multivariable analysis showed that adjuvant therapy tumor category andTNM stage werefactorsTable independent prognosticTo investigate whether neoadjuvant therapy had an effect on OS patients with NEC components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group Table Interestingly KaplanMeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in OS between the two groups Fig 3f p Comparison of OS between patients with differentproportions of NEC components and patients with gastricadenocarcinomaTo investigate whether the level of NEC componentshad an effect on OS in the surgical group GHMiNEN GHMiNEN pure NEC and pure NEC plus GHMiNEN were compared with gastric adenocarcinoma after PSM The results showed that even thegroup with the lowest proportion of NEC componentsthe GHMiNEN group had a poorer OS thanadenocarcinoma Fig 4a P As expected theGHMiNEN pure NEC and pure NEC plus GHMiNEN groups each with relatively high proportionsof NEC components had worse OS than the gastricadenocarcinoma group Fig 4bd P Detailed clinical information after matching isshown in Additional file Tables S1S4PSM was also performed in the neoadjuvant group Incontrast to the results of the surgery group in the pureNEC group containing the highest proportion ofNEC componentstill no significantdifference in OS from gastric adenocarcinoma Fig5d The other three groups with lower NEC contentwere also notfrom gastricadenocarcinoma in terms of OS Fig 5ac Detailedclinicopathologicaland afterPSM are shown in Additional file Tables S5S8characteristics beforethere wassignificantly differentDiscussionAmong gastric neuroendocrine neoplasms the tumorcontaining NEC components is a special type includingpure NEC and mixed tumor containing NEC components The incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated GNENs [ ]received neoadjuvantIn previous study Kim found that in patientschemotherapywho had notprogressionfree survivalPFS of pure GNEC waspoorer than that of gastric adenocarcinoma while thePFS of mixedtype tumors was not significantly differentIn Kimsfrom that of gastric adenocarcinoma []study the mixed type was defined as NET mixed withgastric cancer rather than NEC NET is much less malignant than NEC [ ] This may be the reason whythere was no significant difference in OS between mixedtype and gastric adenocarcinomas In addition mixed tumors with less than or more than of NEC components were not included in that study which webelieve was a deficit of the study PFS is an important indicator for evaluating prognosis in many cases it can reflect the trend of OS Based on Kims research resultswe regarded tumors containing NEC components as awhole and found that the OS of these tumors was poorerthan that of adenocarcinoma in the surgical group Inthe comparison of OS between mixed tumors with different proportions of NEC components and gastricadenocarcinoma the results for pure NEC cases wassimilar to Kims While the OS of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in Kims study Cox multivariable regression analysis showed thattumor categoryneoplasm with NEC component or adenocarcinoma 0cChen BMC Cancer Page of Table Comparison of clinicopathological characteristics before and after PSM in surgical groupPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n P valueMatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomy ± ± Proximal gastrectomy Surgical procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Table Comparison of clinicopathological characteristics before and after PSM in neoadjuvant groupMatched comparisonPatient CharacteristicsUnmatched comparisonPatients with NECcomponents n Age year mean ± SDGender malefemaleBMI mean ± SDAdjuvant therapyYesNoTumor locationUpper thirdMiddle thirdLower thirdEntireTumor size cm¥ cmType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedure LaparoscopicT stageT0T1T2T3T4N stageN0N1N2N3M stageM0M1ypTNM stageIIIIIIIV ± ± Gastricadenocarcinoman ± ± P valuePatients with NECcomponents n ± ± Page of P valueGastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity Score MatchingPatients with NEC components NEC high grade MiNEN high grade MiNEN and high grade MiNEN 0cChen BMC Cancer Page of Table Univariate and multivariate analyses of survival after PSM in surgical groupPatient CharacteristicsUnivariate analysisHR CIMultivariate analysisHR CIP valueAge yearGendermale vs femaleBMIAdjuvant therapyYes vs NoTumor size¥ cm vs cmTumor categoryCarcinoma with NEC component vsGastric adenocarcinoma vsType of gastrectomyTotal gastrectomyDistal gastrectomyProximal gastrectomySurgical procedureLaparoscopic vs TNM stageIIIIIIIVP value tumor size and TNM staging were independent risk factors for prognosis This suggests that the prognosis ofgastric neoplasms with NEC components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of NEC components can alsoimpair prognosis which challenges the current cutoffvalue of The proportion of each component that must theoretically be greater than was set in [] Andsince WHO has also adopted this standard to define MiNEN [] This largely avoids the overdiagnosisof MiNEN in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges In additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines [] Nevertheless it is now being questionedby an increasing number of scholars The componentsin mixed tumors are not evenly distributed For large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely []Park compared the OS between tumors with morethan NEC components and gastric adenocarcinomawith or without less than NEC and they found thattumors with an NEC composition of more than hada worse prognosis This suggests that even a small proportion of malignant components can affect prognosis[] While in Parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith NEC components less than with gastricadenocarcinomas directly nor did they compare allNECcontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyIn our study we regarded tumors containing NECcomponents as a whole and found that the OS of thesetumors was poorer than that of adenocarcinoma in thesurgical group In addition we also found that the OS ofmixed tumors with less than between and more than NEC components or pure NEC wasworse than that of gastric adenocarcinoma Analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of Syn and CgAbetween different NEC content groups only the positiverate of CD56 was found to be higher in the pure NECgroup than that in the GHMiNEN group Therole of CD56 in the diagnosis of NEC is still controversial However Syn and CgA are two wellrecognized 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC and gastric adenocarcinoma in the surgical group aOverall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparison between GHMiNEN andgastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastric adenocarcinoma d Overall survivalcomparison between pure NEC alone and gastric adenocarcinomamarkers Therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing NEC componentsStudies on the molecular mechanism of pathogenesisshow that NEC components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity LOH and mutations at multiple loci and key oncogenes such as TP53 APC and RBgenes All these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] Moreoverin the WHO definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid [] no minimumpercentage for either ingredient is established Thereforewe believe that mixed tumors containing NEC components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma We propose considering mixed tumorscontaining NEC components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiesPreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] In a retrospective study involving patientsMa et alfound that neoadjuvant chemotherapy improves the survival of patients with NEC and HMiNENof the stomach [] Van der Veen reported that 0cChen BMC Cancer Page of Fig Comparison of OS between gastric neoplasm with different proportions of NEC components and gastric adenocarcinoma in theneoadjuvant group a Overall survival comparison between GHMiNEN and gastric adenocarcinoma b Overall survival comparisonbetween GHMiNEN and gastric adenocarcinoma c Overall survival comparison between GHMiNEN plus pure NEC and gastricadenocarcinoma d Overall survival comparison between pure NEC and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing NEC components [] However because only eight patients wereincluded in the neoadjuvant group Vans results arequestionable In our study among patients receivingneoadjuvanttherapy no significant difference in OSbetween mixed tumor and gastric adenocarcinoma wasobserved Even for the pure NEC group with the highestNEC contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsAlthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research In addition we proposed treatingall gastric neoplasms containing NEC components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms In the futurewe will conduct more genomics studies to confirmour ideas This study also has its limitations Due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens As a retrospective study despite our performing PSM in advance selection biascannot be completely avoided In addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cChen BMC Cancer Page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with NEC componentless than so we could only treat all mixed tumors withNEC component as a wholeConclusionsOur study demonstrated that gastric neoplasms withNEC components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma Among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies Therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients Neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12885020072817Additional file Table S1 Comparison of clinicopathologicalcharacteristics before and after PSM of 30GHMiNEN patients insurgical group Table S2 Comparison of clinicopathologicalcharacteristics before and after PSM of GHMiNEN patients in surgicalgroup Table S3 Comparison of clinicopathological characteristics beforeand after PSM of 70GHMiNEN plus pure NEC patients in surgicalgroup Table S4 Comparison of clinicopathological characteristics beforeand after PSM of pure NEC patients in surgical group Table S5 Comparison of clinicopathological characteristics before and after PSM of 30GHMiNEN patients in neoadjuvant group Table S6 Comparison ofclinicopathological characteristics before and after PSM of GHMiNEN patients in neoadjuvant group Table S7 Comparison of clinicopathologicalcharacteristics before and after PSM of 70GHMiNEN plus pure NECpatients in neoadjuvant group Table S8 Comparison of clinicopathological characteristics before and after PSM of pure NEC patients in neoadjuvant groupAbbreviationsAJCC American Joint Committee on cancer CT Computed tomography GHMiNEN Gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm GNEC Gastric neuroendocrine carcinoma HPF High power fieldMiNEN Mixed neuroendocrinenonneuroendocrine neoplasmNEC Neuroendocrine carcinoma NEN Neuroendocrine neoplasmNET Neuroendocrine tumor MRI Magnetic resonance imaging OS Overallsurvival PETCT Positron emission tomography computed tomographyPFS Progressionfree survival PSM Pr sity score matching WHO WorldHealth anizationAcknowledgmentsThanks to Dr Zhongwu Li of the Department of Pathology Peking UniversityCancer Hospital and his colleagues for their assistance in pathologicaldiagnosis and review Thanks to all colleagues in the Department ofGastrointestinal Surgery of Peking University Cancer Hospital and Dr JiangHong from the Statistics Department for their assistance in this studyAuthors contributionsAll authors contributed to the study conception and design JC performeddata collection and wrote the manuscript AW wrote and t revised hemanuscript KJ helped with statistical analysis and prepared the illustrationsZB edited the manuscript JJ conceived the study and reviewed themanuscript All authors read and approved the final manuscriptFundingThis work was supported by the National Science Foundation for YoungScientists of China Beijing Youth Talent Plan QML20191101 andScience Foundation of Peking University Cancer Hospital Thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThe study was approved by the Ethics Committee of Peking UniversityCancer Hospital and the patients written consent was also obtained Writteninformed consent for publication was obtained and stored in PekingUniversity Cancer HospitalConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Matsubayashi H Takagaki S Otsubo T Iiri T Kobayashi Y Yokota T et alAdvanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy Gastric Cancer Park JY Ryu MH Park YS Park HJ Ryoo BY Kim MG Prognosticsignificance of neuroendocrine components in gastric carcinomas Eur JCancer La Rosa S Inzani F Vanoli A Klersy C Dainese L Rindi G Histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms Hum Pathol Ishida M Sekine S Fukagawa T Ohashi M Morita S Taniguchi H et alNeuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis Am J Surg PatholRayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc | Thyroid_Cancer |
The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical managementDehong Che1 Zhirong Yang2 Hong Wei3 Xuedong Wang4 Jiayin GaoID1 Department of Obstetrics and Gynecology The Second Affiliated Hospital of Harbin Medical UniversityHarbin China Department of Ultrasound Harbin Red Cross Center Hospital Harbin China Departmentof Ultrasound The Second Affiliated Hospital of Harbin Medical University Harbin China Department ofObstetrics and Gynecology Longnan Hospital Daqing General Hospital Daqing Chinaa1111111111a1111111111a1111111111a1111111111a1111111111 dsp082901163comAbstract ACCESSCitation Che D Yang Z Wei H Wang X Gao J The Adler grade by Doppler ultrasound isassociated with clinical pathology of cervicalcancer Implication for clinical management PLoSONE e0236725 101371journalpone0236725Editor Linus Chuang University of Vermont LarnerCollege of Medicine UNITED STATESReceived February Accepted July Published August Copyright Che This is an access distributed under the terms of the CreativeCommons Attribution License which permitsunrestricted use distribution and reproduction inany medium provided the original author andsource are creditedData Availability Statement All relevant data arewithin the paper and its Supporting InformationfilesFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existAbbreviations FIGO International Federation ofObstetrics and Gynecology TVCDFI transvaginalObjectiveTo analyze the relationship of Adler grade by transvaginal color Doppler flow imaging TVCDFI and the clinical pathological parameters of patients with cervical cancer and to identify the value of Adler grade in the diagnosis and treatment of cervical cancerMethodsPatients with cervical cancer diagnosed pathologically in our hospital from January to December were included All patients underwent TVCDFI examination and theimages were divided into to III grades according to the Adler grades and the correlationsbetween the Adler classification and clinical pathological parameters clinical stage masssize pathological type squamous cell carcinoma subtype CA125 CA199 were analyzedResultsA total of patients with cervical cancer were included With the increase of Adler severity the clinical stage of cervical cancer increased accordingly the cancer size differed significantly in patients with different Adler grade p There were significant differences inthe level of CA125 CA199 between the squamous cell carcinoma and adenocarcinoma allp005 the Adler grade was positively related with the clinical stage pathological type andsquamous cell carcinoma subtypes of cervical cancer all p005 no correlations werefound among the Adler grade and the cancer size CA125 CA199 all p005 The areaunder ROC curve of the cervical squamous cell carcinoma predicted by Adler grade basedon FIGO results and pathological results was 0811and respectively all p005ConclusionsAdler grades are closely associated with the clinical pathology of cervical cancer which maybe a convenient and effective approach for the assisting assessment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0ccolor Doppler ultrasound WHO World HealthanizationUltrasound cervical cancerIntroductionCervical cancer is one of the most common malignant tumors in gynecology it has beenreported that the morbidity and mortality of cervical cancer ranks second among gynecological malignancies second only to breast cancer [ ] The number of new cases of cervical cancer has been increased every year around the world and the onset age of cervical cancer hasbecome much younger [] Cervical cancer is a malignant tumor which occurs at the junctionof squamous epithelial cells in the cervical vagina or the transitional zone and columnar epithelial cells in the endometrium of the cervical canal [] The etiology is not totally clear and itmay be related to sexual behavior frequency of deliveries and the infection of human papillomavirus [ ] The early detection and treatment of cervical cancer is very essential to theprognosis of patients with cervical cancerIn recent years with the popularization of many effective screening methods the overallmortality rate of cervical cancer patients has declined [] The clinical diagnosis mainlydepends on clinical manifestations and gynecological examinations which requires the combination of multiple auxiliary diagnostic methods including cervical cytology film cervicalmultipoint biopsy The International Federation of Obstetrics and Gynecology FIGO hasdeveloped a clinical staging standard for cervical cancer which referred to the colposcopypathological biopsy [] However when comparing the early diagnosed stage with the patientspostoperative medical examination results the stage of cervical cancer has been much underestimated [] More sophisticated radiological methods such as ultrasound computertomography and magnetic resonance imaging hasnt been included in the stage of FIGO butthey have been the routine examination methods in clinical practice [] Many scholars [] have studied the characteristics of those examinations but so far there is no asto which is the best examination method for assessing cervical cancer staging Its necessary toconduct more studies to identify the role of those tools for early diagnosis of cervical cancerPrevious studies [] have reported that the transvaginal color Doppler ultrasoundTVCDFI can accurately reflect the size of the lesion the extent of infiltration and the bloodsupply and it is widely used in the detection of cancers in breast and thyroid but not in thecervical parts Besides it is well known that vascular patterns such as density of vessels is different for squamous and adenocarcinomas [] Therefore its necessary to show the difference to provide insights into clinical treatment Therefore in order to make clinicians moreclear about the blood supply of cervical cancer and facilitate communication between ultrasound doctors and clinicians we attempted to conduct a preliminary investigation on the correlation between the Adler grade and clinical pathology of cervical cancer to provide insightsinto the diagnosis and treatment of cervical cancer The study design was participantspatients with cervical cancer 2intervention TVCDFI detection and Adler grading 3comparison no applicable 4outcomes Adler secores and antigens CA125 and CA199MethodsEthical considerationsThe study was approved by the Medical Research Ethics Committee of our hospitalNo20180038 and written informed consents were obtained from all the patientsPatientsPatients with cervical cancer diagnosed pathologically in our hospital from January toDecember were selected as study objects in this present study The inclusion criteriawere as follows there were surgical pathology or biopsy detections verifying small cellPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerneuroendocrine tumors by our experienced clinicans and the FIGO cervical cancer stagingwere used for severity differentiation [] no history of other malignant tumors or historyof radiotherapy and chemotherapy complete clinical data no history of cervical surgerysuch as we excluded patients undergone microwave freezing laser electrocautery surgeryThe exclusion criteria were as follows patients with congenital malformations of theuterus patients with coagulopathy patients with severe heart liver and kidney dysfunction patients with inability to cooperate with research or couldnt followupTVCDFI detectionThe color doppler ultrasound system Philips E80 equipped with ultrasonographic probeDC58D were selected for ultrasound detection Before the detection all patients were askedto empty the rectum and bladder When performing the doppler ultrasound detection thepatients took the lithotomy positions Firstly we performed a transvaginal ultrasound examination of the uterus and bilateral accessory areas to determine the location number size shapeboundary echo of the cervical area and its relationship with surrounding tissues Secondly wechose the color Doppler blood flow imaging mode to observe the blood flow inside and aroundthe cervical area related parameters such the blood flow cancer size have been calculated andcollected The staging and TVCDFI done in the same setting and in different datesAdler grade classificationAdler grade classifications were conducted based on the detected blood flow [ ] We classified the blood flow signal of the lesion into four levels Adler refers to that no obvious bloodflow signal Adler I refers to or small blood vessels with a diameter of mm are detectedAdler II refers to that or small blood vessels are detected Adler III refers to that more than blood vessels or the blood vessels are intertwined into a network are detected The Adlergrade classifications were made by one radiologist and one gynecologist coherently and ifthere were any disparity further discussions were conducted for consensusThe detection of carbohydrate antigens CA125 and CA199Before any drug treatment ml venous blood without anticoagulation in the early morningwere collected from patients the blood specimens were left at room temperature for 15minthen centrifuged it and separated the serum then we stored it at environment with ËC for further examination The levels of serum CA125 and CA199 were detected by professional stallwith electrochemical luminescence method The electrochemical luminometer and detectionkit used in the test were produced by Roche and the operation process was strictly performedaccording to the instructionsStatistical analysisSPSS statistical software were used for data analysis The measurement data for normal distribution was expressed as mean± standard deviation and the measurement data for skeweddistribution was expressed as median P25 P75 The tumor size CA125 CA199 were compared using the KruskalWallis H rank test of multiple sample comparisons of which Wilcoxon rank test was further used for pairwise comparisons And t tests were conducted in thecancer size between pathological types or squamous cell carcinoma subtypes Spearman correlation analysis was conducted for correlation analysis And receiver operating characteristicROC curves were drawn for evaluating the diagnosis value of Adler grade for cervical cancer[] p005 was considered as being statistically different in this present studyPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerResultsThe ultrasound characteristics of cervical cancerAs Fig presented the ultrasound characteristics differed hugely among the ultrasoundimages with different Alder grades For Alder the cervical morphology was normal andno obvious blood flow signal were found in the images of color Doppler For Alder I thecervical morphology was slightly thickened and more enhanced intracervical echo couldbe collected one or two small spotlike blood flow signals could be detected For Alder IIuneven or thickened cervical echo distribution could be detected and strip blood flow signals at two to four locations could be seen For Alder III parauterine and extrauterineinvasion and blood metastasis could be found and reticular blood flow signals could bedetectedThe distribution of Adler classification and clinical stage of cervical cancerWe identified potential candidates firstly and patients were excluded As Table presented a total of patients were included for data analysis With the increase of Adler severity classification the clinical stage of cervical cancer increased accordinglyFig The Doppler ultrasound images on cervical cancer with different Adler grade a Adler b Adler I c Adler II d Adler III101371journalpone0236725g001PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable The distribution of Adler classification and clinical stage of cervical cancerAdler gradeFIGO gradeGrade IGrade IIGrade IIIIVAdler Adler IAdler IIAdler IIIIn total101371journalpone0236725t001In totalThe comparison on the cancer size CA125 CA199 with Adler grade andpathological parametersAs Table showed the cancer size differed significantly among the different Adler gradesp but there were no significantly differences in the level of CA125 CA199 amongthe different Adler grades all p005 There were significant differences in the level ofCA125 CA199 between the squamous cell carcinoma and adenocarcinoma all p005 nosignificant difference was found in the cancer size between the squamous cell carcinoma andadenocarcinoma p No significant differences were found in the cancer size CA125and CA199 between the keratinized and nokeratinized squamous cell carcinoma all p005Furthermore as Table showed the Adler grade was positively related with the FIGO stagingpathological type and squamous cell carcinoma subtypes of cervical cancer all p005 nocorrelations were found among the Adler grade and the cancer size CA125 CA199 allp005The diagnosis value of Adler grade for cervical cancerAs Fig showed based on FIGO results the area under the ROC curve of the cervical squamous cell carcinoma predicted by Adler grade was p005 and its sensitivityTable Relationship analysis on the cancer size CA125 CA199 with Adler grade and pathological parametersItemsAdler gradeAdler Adler IAdler IIAdler IIItzpPathological typeSquamous cell carcinomaAdenocarcinomatzpsquamous cell carcinoma subtypesKeratinizedNonkeratinizedtzpCasesCancer sizemmCA125[MP25 P75 UmL]CA199[MP25 P75 UmL]±±± ± ± ± ± 101371journalpone0236725t002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerTable Correlation analysis between Adler grade and clinicopathological parametersClinicopathological parametersFIGO gradeCancer sizeCA125CA199Pathological typesquamous cell carcinoma subtypes101371journalpone0236725t003rpspecificity positive predictive value negative predictive value and Youden index were respectively And based on pathological results the area underthe ROC curve of the cervical squamous cell carcinoma subtype predicted by Adler grade was p005 and its sensitivity specificity positive predictive value negative predictivevalue and Youden index were respectively Thoseresults indicated that Adler grade could provide valuable reference for the diagnosis of cervicalcancerDiscussionsTVCDFI has the advantages of being noninvasive cheap with convenient operation [ ]It can not only clearly display the structure of each layer of the uterus accurately locate andqualitatively diagnose the lesion but also can observe the blood flow of detected area [ ]Dynamic ultrasound is currently one of the most widely used and mature imaging methods inthe diagnosis and treatment of obstetrics and gynecology diseases [] In this present studythe changes of cervical morphology and echo in Adler grade to I were small and Dopplerblood flow signals were less displayed but the blood flow signals in Adler grade II and IIIincreased significantly and the flakelike low echoes were seen inside With the increase ofAdler classification cervical masses continue to increase and color Doppler blood flow signalsare also displayed The results of this present study have indicated that Adler grade is expectedto be useful to reflect the richness of blood flow in cervical cancer which can provide important insights into the diagnosis and treatment of cervical cancerFig The ROC curve on the Adler grade for cervical cancer101371journalpone0236725g002PLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerThe occurrence proliferation invasion and metastasis of malignant tumors may largelydepend on tumor neovascularization [ ] Cervical cancer has characteristics of rapid proliferation and active cell division and growth which is closely related to the proliferation oftumor vessels in it [] On color Doppler ultrasound images there are often abundant different types of blood flow signals in tumor tissues which correspond to its rich vascular networkand are related to the special structure and blood flow characteristics of tumor blood vessels[ ] The Alder grades are mainly concentrated on the blood flow signals which to someextent can reflect the proliferation of vessels and the characteristics of microvessel densityaround the cervical cancer Previous studies [] have reported that the blood signals maypredict the ability of tumor invasion and metastasis to a certain extent which is consistentwith the results of our studySquamous carcinoma is more common in the pathological and histological types of cervicalcancer [] We have found that there was correlation between Adler grade and the histopathological type The World Health anization WHO classifies cervical squamous cell carcinoma into two subtypes keratinized and nonkeratinized according to the presence ofkeratinized morphological features in the tissue [] Currently the studies on the relationshipof keratinized cervical squamous cell carcinoma and patient prognosis has been controversialIts been reported [] that compared with nonkeratinized cervical squamous cell carcinoma the survival rate of keratinized cervical squamous cell carcinoma is significantlyreduced and it is not sensitive to radiotherapy and the prognosis is very poor for untreatedpatients or advanced patients Therefore differentiate the various subtypes of squamous cellcarcinoma of cervix is very helpful in clinical managementIn this study Spearman rank correlation analysis showed that Adler grade was positivelycorrelated with squamous cell carcinoma subtypes Therefore this technique is expected to bea reliable indicator for predicting the prognosis of patients with cervical cancerWith the continuous progress of cancer marker research the role of cancer related markerin the diagnosis and treatment of cancers has become increasingly important [ ] Severalstudies have shown that cervical cancer is also closely related to the marker CA125 and CA199It has been reported [] that the carbohydrate antigens CA125 and CA199 are expressedat high levels in a variety of malignancies In this study the levels of CA125 and CA199 inpatients with adenocarcinoma were significantly higher than those in patients with squamouscell carcinoma The results were consistent with the findings of previous studies [ ] Eventhrough the Spearman correlation analysis shows that there is no correlation between Adlergrade and CA125 and CA199 it may be explained that the samples in this present study is notlarge enough to powerfully detect the difference future studies with larger population samplesare warranted to identify the role of CA125 and CA199 in cervical cancerSeveral limitations must be concerned in this present study Firstly the sample size of thisstudy is not large enough and the clinical staging is relatively rough biases may be existed inthis present study Secondly we did not use the cervical cancer specific markers such as squamous cell carcinoma tumor marker for reference it should be further elucidated in the futurestudies Thirdly it must bring to our attentions that the subjective factors in the Adler classification different operators and different parameter settings may bias the test results Howeverwe have performed related trainings on the TVCDFI before this study to avoid unnecessaryerrors in the process of ultrasound detection and analysisIn the results of this study have favored the value of Adler grade in the diagnosisand treatment of cervical cancer With the continuous development of ultrasound technologyand popularization of clinical ultrasound applications Adler grade should be promoted in theapplication of color Doppler ultrasound for the diagnosis and treatment of cervical cancerPLOS ONE 101371journalpone0236725 August PLOS ONE 0cUltrasound cervical cancerSupporting informationS1 ChecklistDOCXS2 ChecklistDOCXAuthor ContributionsConceptualization Dehong Che Zhirong Yang Jiayin GaoData curation Dehong Che Xuedong Wang Jiayin GaoFormal analysis Dehong Che Zhirong Yang Hong Wei Jiayin GaoInvestigation Dehong Che Zhirong Yang Hong Wei Xuedong Wang Jiayin GaoMethodology Xuedong WangResources Xuedong Wang Jiayin GaoSoftware Dehong CheValidation Dehong Che Jiayin GaoWriting original draft Dehong CheReferences Shrestha AD Neupane D Vedsted P Kallestrup P Cervical Cancer Prevalence Incidence and Mortality in Low and Middle Income Countries A Systematic Review Asian Pac J Cancer Prev Epub 1022034APJCP2018192319 PMID PubMed Central PMCID PMC5980914 Kalliala I Athanasiou A Veroniki AA Salanti G Efthimiou O Raftis N Incidence and mortalityfrom cervical cancer and other malignancies after treatment of cervical intraepithelial neoplasia a systematic review and metaanalysis of the literature Ann Oncol Epub 101016jannonc201911004 PMID Murfin J Irvine F MeechanRogers R Swift A Education income and occupation and their influenceon the uptake of cervical cancer prevention strategies A systematic review J Clin Nurs 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Thyroid surgery in children in a single institution from Osama Ibrahim Almosallama Ali Aseerib Ahmed Alhumaida Ali S AlZahranic Saif Alsobhib Saud AlShanafeybFrom the aDepartment of Surgery College of Medicine Qassim University Buraidah Al Qassim Saudi Arabia bDepartment of Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia cDepartment of Medicine King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia Correspondence Dr Osama Ibrahim Almosallam Department of Surgery College of Medicine Qassim University PO Box Buraidah Al Qassim Saudi Arabia osama_iaahotmailcom ORCID orcid0000000290367564 Citation Almosallam OI Aseeri A Alhumaid A AlZahrani AS Alsobhi S AlShanafey S Thyroid surgery in children in a single institution from Ann Saudi Med Received January Accepted May Published August Copyright Copyright Annals of Saudi Medicine Saudi Arabia This is an access under the Creative Commons AttributionNonCommercialNoDerivatives International License CC BYNCND The details of which can be accessed at httpcreativecommons licensesbyncnd40Funding NoneBACKGROUND Data on thyroid surgery in children are scarceOBJECTIVE Analyze outcome data on thyroid surgery in a pediatric populationDESIGN Medical record reviewSETTING Tertiary health care institutionPATIENTS AND METHODS We collected demographic and clinical data on patients years or younger who had thyroid surgery in the period to Descriptive data are presentedMAIN OUTCOME MEASURES Indications for thyroidectomy thyroid pathology complications length of stay and radioactive iodine treatment and recurrencesSAMPLE SIZE RESULTS Of patients who underwent thyroidectomy procedures were females and the mean age at operation was years and were associated with multiple endocrine neoplasia type There was no history of radiation exposure Eightyone patients had fine needle aspiration FNA which correlated with the final histopathology in of cases Sixtysix patients had malignant cancer papillary of patients who had neck dissection had lymph node metastasis and had distant metastases to the lung Procedures included total thyroidectomy hemithyroidectomy completion and subtotal thyroidectomy Twentythree patients developed hypocalcemia permanent and had unilateral recurrent laryngeal nerve injury permanent Patients were followed up for a mean duration of months median months Of patients with thyroid cancer received radioactive iodine and had recurrence Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic Hypocalcemia and recurrent laryngeal nerve injury are significant complications The recurrence rate in thyroid cancer is LIMITATIONS RetrospectiveCONFLICT OF INTEREST Noneoriginal ANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cThyroid diseases requiring surgery are relatively uncommon in children compared to adults The prevalence of palpable thyroid nodules in children ranges from to Sporadic welldifferentiated thyroid cancer is the most common endocrine malignancy in children accounting for of pediatric cancers in the prepubertal age group and up to of cancers in adolescents aged year2 The most common indication for thyroid surgery in children varies among published studies but thyroidectomy for malignant conditions is rising38 Data in children throughout the world are relatively scarce The objective of this study was to analyze the clinical data and outcome of thyroid surgery in a large series of children treated at a single center at King Faisal Specialist Hospital and Research Center KFSHRC in RiyadhPATIENT AND METHODS With the approval of the Institutional Review Board IRB at KFSHRC the medical records of all patients years old and younger who underwent a thyroid surgery between and were retrospectively reviewed We elected to include patients up to the year to ensure a reasonable followup period Patients for the study were identified by a search of the operating room log for all procedures involving the thyroid gland for the specified age groupDemographic data clinical features and surgical outcomes were collected Specific data that were obtained included age at operation gender family history presenting symptoms history of radiation exposure presence of multiple endocrine neoplasia type MEN thyroid function test presence and size of thyroid nodules by ultrasound presence of lymph nodes metastasis or distant metastasis fine needle aspiration FNA cytology surgical procedure final histopathology and length of followup Outcomes analyzed were postoperative complications including transient or permanent hypocalcemia transient or permanent recurrent laryngeal nerve paralysis wound infection and hematoma length of stay and radioactive iodine treatment and recurrences Thyroid procedures in this series included hemithyroidectomy subtotal total and completion thyroidectomy Surgeries were performed by either an endocrine adult surgeon or a pediatric surgeon No intraoperative nerve monitoring was used Early in the series procedures were performed by adult endocrine surgeons but lately a combined approach was adopted where pediatric surgeons and adult endocrine surgeons collaborated in such cases proceduresthe normal range in our laboratory regardless of symptoms Transient hypocalcemia was identified if it lasted for less than months while permanent hypocalcemia was considered if the serum calcium level remained below normal range and the patient continued on calcium supplementation after months of the surgery All patients with a family history of MEN underwent genetic testing of the RET protooncogene to confirm the diagnosis All patients who underwent completion thyroidectomy had a preoperative and postoperative vocal cords assessment at the Otolaryngology clinic Descriptive data were generated and comparisons were conducted using the t test for continuous data and the chisquare or Fisher exact tests for proportionsRESULTSBetween and patients underwent surgical procedures patients underwent two procedures for thyroid disease at our institution Eighty patients were females The mean age at operation was years median years range years The most common indication for thyroidectomy was thyroid nodule which was present in of cases Table The mean SD size of thyroid nodules was mm There were cases associated with MEN syndromes The final pathology in two patients with MEN syndrome showed medullary thyroid cancer MTC while the remaining patients had prophylactic procedures before development of MTC None of the patients had a history of radiation exposure Eightyone patients FNA which correlated with the final histopathology in of cases There were three cases of toxic adenoma and one case of Graves disease which did not require FNA The remaining cases underwent FNA at another institution and FNA was not repeated at our institution or they came for completion thyroidectomy with documented pathology for malignancy after they had their first surgery in another hospitalThe most common diagnoses included papillary thyroid cancer and multinodular goiter or colloid Table Indications for thyroidectomy in patients IndicationNodulen MEN prophylaxisHyperthyroidismMultinodular goiterCompletion thyroidectomy Hypocalcemia was defined by calcium levels below Data are number original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cnodule Table Surgical procedures included total thyroidectomy hemithyroidectomy completion thyroidectomy and subtotal thyroidectomy Neck dissection was performed in patients Operative complications were observed in patients The most common complication was hypocalcemia transient permanent and Table Thyroid pathology in the patientsPathologyn BenignNormal thyroid tissueColloid noduleCystAdenomaThyroiditisGraves diseaseThyroid cancerPapillaryFollicularMedullaryHurthleAnaplasticTotalData are number Table Benign and malignant lesions in patientsBenignn37Malignantn66 P value Age meanyearsGender malefemalePresence of noduleHypocalcemiaRecurrent laryngeal nerve palsyBleedinghematomaWound infectionTracheal injuryOverall complicationsMean length of stay daysMEN recurrent laryngeal nerve palsy transient permanent all were unilateral Table Of patients with malignant lesions had lymph node metastasis and patients had distant metastases to the lung None of the patients developed postoperative bleeding wound infection or tracheal injury Patients were followed up for a mean of months median range months radioactive iodine treatment was delivered to patients with malignant lesions patients had recurrences were local recurrences and were local and distant recurrences to the lung Three cases received radioactive iodine RAI before and after recurrence One case was low risk before recurrence so did not receive RAI until after recurrence One case had medullary thyroid cancer so did not receive RAI In the remaining five cases there was no clear data whether those patients received RAI before or only after a recurrence All local recurrences underwent resection except for one patient who was lost follow up There was no mortality in this study DISCUSSIONThe most common indication for thyroidectomy in this series was thyroid nodule which correlates with previously published reports in the pediatric population35 Children with thyroid nodules have an estimated fourfold higher risk of developing thyroid cancer compared to adults910 The high incidence of malignancy in this series suggests children with a thyroid nodule should be carefully evaluatedFNA is a valuablemethod for preoperative evaluation of thyroid nodules However there are limitations on the routine use of FNA in children including the need for sedation sampling errors and the limited availability of experienced cytopathologists11 Many previous studies reported high sensitivity and specificity of FNA in evaluating thyroid nodule in children1114 which correlate with our findingsOur data showed lymph node metastasis in of thyroid cancer cases which supports the notion that children with thyroid cancer frequently present with more extensive disease than adults Lymphnode involvement at diagnosis is seen in to of children compared with to of adults with differentiated thyroid cancer1523 Because our hospital is the largest referral center in Saudi Arabia especially for oncology cases this may explain the large number of lymph node and distant metastasis In this cohortThe most common complication reported after thyroidectomy in children is hypoparathyroidism with an incidence ranging between to which original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0ccorresponds with our results of which are reported as hypocalcemia in Table One study found that total thyroidectomy central and bilateral neck dissection Graves disease and malignancy were risk factors for hypocalcemia after thyroid surgery3 In this cohort postoperative hypocalcemia was noted more in malignant cases but it failed to reach statistical significance Moreover there was no significant difference between benign and malignant cases in terms of mean age gender distribution recurrent laryngeal nerve injury or overall complications a finding that was reported previously26 Multiple studies in recent years have found an inverse relationship between surgeon volume and complication rates2728 but similar data in the pediatric population is lacking One study found that highvolume endocrine surgeons have better outcomes and shorter lengths of stay and lower costs after thyroidectomy and parathyroidectomy in children compared to pediatric surgeons general surgeons or otolaryngologists29 Scheumann and colleagues also concluded that a collaborative approach between pediatric and endocrine surgeons would have better outcomes This has led other authors to suggest that a combined approach with endocrine and pediatric surgeons in addition to pediatric endocrinologists may optimize the care of children with surgical thyroid disease given the low number of pediatric patients4 Our data do not allow for comparisons of different approaches given the late adoption of the combined approach The recurrence rate for thyroid cancer in children after thyroidectomy has varied widely in reported studies ranging from to while it was in this cohort Only a few studies explored the predictors of recurrence Lymph node involvement multiple nodules male gender younger age histologic subtype and advanced tumor stage were risk factors associated with recurrence17233033 In this study of patients with malignant lesions received RAI Although there are conflicting data regarding the indications of postoperative RAI treatment in lowrisk patients the current recommendation is that lowrisk patients can be treated without RAI3436There are some limitations to this study The retrospective nature may affect the validity and quality of the data The small number of cases in some categories did not enable us to compare groups and explore predictors relative to these factors On the other hand this study adds to the scarce data on thyroid surgery in pediatric age group Malignancy is the commonest indication for thyroid surgery in children and FNA is highly diagnostic Hypocalcemia and recurrent laryngeal nerve injury are significant complications Cancerrelated death is extremely rare but recurrence is not uncommon and a significant number of patients with malignant cases received RAI treatmentoriginal PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0cREFERENCES Trowbridge FL Matovinovic J McLaren GD Nichaman MZ Iodine and goiter in children Pediatrics Ries LAG Melbert D Krapcho M Stinchcomb DG Howlader N Horner MJ et al SEER Cancer Statistics Review Bethesda National Cancer Institute Based on November SEER data submission Chen Y[h] Masiakos PT Gaz RD Hodin RA Parangi S Randolph GW et al Pediatric thyroidectomy in a high volume thyroid surgery center Risk factors for postoperative hypocalcemia J Pediatr Surg Aug5081316 Wood JH Partrick DA Barham HP Bensard DD Travers HS Bruny JL et al Pediatric thyroidectomy a collaborative surgical approach J Pediatr Surg May4658238 Scholz S Smith JR Chaignaud B Shamberger RC Huang SA Thyroid surgery at Childrens Hospital Boston a 35year singleinstitution experience J Pediatr Surg Mar46343742 Josefson J Zimmerman D Thyroid nodules and cancers in children Pediatr Endocrinol Rev Sep611423 Hameed R Zacharin MR Changing face of paediatric and adolescent thyroid cancer J Paediatr Child Health LugoVicente H Ortiz VN Irizarry H Camps JI Pagán V Pediatric thyroid nodules management in the era of fine needle aspirationJ Pediatr Surg Mussa A De Andrea M Motta M Mormile A Palestini N Corrias A Predictors of Malignancy in Children with Thyroid Nodules J Pediatr Oct167488692 Amirazodi E Propst EJ Chung CT Parra DA Wasserman JD Pediatric thyroid FNA biopsy Outcomes and impact on management over years at a tertiary care center Cancer Cytopathol Partyka KL Huang EC2 Cramer HM Chen S Wu HH Histologic and clinical followup of thyroid fineneedle aspirates in pediatric patients Cancer Cytopathol Sinha CK Decoppi P Pierro A Brain C Hindmarsh P Butler G et al Thyroid Surgery in Children Clinical Outcomes Eur J Pediatr Surg Oct2554259 Kundel A Thompson GB Richards ML Qiu LX Cai Y Schwenk FW et al Pediatric Endocrine Surgery A 20Year Experience at the Mayo Clinic J Clin Endocrinol Metab February Jiang W Newbury RO Newfield RS Pediatric thyroid surgery and management of thyroid nodulesan institutional experience features and over a 10year period Int J Pediatr Endocrinol Burke JF Sippel RS Chen H Evolution of Pediatric Thyroid Surgery at a Tertiary Medical Center Surg Res AlQahtani KH Tunio MA Al Asiri M Aljohani NJ Bayoumi Y Riaz K et al Clinicopathological treatment outcomes of differentiated thyroid cancer in Saudi children and adults J Otolaryngol Head Neck Surg Nov Kluijfhout WP van Beek DJ Verrijn Stuart AA Lodewijk L Valk GD Van der Zee DC et al Postoperative Complications After Prophylactic Thyroidectomy for Very Young Patients With Multiple Endocrine Neoplasia Type Medicine Baltimore 20159429e1108 Raval MV Browne M Chin AC Zimmerman D Angelos P Reynolds M Total thyroidectomy for benign disease in the pediatric patientfeasible and safe J Pediatr Surg Stavrakis AI Ituarte PH Ko CY Yeh MW Surgeon volume as a predictor of outcomes in inpatient and outpatient endocrine surgery Surgery Sosa JA Bowman HM Tielsch JM Powe NR Gordon TA Udelsman R The importance of surgeon experience for clinical and economic outcomes from thyroidectomy Ann Surg Tuggle CT Roman SA Wang TS Boudourakis L Thomas D Udelsman R et al Pediatric endocrine surgery Who is operating on our children Surgery Dec144686977 Park S Jeong JS Ryu HR Lee C Park JH Kang S et al Differentiated Thyroid Carcinoma of Children and Adolescents27Year Experience in the Yonsei University Health System J Korean Med Sci Palmer BA Zarroug AE Poley RN Kollars JP Moir CR Papillary thyroid carcinoma in children risk factors and complications of disease recurrence J Pediatr Surg Wada N Sugino K Mimura T Nagahama M Kitagawa W Shibuya H et al Pediatric differentiated thyroid carcinoma in stage I risk factor analysis for disease free survival BMC Cancer D Danese Gardini A Farsetti A Sciacchitano S Andreoli M Pontecorvi A Thyroid carcinoma in children and adolescents Eur J Pediatr Astl J Chovanec M Lukes P Katra R Dvorakova M Vlcek P et al Thyroid carcinoma surgery in children and adolescents years experience surgery of pediatric thyroid lymph node metastases carcinoma Int J Pediatr Otorhinolaryngol Chaukar DA Rangarajan V Nair N Nadkarni MS Pai PS Dcruz AK et al Pediatric thyroid cancer J Surg Oncol Dzodic R Buta M Markovic I Gavrilo D Matovic M Milovanovic Z et al Surgical management of welldifferentiated thyroid carcinoma in children and adolescents years of experience of a single institution in Serbia Endocr J Scheumann GF Gimm O Wegener G Hundeshagen H Dralle H Prognostic significance and surgical management of locoregional in papillary thyroid cancer World J Surg Shi RL Qu N Yang SW Tumor size interpretation for predicting cervical lymph node metastasis using a differentiated thyroid cancer risk model Onco Targets Ther Zimmerman D Hay ID Gough IR Goellner JR Ryan JJ Grant CS et al Papillary thyroid carcinoma in children and adults longterm followup of patients conservatively treated at one institution during three decades Surgery Collini P Mattavelli F Pellegrinelli A Barisella M Ferrari A Massimino M Papillary carcinoma of the thyroid gland of childhood and adolescence Morphologic subtypes biologic behavior and prognosis a clinicopathologic study of sporadic cases treated at a single institution during a 30year period Am J Surg Pathol BorsonChazot Causeret S Lifante JC Augros M Berger N Peix JL Predictive factors for recurrence from a series of children and adolescents with differentiated thyroid cancer World J Surg Baumgarten HD Bauer AJ Isaza A MostoufiMoab S Kazahaya K Adzick NS Surgical management of pediatric thyroid disease Complication rates after thyroidectomy at the Childrens Hospital of Philadelphia highvolume Pediatric Thyroid Center Journal of pediatric surgery Oct Kurzawinski TR De Coppi P Thyroidectomy in Children InPediatric Surgery pp Springer Berlin Heidelberg Francis G Waguespack SG Bauer AJ Angelog P Benvenga S et al Management Guidelines for Children with Thyroid Nodules and Differentiated Thyroid Cancer The American Thyroid Association Guidelines Task Force on Pediatric Thyroid Cancer THYROID Volume Number original PEDIATRIC THYROID SURGERYANN SAUDI MED JULYAUGUST WWWANNSAUDIMEDNET 0c' | Thyroid_Cancer |
"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as San ye qing is a kind of folk plant Because of its slow growth it usually takes years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang Peoples Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as natural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new eight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiangs dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our teams preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally cm long and cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are cm long and cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4 on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of 4αDGalAp1 4DGalp1 and 4αDGlcp1 residues as backbones and DManp1 36DManp1 and αDAraf1residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52 Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a | Thyroid_Cancer |
Treatment Strategy for Metastatic Spinal Tumors A Narrative ReviewSam Yeol Chang Sujung Mok Sung Cheol Park Hyoungmin Kim BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Seoul Korea Metastatic spinal tumors are common and their rising incidence can be attributed to the expanding aging population and increased survival rates among cancer patients The decisionmaking process in the treatment of spinal metastasis requires a multidisciplinary approach that includes medical and radiation oncology surgery and rehabilitation Various decisionmaking systems have been proposed in the literature in order to estimate survival and suggest appropriate treatment options for patients experiencing spinal metastasis However recent advances in treatment modalities for spinal metastasis such as stereotactic radiosurgery and minimally invasive surgical techniques have reshaped clinical practices concerning patients with spinal metastasis making a demand for further improvements on current decisionmaking systems In this review recent improvements in treatment modalities and the evolution of decisionmaking systems for metastatic spinal tumors are discussedKeywords Spinal metastasis Decisionmaking system Radiosurgery Minimally invasive surgical procedures Separation surgeryIntroductionThe spine has been identified as the most common site for malignant metastasis in the musculoskeletal system and vice versa spinal metastasis is considered the most common malignant lesion in the spine [] The incidence of metastatic spinal tumors has seen an increasing trend due to the growing aging population worldwide and continued improvements in survival rates among cancer patients [] Symptomatic spinal metastasis is often the first clinical manifestation for of cancer patients [] whereas up to of cancer patients may experience spinal metastasis at some point during the course of their disease [] The objectives of surgical treatment in patients with metastatic spine tumors are mostly palliative Spine surgeons make an effort to maintain or improve the patients quality of life during the remainder of their survival by reducing pain and preserving ambulatory function via surgical treatment []Because clinical manifestations and treatment responses vary widely among cancer patients a multidisciplinary decisionmaking process that integrates medical and radiation oncology together with surgery along with assistance from pathology and diagnostic radiology deemed is essential when deciding to conduct surgical treatment for spinal metastasis [] In the literature various decisionmaking systems have been developed and introduced to date in an effort to aid in this decisionmaking process Received Jul Revised Jul Accepted Jul Corresponding author BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Daehakro Jongnogu Seoul KoreaTel Fax Email bschangsnuackrCopyright ¸ by Korean Society of Spine SurgeryThis is an Access distributed under the terms of the Creative Commons Attribution NonCommercial License httpcreativecommonslicensesbync40which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly citedAsian Spine Journal ¢ pISSN eISSN ¢ wwwasianspinejournalAsian Spine JournalASJAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J [] However recent advancements in the treatment of metastatic spine tumors have injected more complexity into this decisionmaking process and demanded the evolution of the decisionmaking system itself these recent advances include the development of stereotactic spine radiosurgery SRS the introduction of minimally invasive surgical techniques and the evolution of various target therapies for individual primary cancers [] In this review we discussed the development and evolution of various decisionmaking systems in spinal metastasis treatment Current concepts and recent trends in radiotherapy and surgery for spinal metastasis are also includedDecisionMaking Systems for Managing Metastatic Spinal TumorsPrognostic factors for metastatic spinal tumorsWhen attempting to choose an appropriate treatment for a patient with spinal metastasis establishing an accurate estimation of the individuals life expectancy is the most crucial To do this one must first identify prognostic factors associated with the survival of patients with spinal metastasis As such many authors have conducted studies to try and identify prognostic factors associated with survival among spinal metastasis patients and have developed various decisionmaking systems in order to estimate survival based on these prognostic factors []In a recently published metaanalysis Luksanapruksa [] have identified independent prognostic factors associated with the survival of patients with spinal metastasis Among these factors nine factors can be classified as relating to the preoperative performance or neurological status of a patientfor example the Karnofsky Performance Score or Eastern Cooperative Oncology Group grade [] Meanwhile four factors involve the presence or the number of metastases spine bone or visceral and two factors were found to be related to primary tumors in the Tomita classification scheme [] Finally male sex and the time interval from cancer diagnosis to the start of radiotherapy are the two remaining prognostic factors independently associated with survival in spinal metastasis patients Among these primary tumor histology the presence and number of metastases and performance status are proposed as the three most important prognostic factors associated with survival in spinal metastasis patients not only in this study but also in most previous investigations []In other studies the patients age and comorbidities assessed using the American Society of Anesthesiologists physical status [] and Charlson Comorbidity Index [] have also been identified to be prognostic factors in spinal metastasis [] Other authors have identified laboratory abnormalities such as leukocytosis and low hemoglobulin and albumin levels as prognostic factors and included these into their decisionmaking systems [] In addition previous systemic treatment or chemotherapy has also been suggested as an independent prognostic factor by multiple authors [] Further not only preoperative chemotherapy but also the presence of available systemic treatments in the postoperative period has been hypothetically regarded as a potential prognostic factor in the literature [] In a recently published study by Chang [] the authors verified the presence of the remaining systemic treatment options to be independently associated with improved postoperative performance status and survivalClassificationbased decisionmaking systemsBased on these prognostic factors numerous decisionmaking systems or scoring systems have been developed and introduced to estimate the life expectancy among spinal metastasis patients [] In these classificationbased decisionmaking systems scores for each prognostic factor identified by multivariate logistic or proportional hazards regression analyses are integrated in order to obtain a total prognostic score that reflects the estimated survival of the patient Surgeons can adopt these prognostic scores to identify patients with an estimated survival profile that is sufficient to warrant surgical treatment Although the prognostic factors included in each system vary primary tumor histology and visceral metastases are included in most systems Table In the New England Spinal Metastasis Score NESMS was introduced by Ghori [] The NESMS was developed using multicenter data and retrospectively validated in the following investigations [] The NESMS consists of modified Bauer score components and score serum albumin level and ambulatory status of the patient More recently the NESMS was validated prospectively in the Prospective Observational Study of Spinal Metastasis Treatment trial which aimed to verify the 0cTreatment of Spinal Metastasis Table Prognostic factors in decisionmaking systemsReferencesBauer [] modifiedTomita [] To kuhashi [] revisedKa tagiri [] revisedGhori [] Paulino [] Primary tumorPerformance statusNo of vertebral metastasesBone metastasisVisceral metastasisPrevious systemic treatmentOther factorsOOOOOOOOOOOOOOOOOOOOOOOOOOBr ain metastasis WBC Hb platelet albumin bilirubin Creactive protein lactate dehydrogenaseSerum albuminAge WBC Hb brain metastasisWBC white blood cell Hb hemoglobin NESMS as a reliable predictive tool in spinal metastasis patients []There have been efforts to develop novel decisionmaking systems using evolving methodologies In the Skeletal Oncology Research Group S compared multiple prognostic survival algorithms including classic nomogram and boosting algorithms using the same retrospective dataset obtained from patients [] In their study the nomogram was found intuitive and demonstrated a comparable level of performance Then in the S used machinelearning algorithms to develop a novel prognostic model for metastatic spinal disease [] which was externally validated in subsequent studies []Although these various classificationbased decisionmaking systems are helpful and widely used for predicting the survival of spinal metastasis patients recent studies have reported that the degree of accuracy of these classic systems eg Tomita Tokuhashi decreases over time especially in cancers with poor prognoses such as lung cancer [] This pitfall of classificationbased decisionmaking systems reportedly stems from the inability of these systems to reflect survival improvement due to recent evolutions in systemic treatment for primary cancers [] Another existing limitation is that these systems cannot directly guide the selection of specific treatments appropriate for patients with spinal metastasisPrinciplebased decisionmaking systemsAs an alternative to these classificationbased decisionmaking systems that are incapable of reflecting recent advances in oncology and guiding specific treatments several authors have proposed principlebased decisionmaking systems These principlebased systems do not score the patient and estimate survival but instead provide advice regarding which treatment is more appropriate in individual cases based on the integration of rapidly evolving treatment modalities including target therapies radiosurgery and minimally invasive surgical techniquesThe neurologic oncologic mechanical and systemic NOMS decision framework was first introduced in [] The NOMS decision framework consists of neurologic N oncologic O mechanical M and systemic S considerations integrating novel multimodal therapies including SRS and minimally invasive surgical techniques [] As a neurological N assessment approach the grading system developed by Bilsky and Smith [] was used while surgical decompression is recommended for highgrade spinal cord compressions During oncological O assessment the responsiveness of spinal metastasis to currently available treatments especially the level of tumor sensitivity to radiotherapy is evaluated Mechanically M instability of the spinal column as determined by the Spinal Instability Neoplastic Score SINS indicates the need for surgical stabilization regardless of the neurologic or oncologic status [] Finally systemic S assessment focuses on the patients ability in tolerating the suggested treatment Meanwhile if the general condition performance status and medical comorbidities of a patient do not allow surgery to be performed radiotherapy is instead recommendedA modification of the NOMS decision framework has also been presented in the literature Paton introAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J duced the LMNOP system as an improvement to the NOMS approach [] With this development these authors added two additional key considerations to the NOMS as follows the location and levels of metastasis L and the patients response to previous therapy P The P in LMNOP stands for not only the response to prior therapy but also includes patient fitness and prognosis which was considered previously as part of the systemic S assessment in the NOMS decision framework The authors emphasized that the response of primary cancer to previous treatments including chemotherapy and radiotherapy is considered a significant factor when determining the appropriate treatment for spinal metastasis patients For instance it is anticipated that a patient diagnosed with symptomatic spinal metastasis at the initial presentation of primary cancer synchronous metastasis who would have multiple potential treatment options is more likely to experience a better prognosis than a patient diagnosed with spinal metastasis despite previous treatment metachronous metastasis Differences in the survival rates between the patients with synchronous and metachronous spinal metastasis have been confirmed in a previous research [] Therefore a more aggressive approach including surgical treatment can be considered for patients with a synchronous spinal metastatic lesion In summary principlebased decisionmaking systems relative to classificationbased systems are able to better incorporate evolving treatment modalities and guide the selection of appropriate treatments for patients in a timely fashion Table Current trends and future directions in the development of decisionmaking systemsAdvances in cancer biology and treatment modalities are necessitating the evolution of decisionmaking systems for spinal metastasis Possible future directions to take to improve decisionmaking systems include the following the use of multicenter or multinational databases the integration of histologyspecific data the application of computational methodologies such as machinelearning algorithms and the combination of classificationbased and principlebased systems Some recent studies are already covering these trendsThe size of the study sample under assessment determines the performance and accuracy of prognostic models Although spinal metastasis is found to be relatively common data from a larger sample population beyond that of just a single institution is usually required to develop a powerful enough prognostic model For this reason recently introduced prognostic models or decisionmaking systems are generated from multicenter databases [] Future prognostic models should also have the freedom to rely on even larger databases such as multinational tumor registriesBiologic therapy including molecular target therapy and immunotherapy is believed to be an emerging gamechanger in modern cancer treatment Genetic subtype analysis of the primary cancer histology which guides selection among t hese therapies has become more essential [] In a revised prognostic system proposed by Katagiri Table The NOMS decision frameworkNeurologic NOncologic OMechanical MSystemic SDecisionLowgrade ESCCno myelopathyHighgrade ESCC±myelopathyRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadioresistantRadioresistantStableUnstableStableUnstableStableUnstableStableStableUnstableUnstablecEBRTStabilization followed by cEBRTSRSStabilization followed by SRScEBRTStabilization followed by cEBRTAble to tolerate surgeryD ecompressionstabilization followed by SRSUnable to tolerate surgerycEBRTAble to tolerate surgeryDecompressionstabilization followed by SRSUnable to tolerate surgeryStabilization followed by cEBRTNOMS neurologic oncologic mechanical and systemic ESCC epidural spinal cord compression cEBRT conventional external beam radiation SRS stereotactic radiosurgery 0c [] the authors considered the availability of molecular target therapy when classifying the primary tumor For example lung cancer treated with targeted drugs was designated as an example of a moderategrowth tumor while lung cancer without targeted drugs is regarded as an example of a rapidgrowth tumor [] This application of genetic profiles to decisionmaking systems is likely to grow more specific and tailored corresponding to the evolution of molecular genetics in the futureAs previously described machinelearning algorithms have been applied in the development of prognostic models Classically prognostic models for spinal metastasis have been developed using logistic or proportional hazards regression analyses As part of its research efforts the S was able to develop prognostic models using machinelearning algorithms such as gradient boosting decision trees random forests and neural networks [] and these algorithms were externally validated elsewhere [] Like in other fields of medicine evolving computational methodologies including machinelearning algorithms should be assessed extensively in terms of their potential in the management of spinal metastasisFinally the combination of classificationbased and principlebased decisionmaking systems should be considered Classificationbased systems or prognostic models seek to estimate the patients remaining survival Based on this survival estimation principlebased systems then may suggest the most appropriate treatment option Until now surgeons and physicians have been employing these two separate systems in the same decisionmaking process A novel decisionmaking system could integrate these two systems together and provide survival estimations and appropriate treatment options simultaneouslyRadiotherapy for Metastatic Spinal TumorsTreatment of Spinal Metastasis single session of SRS achieved durable longterm control of spinal metastasis regardless of histology and tumor size Of note the only significant factor associated with tumor control was the radiation dose These results suggest that SRS can be effective even in cases of metastasis previously considered to be radioresistantSRS can also be a definitive treatment for the management of solitary metastasis without spinal cord compression [] Excellent local control rates of have allowed SRS to replace curative surgeries with high morbidities such as total en bloc spondylectomy for addressing these solitary metastases [] In patients with highgrade spinal cord compression SRS can be applied after separation surgery which will be discussed further in the following section Overall the effectiveness of SRS has been changing the role and extent of surgical treatment and in turn shifting the focus of the treatment of spinal metastasisVertebral compression fracture following stereotactic spine radiosurgeryA pitfall of SRS is an increase in vertebral compression fracture VCF following radiotherapy Risk estimates for VCF after SRS are reported to be up to as compared with just in relation to conventional radiotherapy [] The occurrence of VCF is dosedependent and caution is required if the radiation dosage exceeds Gy per fraction in highrisk patients [] Highrisk patients of older ages with lytic lesions andor with spinal malalignment can reportedly benefit from undergoing preventive stabilization surgery before SRS When determining the necessity of stabilization surgery before SRS SINS can be used to identify potentially unstable lesions [] SINS will be further covered in the following sectionStereotactic spine radiosurgery is triggering a paradigm shiftTiming of radiotherapy after surgeryEvidence in the literature supports that radiosurgery is a safe and effective modality for local tumor control with low associated complication rates in patients with spinal metastasis [] Technical improvements including intensitymodulated and imageguided radiation delivery and processing software have allowed SRS to be a gamechanger in the treatment of spinal metastasis [] A recent study by Yamada [] reported that a highdose Adequate timing of radiotherapy following surgery whose determination is related to the risk of wound complications continues to be debated among spine surgeons and radiation oncologists It is also controversial whether the interval can be shortened in patients receiving SRS Lee [] sent questionnaires to radiation oncologists and spine surgeons to gather opinions on the optimal timing of surgery and radiotherapy in spinal metastasis Based on the procured comments the auAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J thors recommended that the interval be at minimum weeks regardless of radiation modalities Interestingly as compared with radiation oncologists surgeons tended to favor a shorter interval of time between surgery and radiotherapy when SRS is performed although there was no statistically significant difference in this regard []A recently published systemic review also advocated for weeks with a minimum of days between surgery and radiotherapy [] When the rates of wound complications were compared between SRS and conventional radiotherapy many studies reported reduced wound complications in SRS patients [] However due to limited highlevel evidence no definite conclusion was made regarding whether the interval could be reduced in patients undergoing SRSSurgery for Metastatic Spinal TumorsSurgical indicationsThe objective of surgical treatment in spinal metastasis was to provide pain relief support neurological improvement and in turn enhance the quality of life during the remaining survival period Clinical benefits of direct surgical decompression in patients with metastatic spinal cord compression MSCC have been well described in the literature [] The most important prerequisite for surgical treatment in spinal metastasis has been identified as the sufficient enough estimated survival time to make surgery a reasonable approach Researchers have largely recommended that a minimum of to months of remaining survival should exist when considering whether to perform surgery [] At this point a number of prognostic scoring systems previously described are being used to estimate a patients survival The patient should also have a good enough general condition or performance status to in order to endure surgery If these conditions are satisfied then surgery can be performed in patients with symptomatic MSCC or mechanical instabilityIn spinal metastasis the instability is assessed by the SINS [] Table The SINS is an independent and unique tool that integrates clinical and radiological components to help surgeons decide whether to conduct surgery for stabilization A score of to points suggests impending instability while that of points or more indicates existing spinal instability which requires stabilization As previously mentioned the SINS has also been incorporated into principlebased decisionmaking systems such as the NOMS and LMNOP systems [] Recent studies have reported the reliability and accuracy of the SINS system in predicting spinal adverse events including VCF especially in those patients who received radiotherapy [] although some components may still require revision []Separation surgery and minimally invasive surgeryWhen considering the surgical techniques used for spinal metastasis the literature shows a trend toward the adoption of less invasive techniques which is thought to have Table Spinal Instability Neoplastic Score systemComponentLocationJunctional occiputC2 C7T2 T11L1 L5S1Mobile spine C3C6 L2L4Semi rigid T3T10Rigid S2S5PainaYesOccasional pain but not mechanicalPainfree lesionBone lesionLyticMixed lyticblasticBlasticRadiographic spinal alignmentSubluxationtranslation presentDe novo deformity kyphosisscoliosisNormal alignmentVertebral body collapse collapse collapseNo collapse with body involvedNone of the abovePosterolateral involvement of spinal elementsbBilateralUnilateralNone of the aboveScoreaPain improvement with recumbency andor pain with movementloading of spine bFacet pedicle or costovertebral joint fracture or replacement with tumor 0cTreatment of Spinal Metastasis primarily resulted from recent advances in radiotherapy as previously described [] With the use of advanced radiation techniques surgeons can minimize surgical morbidities by avoiding extensive debulking surgery [] Fig During the separation surgery circumferential spinal cord decompression is performed only to the extent required to facilitate safe radiosurgery In a study by Laufer [] separation surgery followed by postoperative SRS resulted in a low local progression rate Other studies have also reported that this hybrid surgeryradiosurgery approach is a safe and effective treatment option for MSCC []BCADFig A 68yearold male with spinal metastasis of renal cell carcinoma at T9 The patient also had lung metastasis A Preoperative MRI shows spinal cord compression and involvement of posterior elements and left rib head at the T9 level B Following a separation surgery without spinal instrumentation MRI at postoperative 2weeks shows a decompressed spinal canal but residual metastatic tumor around the left 9th rib head C In the postoperative 1year MRI the patient showed a nearcomplete response following a single session stereotactic radiosurgery 18Gy1 fraction which was performed weeks after the separation surgery MRI magnetic resonance imaging D Planning images for the postoperative stereotatic radiosurgery following a separation surgery Asian Spine Journal 0cSam Yeol Chang et alAsian Spine J Minimalaccess surgery is also used in treating spinal metastasis while reducing surgical morbidities In the anterior approach retractor systems and thoracoscopic assistance have been described [] Minimally invasive posterior approaches for decompression and corpectomy have also been introduced and reviewed in the literature [] Other minimally invasive techniques for spine surgery such as the intraoperative stereotactic navigation system and percutaneous pedicle screw instrumentation are being incorporated into spinal metastasis surgery as well []Studies comparing minimally invasive surgery and surgery showed that minimally invasive surgery provided equivalent or superior outcomes with reduced surgical morbidity and complications in spinal metastasis patients [] However because the quality of evidence is deemed low in the current literature no definite conclusion regarding the superiority of minimally invasive surgery over surgery can be derived and no strong recommendations have been made at this point []Role of curative surgery en bloc resectionMost surgeries for spinal metastasis are found palliative and the role of en bloc resection in spinal metastasis is decreasing even further due to improvements in radiotherapy Generally curative surgical resection of spinal metastasis has been considered in the context of a single metastasis of a slowgrowing tumor such as in renal cell thyroid and breast cancers Fig Favorable outcomes in this regard have been reported in the literature [] However some authors recently reported that curative ABECDFig A 63yearold male with spinal metastasis of thyroid carcinoma at T8 A Preoperative MRI shows pathologic fracture and spinal cord compression at the T8 level B Postoperative Xray at month shows removal T8 vertebra and reconstruction with an expandable cage following total en bloc spondylectomy C MRI at postoperative 3years shows a widely decompressed spinal canal with no tumor recurrence D Postoperative Xray at postoperative 5years shows wellmaintained instrumentation E Bone scan at postoperative 5years shows no evidence of bone metastasis MRI magnetic resonance imaging 0csurgical resection en bloc spondylectomy did not impact the oncologic outcomes of spinal metastasis patients [] Therefore a more careful and thorough decisionmaking process is required before performing curative resection surgery for spinal metastasis especially when the extended role of radiosurgery is consideredPostoperative complications and preventive measuresThe overall complication rate following surgery for spinal metastasis ranges from to in the literature [] Because surgery for spinal metastasis is performed to improve the quality of life of a patient surgeons should try to minimize all possible surgical and medical complications by implementing multidisciplinary interventions Among diverse complications those that require additional attention when found in spinal metastasis patients eg wound infection instrumentation failure and intraoperative bleeding are briefly discussed hereThe incidence of surgical site infection SSI is determined to be higher in spinal metastasis surgery reaching up to as compared with during other spine surgeries [] SSI has also been identified as the most common cause for reoperation in of reoperations following surgery for spinal metastasis [] Poor nutrition and exposure to adjuvant therapies chemotherapy and radiotherapy put spinal metastasis patients at risk for SSI [] Surgeons should provide adequate nutritional support perioperatively and secure a sufficient time interval between radiation and surgery as previously discussed to minimize SSIs Additional risk factors such as smoking obesity and medical comorbidities should also be considered []The second cause for reoperation in spinal metastasis surgery is the failure of instrumentation [] Risk factors associated with instrumentation failure include the number of operated levels prior chest wall resection and history of radiotherapy [] The necessity of additional fusion procedures while performing surgery for spinal metastasis is debated but highlevel evidence remains to be lacking at this time [] In future studies we suggest that instances of early and late failure be distinguished when assessing instrumentation failure because the mechanisms of failure between the two types seem to differ ie insufficient stability of the construct in the context of early failure versus the progression of deformity or lack of fusion in the context of late failureTreatment of Spinal Metastasis Intraoperative bleeding during spinal metastasis surgeries can be massive and can further lead to serious complications such as cardiovascular or cerebral events [] For the spinal metastasis of hypervascular tumors such as renal cell hepatocellular and thyroid cancers preoperative embolization is recommended Previous studies have verified the effectiveness of preoperative embolization in reducing intraoperative bleeding in spinal metastasis surgeries [] Surgery should be performed within hours following embolization to avoid the diminished effect of preoperative embolization []ConclusionsThe determination of appropriate treatment for a patient with spinal metastasis is a challenging task that requires multidisciplinary considerations Recent advances in radiotherapy and surgery for spinal metastasis have brought about improvements in the management of these patients Evolving decisionmaking systems are also crucial contributors to stateoftheart care of patients with metastatic spinal tumorsConflict of InterestNo potential conflict of interest relevant to this was reportedReferences White AP Kwon BK Lindskog DM Friedlaender GE Grauer JN Metastatic disease of the spine J Am Acad Orthop Surg Laufer I Sciubba DM Madera M Surgical management of metastatic spinal tumors Cancer Control Schiff D ONeill BP Suman VJ Spinal epidural metastasis as the initial manifestation of malignancy clinical features and diagnostic approach Neurology Klimo P Jr Schmidt MH Surgical management of spinal metastases Oncologist Nathan SS Healey JH Mellano D Survival in patients operated on for pathologic fracture implications for endoflife orthopedic care J Clin Oncol Curtin M Piggott RP Murphy EP Spinal metaAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J static disease a review of the role of the multidisciplinary team Orthop Surg Ahmed AK Goodwin CR Heravi A Predicting survival for metastatic spine disease a comparison of nine scoring systems Spine J Barzilai O Fisher CG Bilsky MH State of the art treatment of spinal metastatic disease Neurosurgery Tomita K Kawahara N Kobayashi T Yoshida A Murakami H Akamaru T Surgical strategy for spinal metastases Spine Phila Pa Tokuhashi Y Matsuzaki H Oda H Oshima M Ryu J A revised scoring system for preoperative evaluation of metastatic spine tumor prognosis Spine Phila Pa Bauer HC Wedin R Survival after surgery for spinal and extremity metastases prognostication in patients Acta Orthop Scand Van der Linden YM Dijkstra SP Vonk EJ Marijnen CA Leer JW Dutch Bone Metastasis Study Group Prediction of survival in patients with metastases in the spinal column results based | Thyroid_Cancer |
role of miR1179 in the development of cancer has been proved by different studies However the expression profile and role of miR1179 is yet to be explored in human oral cancer Consistently this study was undertaken to explore the molecular role of miR1179 in regulation of the human oral cancer development and progression The results showed miR1179 to be significantly p overexpressed in all the oral cancer cell lines relative to normal cells The repression of miR1179 transcript levels not only suppressed the proliferation of oral cancer cells but also increased their sensitivity to vincristine The decline in proliferative rates was attributed to induction of autophagy in oral cancer cells as confirmed by transmission electron microscopic analysis Western blot analysis showed that the expression of LC3BII increased and that of beclin decreased while LC3BI expression remained constant upon miR1179 inhibition Inhibition of miR1179 caused significant decrease in the migration and invasion of the oral cancer cells The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition At molecular level the miR1179 was shown to exert its anticancer effects via deactivation of MEKERK and PI3KAKT signalling cascades In the findings point towards the potential of miR1179 in the treatment of oral cancerKeywords Oral cancer Micro RNA Proliferation Autophagy MetastasisIntroductionDespite advancement in science and technology the current strategies employed for the treatment of human oral cancer are still far from descent As such presently the oral cancer is still ranked 6th most prevalent type of cancer globally Peers et a0al The survival rates of oral cancer are comparatively lower than breast and prostate cancers The overall 5year survival rate of oral Correspondence MonicaHendrixaxoyahoocom Yanmei Gao and Hanmei Xu contributed equally to this workDepartment of Stomatology Affiliated Hospital of Jilin Medical University NO of Road Huashan Fengman Area Jilin Chinacancer is only as against and for breast and oral cancers respectively Gupta et a0al Additionally the recurrence of human oral cancer further adds to the problem Borsetto et a0al Hence it is need of the hour to look for the alternative approaches for the management of oral cancer As revealed by the recent research reports the molecular regulators of human cancers have emerged as vital targets to be studied for their usage against these deadly ailments Cao et a0 al Among these molecular agents the small RNA entities called microRNAs miRs which do not code for proteins but have molecular regulatory roles have been shown to be involved in the development and tumorigenesis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 0cGao a0et a0al AMB Expr Page of of number of human cancers including the human oral cancer Gaezon et a0 al Wu et a0 al The miRs have not only been shown to regulate the proliferation of human cancers but have also been shown to have influence the metastasis to neighbouring tissues Peng et a0al MicroRNA1179 miR1179 has been implicated in the regulation of proliferation and metastasis of different human cancers Jiang et a0al Krutovskikh et a0al Song et a0 al Lin et a0 al The miR1179 has been shown to inhibit the growth of the glioblastoma cells by inducing cell cycle arrest Xu et a0al The inhibition of metastasis of hepatocellular carcinoma has been reported to be due to interaction of miR365 with ZEB2 Gao et a0al In yet another study miR targets HMGB1 to suppress the proliferation of gastric cancer cells Li and Qin Nonetheless the role and therapeutic implications of miR1179 has not be reported This study was therefore designed to investigate the role of miR1179 in human oral cancer cells via modulation of the MEKERK and PI3KAKT signalling pathwaysMaterials and a0methodsCell lines and a0culture conditionsThe oral cancer cell lines SCC4 SCC9 SCC15 and SCC25 along with normal EBTr cell line were procured from ATCC USA The culturing of cell lines was performed using Dulbeccoʼs modified Eagleʼs medium DMEM Thermo Scientific The cell lines were maintained in a CO2 incubator at a0°C with CO2 concentration and relative humidity of TransfectionTo stably transfect the oral cancer cells with miRNC and miRinhibitor Lipofectamine Thermo Scientific was used and the procedure was carried as per the manufacturer guidelinesExpression analysisThe RNeasy Mini Kit Qiagen and miScript Reverse Transcription Kit Qiagen were respectively used to isolate the RNA from cancer and normal cell lines and for cDNA synthesis The SYBR Green mix Thermo Scientific was used for performing the expression analysis of miR1179 through quantitative realtime polymerase chain reaction qRTPCR on QuantStudio real time PCR Thermo Scientific The cycling conditions were as follows °C for a0 s followed by cycles of °C for a0s and °C for a0min The expression values were quantified using ddCt method The real primer sequences were miRF ² GCG GAA GCA TTC TTT CAT ² and miRR ² CAA GGG CTC GAC TCC TGT ²Cell viability assayTo assess the proliferation rates of the oral cancer cells transfected with miRNC and miRinhibitor for a0 h and administered withwithout a0 µM vincristine the cells were cultured in 96well plate for a0h a0h or a0h at °C Following this the 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT reagent Thermo Scientific was added at final concentration of After a0 h a0 µl dimethyl sulfoxide DMSO was added for dissolving the crystals of formazan Absorbance at a0 nm was taken using spectrophotometer to determine the proliferation rates of oral cancer cellsElectron microscopyFor the assessment of induction of cell autophagy the cancer cells transfected with miRNC or miRinhibitor for a0 h were examined using Transmission electron microscope TEM Trypsin treatment was used to make the collection of cancer cells which were then washed and then fixed with glutaraldehyde in phosphate buffer a0m The Osmium tetroxide was then used for postfixing of cells The cells were then treated with ethanol and embedded in resin The ultramicrotome was used for section cutting which was followed by electron microscopyTranswell chamber assayThe migration and invasion of oral cancer cells transfected with miRNC or miRinhibitor were determined by using Transwell chamber without or with matrigel coating Here the cell suspension containing approximately cells was poured into the upper portion of transwell chamber and lower portion was supplemented with a0µl DMEM medium with fetal bovine serum FBS Following a0 h incubation at a0 °C and CO2 concentration the cancer cells from the surface of membranes upper side were swabbed away with cotton swabs while the cells sticking to lower surface of membrane were fixed with ethanol and the stained with crystal violet The cells were then examined and photographs were taken using light microscopeWestern blottingFor the estimation of protein concentrations the western blotting technique was used Precisely after transfection with miRNC or miRinhibitor the oral cancer cells were cultured for a0h at a0°C Centrifugation was used to collect the cells which were then washed using PBS buffer This was followed by treatment with RIPA buffer containing a0 mM TrisHCl pH a0 mM NaCl Triton X100 SDS a0mM EDTA a0mM Na2HPO4 a0mM NaF a0mM NaVO4 a0mM phenyl 0cGao a0et a0al AMB Expr Page of methylsulfonyl fluoride and protease cocktail inhibitor The total protein concentrations were estimated using Bradford assay Equal protein concentrations were loaded on the Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDSPAGE from each sample The gel was blotted to PVDF membrane followed by treatment with primary antibodies Santa Cruz Biotechnology Inc Dallas TX USA overnight at °C The blots were washed in tris buffered saline TBS incubated with horseradish peroxidaseconjugated secondary antibody Santa Cruz Biotechnology Inc for a0h at °C washed again three times with TBS and chemiluminescence was captured on hyperfilm following incubating the blots in enhanced chemiluminescence reagentStatistical analysisThe experiments were performed in triplicate and expressed as mean ± standard deviation SD The Students ttest was performed through GraphPad Prism software The pvalues were taken as statistically significant differenceResultsmiR is a0upregulated in a0oral cancer cellsThe qRTPCR analysis showed miR1179 to be significantly upregulated in all the oral cancer cell lines studied SCC4 SCC9 SCC15 and SCC25 relative to normal oral cell line EBTr The maximum transcript upregulation of miR1179 was observed in SCC9 oral cancer cells 65fold and SCC25 cells 45fold relative to normal EBTr cells Fig a01a As such SCC9 and SCC25 cell lines were taken for further experimentationmiR inhibition suppresses the a0proliferation and a0enhances chemosensitivity of a0oral cancer cellsTo reveal the molecular functionality of miR1179 in oral cancer the suppression of miR1179 was achieved by transfecting the SCC9 and SCC25 cancer cells with miR1179 inhibitor Using miRNC transfected as control the repression of miR1179 transcript levels was confirmed by qRTPCR method which showed significant p decrease of miR1179 expression in SCC9 and SCC25 cells Fig a01b Additionally the results showed that suppression of miR1179 resulted in remarkable decline of proliferation rates of SCC9 and SCC25 cancer cells Fig a01c Interestingly the antiproliferative effects of vincristine were shown to be greatly enhanced when SCC9 and SCC25 cancer cells were transfected with miRinhibitor construct Fig a0 Together the results clearly indicate that miR1179 inhibition suppresses proliferation and enhances the chemosensitivity of the human oral cancer cellsFig miR1179 regulates the proliferation of human oral cancer cells a Expression of miR1179 in human oral cancer and normal cells b Expression of miR1179 in miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells c Cell viability of miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 enhances the Vincristine sensitivity of the human SCC9 and SCC25 oral cancer cells Individual experiments were performed in triplicate and expressed as mean ± SD P by the western blotting of LC3BI LC3BII and beclin autophagy related proteins The LC3BI protein levels remined constant beclin protein levels declined and the LC3BII protein levels increased in miR1179 inhibitor transfected SCC9 and SCC25 oral cancer cells Fig a03b Hence it is evident that the transcript repression of miR1179 in human oral cancer cells declines the cell proliferation rates via induction of cell autophagymiR suppression reduces oral cancer cell metastasisThe assessment of migration and invasion capabilities of SCC9 and SCC25 oral cancer cells transfected with miR inhibitor or miRNC constructs by transwell chamber assay showed that the miR1179 inhibitor mediated suppression of miR1179 expression resulted in significant decline in the migratory and invasive potential of SCC9 and SCC25 cancer cells Fig a0 The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition Taken together the results indicate that miR1179 has a regulatory role on cancer cell metastasismiR modulates MEKERK and a0PI3KAKT pathways in a0oral cancerIn order to analyse the molecular mechanics of miR1179 in oral cancer the repression of miR1179 was made in SCC9 and SCC25 oral cancer cells The assessment of MEKERK signalling pathway revealed that the decline in miR1179 expression reduced the phosphorylatedMEK and ERK pMEK and pERK protein levels Fig a0 5a However the protein levels of nonphosphorylated versions of MEK and ERK remained almost unchanged Similarly the reduction in phosphorylated protein versions of PI3K and Akt proteins pPI3K and pAkt was Fig Inhibition of miR1179 induces autophagy in oral cancer cells a Ultrastructural analysis of the miRNC and miR1179 transfected SCC9 and SC25 cells b Western blot analysis of miRNC and miR1179 transfected SCC9 and SCC25 cells showing the expression of LC3B I II and Beclin Individual experiments were performed in triplicateInhibition of a0miR induces autophagy in a0the a0oral cancer cellsThe electron microscopic examination of SCC9 and SCC25 human oral cancer cells transfected with miR inhibitor or miRNC clearly revealed the presence of autophagy vesicular structures in miRinhibitor transfected cancer cells Fig a0 3a However such structures were totally lacking from the cancer cells transfected with miRNC The results were further supported 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 inhibits the migration and invasion of miRNC and miR1179 transfected SCC9 and SC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P observed under miR1179 repression Fig a05b The protein level of PI3K and Akt remained unchanged Taken together the results reveal that the suppression of miR expression in oral cancer results in the blocking of phosphorylation of MEK ERK PI3K and Akt proteinsDiscussionOral cancer is one of the lethal human disorders and this malignancy is responsible for a considerable level of human mortality and morbidity Warnakulasuriya Peterson Overall the oral cancer ranks 6th in terms of its incidence rates globally One more worrying fact about human oral cancer is its recurrence and development of drug resistance Silva et a0 al So an urgency is felt in the scientific community to devise more robust measures for the oral cancer management Recently miRs have emerged as potent regulators of various human cancers as they have been seen to have profound role in human physiology and disease development Gong et a0al The deregulation of miR1179 has been implicated to be associated with a number of human cancers Peng et a0al In the present study miR1179 was found to be upregulated in oral cancer cells which is in accordance with several previous studies Peng et a0 al Krutovskikh et a0 al Further miR1179 downregulation was previously been shown to decline the viability of ovarian cancer cells Zhihong et a0al Similar observations were made from the results of the current study The miR1179 repression was found to inhibit proliferation and enhance the drug sensitivity of oral cancer cells to vincristine Such implications have been drawn also previously Zhihong et a0 al The characterization of miR1179 in oral cancer in this study revealed that autophagy is induced in oral cancer cells when miR1179 transcript levels are repressed The autophagy was confirmed by the presence of autophagy vesicles which was further implicated by the enhancement of LC3II conjugated protein expression level and declining of LC3I and Beclin expression The miR repression in oral cancer cells further declined the migration and invasion rates of cancer cells which is in conformity with the previous studies on miR1179 Jiang et a0al Lastly among the most important molecular events of cancer progression the activation of MEKERK 0cGao a0et a0al AMB Expr Page of upregulated in human oral cancer cells Inhibition of miR1179 overexpression resulted in decline of proliferation and metastasis and enhancement of chemosensitivity of human oral cancer cells Additionally miR1179 also resulted in the blockage of the MEKERK and PI3KAKT signalling pathway pointing towards the therapeutic implications of miR1179 in oral cancer treatmentAcknowledgementsWe acknowledge the Affiliated Hospital of Jilin Medical University Jilin China Shandong China for providing necessary laboratory faciality and supportAuthors contributionsYG and TP designed the protocol of the study YG and HX performed the experimental work and collect the data for presented study YG and HX involve in the statistical analysis TP supervised the work and drafted the manuscript although all author contributes for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived June Accepted August ReferencesBorsetto D Higginson JA Aslam A AlQamachi L Dhanda J Marioni G Franchella S Frigo A Praveen P Martin T Parmar S Factors affecting prognosis in locoregional recurrence of oral squamous cell carcinoma J Oral Pathol Med Cao M Tang Y Tang Y Liang XH Noncoding RNAs as regulators of lymphangiogenesis in lymphatic development inflammation and cancer metastasis Front Oncol Gao HB Gao FZ Chen XF MiRNA1179 suppresses the metastasis of hepatocellular carcinoma by interacting with ZEB2 Eur Rev Med Pharmacol Sci Garzon R Calin GA Croce CM MicroRNAs in cancer Ann Rev Med Gong H Liu CM Liu DP Liang CC The role of small RNAs in human diseases potential troublemaker and therapeutic tools Med Res Rev Gupta N Gupta R Acharya AK Patthi B Goud V Reddy S Garg A Singla A Changing Trends in oral cancera global scenario Nepal J Epidemiol Huang M Huang B Li G Zeng S Apatinib affect VEGFmediated cell proliferation migration invasion via blocking VEGFR2RAFMEKERK and PI3KAKT pathways in cholangiocarcinoma cell BMC Gastroenterol Jiang L Wang Y Rong Y Xu L Chu Y Zhang Y Yao Y miR1179 promotes cell invasion through SLIT2ROBO1 axis in esophageal squamous cell carcinoma Int J Clin Exp Pathol Krutovskikh VA Herceg Z Oncogenic microRNAs OncomiRs as a new class of cancer biomarkers Bioessays Fig Inhibition of miR1179 blocks MEKERK and PI3K and AKT signalling pathways a Western blots showing the effects of miR1179 inhibition on expression of MEK pMEK ERK and pERK proteins in SCC9 and SCC25 cells b Western blots showing the effects of miR1179 inhibition on expression of PI3K pPI3K AKT and pAKT proteins in SCC9 and SCC25 The experiments were performed in triplicateand PI3KAKT pathways hold a prime essence as these pathways enable the transcription of various downstream regulators of cancer growth and proliferation Huang et a0al These pathways have been shown to be aberrantly activated in different cancer types For example PI3KAKT pathway has been shown to be activated in pancreatic cancer Lan et a0 al In thyroid cancer cells PI3KAKT and MAPKERK pathway has been shown to significantly overexpressed Su et a0 al Similarly MEKERK pathway has shown to be activated in ovarian cancer Zhang et a0al As such the blockage of these signalling pathways is a vital asset to keep the cancer progression at check Zhihong et a0 al The miR1179 suppression renders the MEKERK and PI3KAkt pathways to proceed at fairly diminished rates by preventing the phosphorylation of crucial signalling components like MEK ERK PI3K and AKT Summing up the current study deduced the molecular functionality of miR1179 and implicated its molecular targeting in the management of human oral cancer To conclude the present study showed miR1179 to be significantly 0cGao a0et a0al AMB Expr Page of Lan CY Chen SY Kuo CW Lu CC Yen GC Quercetin facilitates cell death and chemosensitivity through RAGEPI3KAKTmTOR axis in human pancreatic cancer cells J Food Drug Anal Lin C Hu Z Yuan G Su H Zeng Y Guo Z Zhong F Jiang K He S MicroRNA1179 inhibits the proliferation migration and invasion of human pancreatic cancer cells by targeting E2F5 Chem Biol Interact Li Y Qin C MiR1179 inhibits the proliferation of gastric cancer cells by targeting HMGB1 Hum Cell Peng X Guo W Liu T Wang X Tu XA Xiong D Chen S Lai Y Du H Chen G Liu G Identification of miRs143 and145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT PLoS ONE 275e20341Peres MA Macpherson LM Weyant RJ Daly B Venturelli R Mathur MR Listl S Celeste RK GuarnizoHerre±o CC Kearns C Benzian H Oral diseases a global public health challenge Lancet Petersen PE Oral cancer prevention and controlthe approach of the World Health anization Oral Oncol Silva SD Hier M Mlynarek A Kowalski LP AlaouiJamali MA Recurrent oral cancer current and emerging therapeutic approaches Front Pharmacol Song L Dai Z Zhang S Zhang H Liu C Ma X Liu D Zan Y Yin X MicroRNA1179 suppresses cell growth and invasion by targeting spermassociated antigen 5mediated Akt signaling in human nonsmall cell lung cancer Biochem Biophy Res Co Su X Shen Z Yang Q Sui F Pu J Ma J Ma S Yao D Ji M Hou P Vitamin C kills thyroid cancer cells through ROSdependent inhibition of MAPKERK and PI3KAKT pathways via distinct mechanisms Theranostics Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol Wu BH Xiong XP Jia J Zhang WF MicroRNAs new actors in the oral cancer scene Oral Oncol Xu X Cai N Zhi T Bao Z Wang D Liu Y Jiang K Fan L Ji J Liu N MicroRNA1179 inhibits glioblastoma cell proliferation and cell cycle progression via directly targeting E2F transcription factor Am J Cancer Res Zhang R Shi H Ren F Feng W Cao Y Li G Liu Z Ji P Zhang M MicroRNA3383p suppresses ovarian cancer cells growth and metastasis implication of Wntcatenin beta and MEKERK signaling pathways J Exp Clin Cancer Res Zhihong Z Rubin C Liping L Anpeng M Hui G Yanting W Zhenxiu S MicroRNA1179 regulates proliferation and chemosensitivity of human ovarian cancer cells by targeting the PTENmediated PI3KAKT signaling pathway Arch Med Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | Thyroid_Cancer |
"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ ] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ¥ mmolL onehour bloodglucose postoral sugar ¥ mmolL or twohour bloodglucose postoral sugar ¥ mmolL Preterm birth wasdefined as delivery at ¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Students ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ¥ y y ¥ y y ¥ y y ¥ y Low birth weight y ¥ y Macrosomia y ¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child NutrYun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency 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Valkama SM Helve OM Hytinantti TK Makitie OM AnderssonS Maternal vitamin D status gestational diabetes and infant birth size BMCPregnancy Childbirth Khaing W Vallibhakara SA Tantrakul V Vallibhakara O Rattanasiri S McEvoyM Attia J Thakkinstian A Calcium and vitamin D supplementation forprevention of preeclampsia a systematic review and network metaanalysisNutrients 2017910E1141 Zhang Y Gong Y Xue H Xiong J Cheng G Vitamin D and gestationaldiabetes mellitus a systematic review based on data free of Hawthorneeffect BJOG Zhang MX Pan GT Guo JF Li BY Qin LQ Zhang ZL Vitamin D deficiencyincreases the risk of gestational diabetes mellitus a metaanalysis ofobservational studies Nutrients Aghajafari F Nagulesapillai T Ronksley PE Tough SC O'Beirne M Rabi DMAssociation between maternal serum 25hydroxyvitamin D level andpregnancy and neonatal outcomes systematic review and metaanalysis ofobservational studies BMJ 2013346f1169 Qin LL Lu FG Yang SH Xu HL Luo BA Does Maternal Vitamin D DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet GynecolJolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril 1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132 Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J MedLiu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
"Severe iodine deficiency impacts fertility and reproductive outcomes The potential effects of mildtomoderate iodine deficiency are not well known The aim of this study was to examine whether iodine intake wasassociated with subfecundity ie months trying to get pregnant foetal growth and adverse pregnancyoutcomes in a mildtomoderately iodinedeficient populationMethods We used the Norwegian Mother Father and Child Cohort Study MoBa and included pregnancieswith data on iodine intake and pregnancy outcomes Iodine intake was calculated using an extensive foodfrequency questionnaire in midpregnancy In addition urinary iodine concentration was available in a subsampleof pregnancies Associations were modelled continuously by multivariable regression controlling for a range ofconfounding factorsResults The median iodine intake from food was μgday and the median urinary iodine was μgLconfirming mildtomoderate iodine deficiency In nonusers of iodine supplements n low iodine intake μgday was associated with increased risk of preeclampsia aOR CI at vs μgday p overall preterm delivery before gestational week aOR at vs μgday p overall and reduced foetal growth SD difference in birth weight zscore at vs μgday p overall but not with early preterm delivery or intrauterine death In planned pregnanciesn having an iodine intake lower than μgday was associated with increased prevalence ofsubfecundity aOR at μgday vs μgday p overall Longterm iodine supplementuse initiated before pregnancy was associated with increased foetal growth SD on birth weightzscore p and reduced risk of preeclampsia aOR p but not with the other adverseContinued on next page Correspondence annelisebrantsaeterfhino2Department of Environmental Health Division of Infection ControlEnvironment and Health Norwegian Institute of Public Health PO Box Sk¸yen NO0213 Oslo NorwayFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cAbel BMC Medicine Page of Continued from previous pagepregnancy outcomes Urinary iodine concentration was not associated with any of the dichotomous outcomes butpositively associated with foetal growth n p overall Conclusions This study shows that a low iodine intake was associated with restricted foetal growth and a higherprevalence of preeclampsia in these mildtomoderately iodinedeficient women Results also indicated increasedrisk of subfecundity and preterm delivery Initiating iodine supplement use in pregnancy may be too lateKeywords Mildtomoderate iodine deficiency Iodine intake Iodine supplement Pregnancy cohort Foetal growthPreeclampsia Preterm delivery Subfecundity The Norwegian Mother Father and Child Cohort Study MoBaBackgroundIodine is an essential micronutrient and an integral partof the thyroid hormones triiodothyronine T3 and thyroxine T4 The thyroid hormones regulate multiplemetabolic processes that are important in growth metabolism and reproduction Thyroid dysfunction hasbeen linked to menstrual disturbances reduced fecundityto become pregnant miscarriagegestationinduced hypertension preterm delivery andreduced foetal growth []abilityieIodine deficiency is highly prevalent in both low andhighincome countries even though deficiency is easilypreventable through salt iodization strategies as recommended by the World Health anization WHO []Worldwide iodine nutrition is recognized as one of thekey determinants of thyroid dysfunction [] Recent findings in two populationbased cohort studies have indicated that even mildtomoderate iodine deficiency mayaffect thyroid function in pregnant women [ ] Inaddition an abrupt increase in iodine intake caused byintroduction of salt iodization programmes or iodinesupplement use might temporarily affect thyroid function in populations that are mildtomoderately iodinedeficient [ ] While it is well documented that severeiodine deficiency poses reproductive risksincludingabortions stillbirths and impaired neurodevelopmentthe potentialimpact of mildtomoderate iodine deficiency on fertility and pregnancy outcomes remainslargely unknown []It has been well known for many decades that iodinedeficiency reduces fecundity in livestock [] but we haveidentified only one study that has investigated this association in humans [] The study sample included women trying to get pregnant and a low urinary iodineconcentration UIC μgg creatinine wassignificantly associated with delayed conception compared to aUIC within the normal range ¥ μgg creatinine []A few studies have investigated associations betweeniodine status and risk of adverse pregnancy outcomesie preeclampsia preterm delivery pregnancy loss andor birth anthropometrics in mildtomoderate iodine deficiency [] but the studies were underpowered toidentifyforpotential minorchangesrisksinthe effect ofdichotomous outcomes and most have reported nullfindings For birth weight some studies have reportedreduced birth weight in mildtomoderate iodine deficiency [ ] but a recent systematic review found noevidence ofiodine supplement or saltiodization on prenatal growth in mildtomoderate iodine deficiency [] However the quality of the evidencewas assessed as very low [] Therefore there is still amajor knowledge gap as to whether mildtomoderateiodinepregnancyoutcomesdeficiencyfertilityaffectsandIn the present study we used data from the Norwegian Mother Father and Child Cohort Study MoBaa large pregnancy cohort with detailed informationabout food intake supplement use and a number ofobstetric outcomes [] We have previously documented that the MoBa pregnant women were mildtomoderately iodine deficient at a group level definedby WHO criteria [] and that there was a large variation in iodine intake between participants due to fewfood sources mainly milk and fish and supplementuse [] We also found that iodine intake was associated with thyroid function in pregnancy and that alow maternaliodine intake in pregnancy was associated with poorer child neurocognitive development atages and years [ ] In MoBa three different exposures are available as measures of iodine intake calculated iodine intake from food reported useof iodinecontaining supplements and UIC in a subsample of women Consequently this large prospective study offered a unique opportunity to add newknowledge about the role of mildtomoderate iodinedeficiency on subfecundity and adverse pregnancyoutcomesMethodsThe aim of the current study was to examine if iodine intake was associated with subfecundity ie months trying to get pregnant stillbirth preeclampsia preterm delivery and birth anthropometrics in alarge cohort of mildtomoderately iodinedeficientwomen 0cAbel BMC Medicine Page of Subjects and designThis study is based on MoBa a prospective populationbased pregnancy cohort initiated and maintained by theNorwegian Institute of Public Health [] Women pregnant in their first trimester were recruited from all overNorway during years to and were asked toanswer questionnaires available in Norwegian only atregular intervals during pregnancy and after birth Pregnancy and birth records from the Medical Birth Registryof Norway MBRN are linked to the MoBa database[] The women consented to participation in ofthe pregnancies The cohort now includes children mothers and fathers The currentstudy is based on version of the qualityassured datafiles released for research in and restricted to participants recruited from to because the MoBafood frequency questionnaire FFQ was included in thedata collection from March To be included in thecurrent study participants had to have responded to ia baseline questionnaire Q1 around gestational weekGW covering general health and sociodemographicinformation and ii the FFQ Q2 around GW andiii to be registered in MBRN with a singleton deliveryWe excluded women who reported use of thyroid medication at any time during pregnancy Given the largesample size and low rates of missing values ¤ onlypregnancies with information on all covariates were included FFQs with more than three blank pages or withcalculated energy intakes outside the range MJday were excluded [] Some women participate inMoBa with more than one pregnancy The final studypopulation comprisedpregnancieswomen for the analyses of pregnancy outcomes and planned pregnancies for the analysis of subfecundity UIC was available in a subsample of pregnanciesand was measured in GW A flow chart of inclusion isillustrated in Fig Exposure variable iodine intakeThe MoBa FFQ is a comprehensive semiquantitativequestionnaire specifically designed and validated forMoBa [ ] Participants responded to the FFQaround GW and were asked to report their averageintake since becoming pregnant GW Food frequencies were converted to food amounts using standard Norwegian portion sizes and daily intakes of energyand nutrients were calculated using FoodCalc [] andthe Norwegian food composition table [] Data on thecontent of more than food supplements was collected from suppliers [] Participants with unrealisticenergy intakes in the FFQs ie or MJday ormore than three blank pages were excluded from thestudy sample Iodine intake measured by the FFQ haspreviously been validated and it shows good agreementwith days weighed food diary [] and urinary iodineconcentration UIC [ ] Use of supplements was reported in the FFQ and in the general questionnaires fordifferent time periods Timing of the first reported usewas coded in four categories never weeks beforepregnancy GW and GW Although we assessed iodine intake in pregnancy withan FFQ covering the average food intake in the first months of pregnancy we propose that this iodine intakeFig Flow chart of inclusion Only complete cases were included had missing values on one or more covariates Asterisk indicates that thefood frequency questionnaire FFQ was in use from 0cAbel BMC Medicine Page of also can serve as an indicator of habitual iodine intakeprior to pregnancyUrine samples were collected at the routine ultrasoundexamination offered free of charge to all Norwegianwomen in GW UIC was determined at the NationalInstitute for Health and Welfare in Helsinki Finland byinductively coupled plasmamass spectrometry using anAgilent ICPMS system Agilent Technologies IncSanta Clara CA USA The limit of quantification was μgL and the linearity was excellent up to μgLr The coefficient of variation was Pregnancy and birth outcomesAll outcomes except subfecundity were based on information in the MBRN The subfecundity outcome whichapplied only to planned pregnancies was based on reported time months to conception reported in the general questionnaire in GW Subfecundity was defined as months trying to getpregnant for planned pregnancies of the womenhad reported that pregnancy was planned and also reported time to pregnancy The wording of the questionwas How many months did you have regular intercourse without contraception before you became pregnant Women with in vitro fertilization were notexcludedIntrauterine death was defined as death before birth or death during birth It also includedregistered intrauterine deaths where the time of deathwas not specified Abortions oflive foetuseswere not includedPreeclampsia was defined if any of the following conditions were checked off in the pregnancy record iHELLP syndrome ie haemolysis elevated liver enzymes and low platelet count ii eclampsia iii earlyivonset preeclampsia diagnosed before weeksmild preeclampsia orsevere preeclampsiaInNorway all pregnant women receive free antenatal careBlood pressure measurement and proteinuria analysisare carried out at each antenatal visit According toguidelines issued by the Norwegian Society of Obstetricsand Gynaecology the diagnostic criteria for preeclampsia are blood pressure after weeks gestationcombined with proteinuria greater than dipstick onat least two occasionsvPreterm delivery was defined as delivery before GW and as early preterm when delivered before GW Gestational age in days was determined based onthe routine ultrasound examination given free of chargeto all women in GW or it was calculated based ontime from the first day of the last menstruation periodin the few women where ultrasound data was missing Preterm delivery was also categorized by deliveryinitiationie spontaneous preterm delivery pretermlabour or preterm prelabour rupture of the membranesor iatrogenic preterm delivery induced or primary caesarean delivery on maternal or foetal indicationsBirth weight was examined as four outcomes crudebirth weight gram standardized birth weight zscoreie birth weight adjusted for child sex and gestationalage based on all deliveries in MBRN small for gestational age SGA gestational age and sexadjusted zscore percentile and large for gestational ageLGA gestational age and sexadjusted zscore percentile Outcomes on birth weight and gestational ageat birth were recoded to missing if birth weight for gestational age and sex was ± standard deviations fromthe mean n since these data suggested misreporting of either birth weight or gestational lengthHead circumference was examined as a crude measurecm Recorded head circumference cm wassuspected as misreporting and recoded to missingPlacenta weight was examined as a crude measuregram Recorded placenta weight g wassuspected as misreporting and recoded to missingOther variablesCovariates were included in the statistical modelsbased on previous knowledge and directed acyclicgraphs DAGs see Additional file Figure S1 Maternal age at the time of birth was obtained from thebirth registry Maternal prepregnancy body massindex BMI education ¤ ¥ years marital status marriedcohabitant yesno parity previous pregnancies ¥ weeks ¥ history ofchronic illness asthma diabetesinflammatory boweldisease rheumatic disease epilepsy multiple sclerosisor cancer before or during pregnancy yesno smoking before pregnancy no occasional daily use ofin vitro fertilization in current pregnancy yesnoand use of a folic acid supplement within the intervalfrom weeks before to weeks after conception yesno were obtained from questionnaire GW Maternal energy intake fibre intake as marker of ahealthy diet use of probiotic milk products yesnoand total intake of the omega3 fatty acids EPA andDHA were calculated based on the FFQ GW Also use of dietary supplements other than the onescommonly recommended for pregnant women ieother than vitamin D folic acid and iron was obtained from the FFQ yesno Information on smoking in pregnancy was obtained from questionnaire andif available questionnaires GW and childs age months three categories no reportedsmoking in pregnancy reported occasional smokingor stopped smoking before GW and daily smokingat any time in pregnancy and had not stopped smoking before GW 0cAbel BMC Medicine Page of Statistical methodsStatistical analyses were performed in STATA version Stata Corp College Station TX Associations were estimated by linear regression analysesfor continuous outcomes and logistic regression fordichotomous outcomesIn sensitivity analyses forthe outcome subfecundity Cox regression was usedto modelcontinuousvariableto pregnancytimeasaAssociations between iodine from food and outcomes and UIC and outcomes were modelled flexiblywith restricted cubic splines Since some motherswere included with more than one pregnancy we specified person clusters by using the option vcecluster person_ID in models in STATA which relaxes the assumption of independence of the observations and produces robust estimates of variance pvalues are reported for overall associations betweencontinuous exposures and outcomes testing H0 noassociation by testing the coefficients of all splinetransformations equal to zero In addition tests fornonlinearity were performed by testing the coefficients of the second and third spline transformationsequal to zero Covariates were included in the modelsbased on DAGs Continuous covariates eg maternalage BMI and energy intake were modelled flexiblyby restricted cubic splines if there was evidence ofnonlinear associations determined by inspecting theestimated associations while controlling for other covariates in the model otherwise they were modelledlinearly We reportthe specific covariates for eachoutcome in the respective tables and figures Tabularresults of the graphs included in this paper are provided in Additional file Tables S1S3firstneverreported useIodine supplement use was modelled as any reported iodine supplement use in GW and timing ofstarted beforeconception started in GW started in GW Potential effect modification by iodine intake from foodwas explored including an interaction term between iodine from food modelled by restricted cubic splines andthe supplement use variable Potential interactions wereexplored by testing all interaction coefficients equal tozero If the interaction terms were not statistically significant iodine from food was not included in the finalmodels Women in the control group were all nonusersof iodinecontaining supplements In the sensitivity analysis we restricted the control group to women who hadreported use of dietary supplements other than thestandard recommended ones The use of this restrictedcontrol group could control for the behaviour of takingan extra vitaminmineral supplement and could to someextentin themultisupplementsother nutrientscontrolalsoforWe did not include power calculations as no relevantcomparableestimates wereavailableineffectpopulationsA p value was considered statistically significantand results are reported including robust confidence intervals CI Only participants with completedata on all covariates were included in the analyses dueto the low rate of missing values in total of eligibleparticipants had missing on one or more covariatesResultsThe median calculated iodine intake from food was μgday IQR μgday Table Seventyfourper cent had an iodine intake from food lower than theestimated average requirement for pregnant women defined by the Institute of Medicine ie μgday[] and only reached the recommended intake inpregnancy by the WHO ie ¥ μgday [] withoutincluding supplements The median UIC measured inn was μgL and had UIC μgLThis is well below the WHO recommendation iemedian UIC ¥ μgL for pregnant women and median ¥ μgL for nonpregnant []Some groups of women could be identified as having aparticularly low UICfor example all nonusers ofiodinecontaining supplements of all participantsmedian UIC μgL Furthermore noniodine supplement users who consumed less than dL milkyoghurtper day of all participants had a median UIC of μgL and those who excluded dairy products entirelyfrom their diet of all participants had a medianUIC of μgLIodine intake from food correlatedstrongly with the reported intake of milkyoghurtSpearman r and moderately with the intake oflean fish Spearman r The women with availableUIC measurements of the total study populationhad a similar calculated iodine intake from food by theFFQ and equal frequency distribution of reported iodinesupplementthe women without UICmeasurementsuseasThere were marginal variations in iodine intake fromfood use of iodinecontaining supplements and UIC bybackground characteristics Table Table S4 in Additionalfile shows background characteristics bycategories of iodine intake from food and use of iodinecontaining supplements Iodine from food was weaklycorrelated with fibre intake Spearman r afteradjusting for energy intake indicating a weak negativeassociation with this indicator of a healthy dietParticipants in MoBa that were excluded from thestudy sample due to missing values on one or more ofthe covariates n did not differ in iodine intake from food UIC or any of the outcomes prevalenceofpreeclampsiasubfecundityintrauterinedeath 0cAbel BMC Medicine Page of Table Iodine exposures by characteristics of the study population n Study populationStudy sample n Maternal age at delivery mean SD years ¥ Prepregnancy BMI mean SD kgm2 Parity or moreMaternal education ¤ years years yearsMarriedcohabitant YesNoSmoking in pregnancy NoOccasionallyDailyChronic illness NoYesCouples incomeLowMediumHighMissingIodine supplement in pregnancy NoYesReported use in GW Vitamin D supplement Multivitaminmultimineral Folic acid beforeearly pregnancy Maternal energy intake median IQR MJ Iodine from foodmedian IQR μgday Iodine supplementGW UICa n median IQR μgL 0cAbel BMC Medicine Page of Table Iodine exposures by characteristics of the study population n ContinuedStudy populationIodine from foodmedian IQR μgdayIodine supplementGW UICa n median IQR μgLIodine from food median IQR μgday ¥ 96b 114b 129baUrinary iodine concentration UIC was measured in a subsample of n pregnant women in mean gestational week SD Iodine intake from foodand use of iodinecontaining supplements were comparable in this subgroup versus the whole study samplebRestricted to nonusers of iodinecontaining supplementspreterm delivery or birth weight zscore The prevalence of iodine supplement use was lower in excludedparticipants vs p Descriptive statistics of the pregnancy and birth outcome variables is provided in Table There were someoverlaps between the outcomes Of the preeclampticpregnancies were preterm deliveries and resulted in SGA infants while of preeclampticpregnancies were both preterm and SGASubfecundityThe association between iodine intake from food and subfecundity was Ushaped and iodine intake in the intervalbetween approximately and μgday was associatedwith the lowest likelihood of subfecundity Fig Compared with an intake of μgday reference OR the aOR at μgday was CI and at μgday aOR was CI p overall There was no data in MoBa on supplement useTable Pregnancy and birth outcomes n before months prepregnancy thus supplement use wasnot included as a variable in the model for subfecundityand women were included in the analysis regardless oftheir reported iodine supplement use later However insensitivity analyses women who reported use of iodinecontaining supplements in the time period weeks before conception were excluded and this did notchange the results results not shown Time to pregnancyin months was also modelled as a continuous variable byCox regression and the findings were consistent with theresults for subfecundity In the subsample of women withUIC measurements GW who did not report currentsupplement use at the time of UIC sampling there was noassociation between UIC in pregnancy and prevalence ofsubfecundity n p Intrauterine death preeclampsia and preterm deliveryIn nonusers of iodinecontaining supplementsiodineintake from food lower than μgday was associatedStudy sampleaMedian IQRTime to pregnancy monthsbGestational length weeksBirth weight gBirth weight zscore by gestational age and sexPlacenta weight gHead circumference cmSubfecundity months bIntrauterine deathPreeclampsiaPreterm delivery GW Spontaneous preterm delivery weeksEarly preterm delivery weeksSmall for gestational age percentileStudy sampleaLarge for gestational age percentileaSmall differences in numbers are explained by missing databOnly for planned pregnancies with available data on time to pregnancy cPrevalence in the subsample with UIC measurements n Number with outcome range Percent with outcome 100c 0c 27c 29c 22c 004c 82c 106c 0cAbel BMC Medicine Page of Table For preeclampsia longterm supplement useie use initiated before pregnancy was associated witha decreased prevalence aOR CI p and the effect estimate was not attenuatedwhen restricting the reference group Regarding pretermdelivery the results were not consistent Longterm supplement use was associated with an increased risk ofpreterm delivery also when restricting to spontaneouspreterm delivery whereas more shortterm supplementuse initiated in pregnancy was associated with a decreased risk of early preterm deliveryThere was no evidence of effect modification by habitual iodine intake from food for any of the associationsstudied between iodine supplement use and outcomesAlso supplement use was not associated with iodine intake from food Thus models presented were not adjusted for iodine intake from foodIn light of results in previous studies in MoBa [ ]we additionally tested a potential confounding effect ofuse of milk products containing probiotic bacteria yesno in the models with the outcomes preeclampsia andpreterm delivery and for preeclampsia a potential confounding effect of vitamin D supplement use The resultsdid not change and therefore these variables were notincluded in the final modelsChild anthropometrics at birthA low iodine intake from food less than μgdayas well as a low UIC below μgL was associatedwith lower birth weight and lower birth weight zscoreadjusted for gestational length child sex and standardized Fig Compared with an intake of μgdayreference mean zscore was SD lower at μgday CI and SD lower at μgday CI p overall In fullterm babies born in GW a SD difference in zscore corresponds to g A low iodine intake from foodwas also associated with a reduced risk of being LGAie having a birth weight in the top percentile forchild sex and gestational age at birth and an increasedrisk of being SGA percentile Fig Results didnot change when restricting the definition of SGA to thebelow percentiles or on birth weight zscore resultsnot shown The curve shapes indicated similar associations for UIC but they did not reach statistical significance Fig Iodine intake from food was also associated with placenta weight and head circumference but when adjusting for child birth weight the associations were nolonger present Additional file Figure S6 Again thecurve shapes indicated similar associations for UIC butthey did not reach statistical significance results notshownFig Habitual iodine intake from food GW and estimatedprevalence of subfecundity months trying to get pregnant inplanned pregnancies n subfecundity Theassociation was modelled by logistic regression adjusting formaternal age BMI parity education smoking before pregnancyenergy intake and fibre intake The curve represents the estimatedprevalence when all covariates are set to their means and theshaded area illustrates the robust confidence interval Thehistogram shows the distribution of the exposure For the crudeassociation see Additional file Figure S2with increased prevalence of preeclampsia and pretermdelivery but not with early preterm delivery or intrauterine death Fig Compared to an intake of μgdayreference an intake of μgday was associated withan increased risk of preeclampsia aOR CI and an increased risk of preterm deliveryaOR CI At an intake of μgday the adjusted odds ratio of preeclampsia was CI and for preterm delivery it was CI Fig For preterm delivery less than week excludingparticipants with preeclampsia did not change the resultsresults not shown When subdividing into spontaneousand iatrogenic preterm delivery the shape of the associations looked similar but only remained significant foriatrogenic PTD p Additional file Figure S4We did not examine associations between UIC andintrauterine death prevalence or early preterm delivery prevalence due to lack of power to detectpotential differences in the subsample of women withavailable UIC data We found no associations betweenUIC and preterm delivery or preeclampsia Additionalfile Figure S5Use of an iodinecontaining supplement in GW was borderline associated with a reduced risk of intrauterine death however the association was attenuatedwhen restricting the reference group ie nonuse ofiodinecontaining supplements to only include womenwho reported use of dietary supplements other than theones commonly recommended for pregnant women 0cAbel BMC Medicine Page of Fig Iodine from food and adverse pregnancy outcomes in nonusers of iodinecontaining supplements Sample size intrauterine death n intrauterine deaths preeclampsia n preeclampsia and preterm delivery n preterm and earlypreterm The associations were modelled by logistic regression adjusting for maternal age BMI parity education smoking in pregnancy energyintake and fibre intake For the crude associations see Additional file Figure S3Use of an iodinecontaining supplement was associated with increased birth weight but the estimates wereattenuated when restricting the reference group to participants using nutrient supplements other than thestandard recommended ones Table Then only longterm use initiated before pregnancy remained statistically significant However use ofiodinecontainingsupplements was associated with a reduced risk of beingSGA aOR p and longtermuse was associated with an increased risk of being LGAaOR p Overall the results indicated that use of iodine supplements increased birthweight and especially longterm use SD or gin fullterm babiesDiscussionThe main finding in this uniquely large pregnancy cohortis that a low iodine intake lower than μgday wasconsistently associated with reduced foetal growth acrossall three measures of exposure ie iodine from food UICand iodine supplement use This strengthens the evidenceof a causal relationship Also both a low iodine intakefrom food lower than μgday and no iodine supplement use were associated with an increased risk of preeclampsia This association was not detected for UIC butUIC was only measured in a subsample of women anda single spot UIC provides a very poor measure of iodine status at the individuallevel Thus the analyseswith UIC as the measure of exposure were underpowered to detect small differences in risk for dichotomousoutcomes A low iodine intake from food was associated with an increased risk of preterm delivery but theresults for supplement use on this outcome were notconsistent We also found indications that low iodineintake from food was associated with an increased riskof subfecundity but the design of this study includingonly women who had already succeeded in becomingpregnant and measuring the exposure after the outcome was far from optimal to study the associationwith this outcome 0cAbel BMC Medicine Page of Table Use of iodinecontaining supplements and pregnancy and birth outcomesNumberCrude modelsOdds ratio CIAdjusted modelsaRestricted controlsbp value Odds ratio CI p value Odds ratio CIIntrauterine death Any iodine supplement use GW First report of iodine supplementNever nonsupplement userBefore pregnancycGW GW 13451b ref ref ref Preeclampsia Any iodine supplement use GW First report of iodine supplementNever nonsupplement userBefore pregnancycGW GW 13451b ref ref ref Preterm delivery GW Any iodine supplement use GW First report of iodine supplementNever nonsupplement userBefore pregnancycGW GW 13405b ref ref ref Early preterm delivery GW Any iod | Thyroid_Cancer |
"Proteasomes are found in both the cell nucleus and cytoplasm and play a major role in the ubiquitindependent and independent nonlysosomal pathways of intracellular protein degradation Proteasomes are alsoinvolved in the turnover of various regulatory proteins antigen processing cell differentiation and apoptosis Todetermine the diagnostic value of serum proteasome in antineutrophil cytoplasmic antibody ANCAassociatedvasculitis AAV we investigated patients with AAV at various stages of the diseaseMethods Serum 20Sproteasome was measured by ELISA in patients with MPOANCAassociated microscopicpolyangiitis MPA and renal involvement Thirty of the patients provided serum samples before the initialtreatment and provided samples during remission provided samples at both time pointsResults The mean serum 20Sproteasome level was significantly higher in the activevasculitis patients ± ngmL n compared to the inactivevasculitis patients ± ngmL n p and controls ± ngmL p There were significant positive correlations between the serum 20Sproteasome level and the Birmingham Vasculitis Activity Score BVAS r p the ANCA titer r p the white blood cell WBC count r p the platelet count r p and the serum Creactive protein CRP level r p There were significant negative correlationsbetween the serum 20Sproteasome level and both the hemoglobin concentration r p and theserum albumin level r p In a multiple regression analysis there was a significant positivecorrelation between the serum 20Sproteasome level and only the BVAS results p In a receiveroperating curve analysis the area under the curve for the serum 20Sproteasome level was which is higherthan those of the WBC count and the serum CRP level Conclusion The serum level of 20Sproteasome may be a useful marker for disease activity in AAVKeywords ANCAassociated vasculitis 20Sproteasome Disease activity Microscopic polyangiitis Proteasome Correspondence khiratokyomedacjp1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki JapanFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMaruyama BMC Rheumatology Page of BackgroundProteasomes are located in both the nucleus and cytoplasm of cells and they play a major role in theubiquitindependent and ubiquitinindependent nonlysosomal pathways of intracellular protein degradation[ ] Proteasomes are also involved in the turnover ofvarious regulatory proteins eg ratelimiting enzymes[] and proteins for cellcycle control [] or transcriptional regulation [] antigen processing [] cell differentiation [] and apoptosis [] The 26S proteasome is amulticatalytic enzyme with a highly ordered structurecomposed of at least different subunits arranged intwo subcomplexes a 20S core and a 19S regulator p [] The 20Sproteasome is composed of four ringsof nonidentical subunits two rings are composed ofseven alpha subunits and the other two rings are composed of seven beta subunits Three of the seven betasubunits have proteolytic sites the 1 2 and 5 subunits are associated with caspaselike trypsinlike andchymotrypsinlike activities respectively [] These 12 and 5 subunits cleave peptide bonds at postacidicbasic and hydrophobic amino acid residues respectively [] However subunits 1 2 and 5 could be replaced with 1i 2i and 5i by interferongamma IFNÎ and this IFNÎinducible proteasome isotype is calledthe immunoproteasome []The serum proteasome levels of patients with malignanttumors are elevated because the proteasome is overexpressed in tumor cells In patients with multiple myelomaserum proteasome concentrations have been shown to beassociated with disease severity and activity [] theserum proteasome concentrations were significantly elevated in patients with multiple myeloma compared tocontrols in multiple myeloma versus monoclonal gammopathies of undetermined significance MGUS and in active versus smoldering multiple myeloma [] Similarlyelevated serum proteasome levels were also reported inautoimmune diseases characterized by Bcell abnormality[] In the present study to determine the diagnosticvalue of the serum proteasome concentration in antineutrophil cytoplasmic antibody ANCAassociated vasculitisAAV we investigated patients with myeloperoxidaseMPOAAV at various stages of the diseasePatientsPatients and controlsWe analyzed the cases of patients with MPOANCAassociated microscopic polyangiitis MPA and renal involvement The diagnosis of MPA was based on theEuropean Medicines Agency algorithm [] and patientswith other types of systemic vasculitis including eosinophlic granuromatosis with polyangiitis granulomatosis with polyangiitis and antiglomerular basementdisease were excludedOf the MPOAAV patients provided serumsamples before the initial treatment and providedsamples during remission provided samples both before the initial treatment and during remission The Birmingham Vasculitis Activity Score BVAS was used toevaluate patients disease activity and remission was defined as a BVAS of As controls healthy volunteersand patients with chronic kidney disease CKD wereinvestigated The causes of CKD were nephrosclerosisn chronic glomerulonephritis n diabeticnephrosclerosis n and autosomal dominant polycystic kidney disease n Sample collection and analysisThe serum samples measured by a commerciallyavailable enzymelinked immunosorbent assay ELISAkit Enzo Life Science Plymouth Meeting PA USin duplicate In brief 96well microtiter plates werecoated with a mouse anti20Sproteasome alpha6subunit monoclonal antibody and left overnight at °C followed by blocking with phosphatebuffered saline PBS containing bovine serum albumin for h atroom temperature RT A serum sample was thenadded to each well and the plates were incubated for h at RT A rabbit anti20Sproteasome polyclonalantibody was then added to each well and the plateswere incubated for h at RT followed by incubationwith a horseradishperoxidaselabeled goat antirabbitIgG antibody for h at RT The plates were finallyincubated with chromogen tetramethylbenzidine andhydrogen peroxide for min at RT and then addedwith N hydrochloride acid solutionBetween these steps the plates were washed five timeswith Trisbuffered saline The plates were immediatelyread on a microplate reader Sunrise Remote® TecanJapan Kanagawa Japan set at nm with nm as areference wavelength The inter and intraassay variationswere Statistical analysesAll statistical analyses were performed using PASW Statistics software ver IBM Japan Tokyo for Windows The data are expressed as means ± standarddeviations or as numbers with percentages of the totalThe chisquare test with Yates continuity correctionand Fishers exact test were used for differences in categorical variables and posthoc comparisons Bonferronicorrection were performed to detect differences amongthree groups The MannWhitney Utest was used fortwogroup comparisons and we conducted an analysisof variance ANOVA to assess differences among threeor more groups posthoc comparisons were made usingthe BonferroniDunn test Correlations were determinedusing Spearmans univariate correlation test and a linear 0cMaruyama BMC Rheumatology Page of regression analysis The multiple linear regression analysis included the covariates shown to be significantly associated with the serum 20Sproteasome levelin thecorrelation analysis and the data are expressed as standardized regression coefficients We applied comparative receiveroperatingcharacteristic ROC curvesand the area under the curve AUC to assess the diseaseactivity accuracy of the the serum 20Sproteasome leveland inflammatory variables Pvalues were accepted assignificant at but in the comparisons of three ormore groups the critical pvalue was divided by thenumber of comparisons being madeResultsThe subjects characteristicsThe characteristics clinical symptoms and laboratorydata among the subjects of the three groups the activevasculitis patients the inactivevasculitis patients andthe controls are shown in Table At the testing therewas no patients treated with any immunosuppressant inboth active and inactive vasculitis but allinactivevasculitis patients had treated with corticosteroids dosesof prednisolone ± mgdaySerum 20Sproteasome levelsAs illustrated in Fig the mean level of serum proteasome in the activevasculitis patients ± ngmL was significantly higher than those in the inactivevasculitis patients ± ngmL p andthe controls ± ngmL p There weresignificant positive correlations between the serum 20Sproteasome levels and the BVAS results r p the MPOANCA titers r p theWBC counts r p the platelet countsTable Characteristics of subjectsAge yearsGender malefemaleBirmingham vasculitis activity scoreClinical symptomsFeverWeight lossArthralgiaEpiscleritis or uvitisSinusitisHearing lossAlveolar hemorrhageInterstitial lung diseaseArrhythmiaPericarditisHeart failureRapidly progressive glomerulonephritisPeripheral nerve damageLaboratory dataANCA titer UmLWhite blood cell mm3Hemoglobin conc gdLPlatelet count 104mm3Serum albumin gdLSerum creatinine mgdLSerum Creactive protein mgdLSerum 20Sproteasome mgdLDoses of prednisolone mg daily P vs Inactivevasculitis P vs ControlsMPOANCA associated vasculitisActivevasculitis n ± Inactivevasculitis n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Controlsn ± ± ± ± ± ± ± ± 0cMaruyama BMC Rheumatology Page of Fig The serum levels of 20Sproteasome Closed circles means bars standard deviations circles the values for individual patientsr p and the serum CRP levels r p There were significant negative correlations between the serum 20Sproteasome levels and boththe hemoglobin concentrations r p and the serum albumin levels r p In the multiple regression analysis there was a significant positive correlation between the serum 20Sproteasome levels and only the BVAS results p Table In the activevasculitis patients there was no associationbetween the serum 20S proteasome levels and clinicalsymptoms except for pulmonary involvement Supplementary file S1 The mean serum 20S proteasome level inthe activevasculitis patients with interstitial lung diseasen ± ngmL was significantly higherthan those in the activevasculitis patients withoutpulmonary involvement n ± ngmLp and those in activevasculitis patients withalveolar hemorrhage n ± ngmL p There was no association between the serum 20Sproteasome levels and the percentages of crescent formation renal histological classification Berdens classification [] or renal symptoms patients with chanceproteinuriahematuria and patients with rapidly progressive glomerulonephritisThe diagnostic potential for disease activityThe optimum cutoff levels for the disease activity ofvasculitis were identified from the ROC curves for theWBC count 7250mm3 serum CRP level mgdL and serum 20Sproteasome level ngmLFig The area under the curve AUC for the serumTable Correlation between the serum 20Sproteasome level and clinical parametersAgeBirmingham Vasculitis Activity ScoreANCA titerWhite blood cellHemoglobin concPlatelet countSerum albuminSerum creatinineSerum Creactive proteinUnivariate analysisrPvalue Multivariate analysisPvalue 0cMaruyama BMC Rheumatology Page of polymyositis serum proteasome levels were correlatedwith serum creatinine kinase levels and serum proteasome levels were associated with disease activity [] Inthe present study elevated serum 20Sproteasome levelswere also demonstrated in patients with AAV Althoughthere was no relationship between the MPOANCA titersand the serum 20Sproteasome levels these elevationswere associated with disease activity ie the BVASTherefore the serum level of 20Sproteasome may be auseful marker for disease activity in AAVThe mechanisms that underlie the elevated serum proteasome observed in patients with AAV are not yetknown Several serum biomarkers are filtrated at theglomerulus and reabsorbed and catabolized by proximaltubular cells and the serum levels of such biomarkers inpatients with renal insufficiency are elevated due to lowurinary filtration Because we found no relationship between serum 20Sproteasome levels and serum creatinine in AAV patients we conclude that elevated serumproteasome is not associated with renal functionIn a previous investigationthe serum proteasomelevels in patients with multiple myeloma were elevateddue to overexpression in tumor cells but the mechanisms underlying these elevations in autoimmune diseases were not clarified [] On the other hand thestructure and function of serum proteasome in healthydonors and patients with autoimmune diseases SLE andRA were maintained in the same manner as the intracellular forms [] However rings of proteasomes inthe serum of patients with autoimmune diseases weredifferent from those in healthy donors and those ringscontained immunosubunits 2i and 5i [] Consideringthat proteasomes from nonimmunocompetent cells donot contain immunosubunits [] it was speculated thatserum proteasome in patients with autoimmune diseasesmay have its fraction structure added by an immunocompetent cell origin that is different from that in normal individuals Therefore the elevated serum proteasome levelsin AAV may also be associated with the activation of immunocompetent cells Further investigations are neededto clarify the mechanism by which the proteasome isreleased into the circulationBortezomib a proteasome inhibitor prevents the degradation of proteins marked by ubiquitination by inhibiting the 26S proteasome [] The main effects ofbortezomib are NFκB inhibition inhibition of cell proliferation by the stabilization of cyclindependent kinasesFig The comparative ROC curves for three measurements ofdisease activity Solid line serum levels of 20Sproteasome Dashdotted line WBC counts Dashed line serum CRP levels Dotted linereference line20Sproteasome level was which is higher thanthose of the WBC count and the serum CRP level On the ROC curve the serum 20Sproteasomehad sensitivity and specificity for the diseaseactivity Although the specificity of the serum 20Sproteasome level was less than that of the serum CRPlevelserum 20Sproteasome level was superior to that of the serumCRP level Table sensitivity ofthetheDiscussionPrevious studies have demonstrated that the serum20Sproteasome levels are elevated in individuals with autoimmune diseases In patients with various autoimmune diseases including systemic lupus erythematosusSLE polymyositis Sjögrens syndrome antiphospholipidsyndrome rheumatoid arthritis RA systemic sclerosisautoimmune hepatitis and myasthenia gravis the serumproteasome levels were higher than in the controls[] The levels were especially and significantly high inthe patients with SLE polymyositis Sjögrens syndromeRA and autoimmune hepatitis [] In patients withWhite blood cell countTable Comparative ROC curves for parameters of disease activity confidence intervalArea under the curveSerum Creactive proteinSerum 20SproteasomePvalue Optimal cutoff levelsSensitivity Specificity 0cMaruyama BMC Rheumatology Page of the induction of apoptosis by the activation of cJunNH2terminal kinase the stabilization of proapoptotic proteinsand transcription factors and tumor suppressors and theinduction of cell death by activation of the terminalunfolded protein response [] Bortezomib has been approved for clinical use in patients with multiple myelomaand bortezomib treatment has implications for antibodymediated immune diseases as well []The efficacy of bortezomib was demonstrated in amouse model of MPOANCAassociated glomerulonephritis [] That is in antiMPOassociated glomerulonephritisinduced by immunizing MPOdeficientmice with murine MPO followed by irradiation and thetransplantation of wildtype bone marrow proteinuriaalbuminuria and hematuria were significantly reducedcompared to the controls by both standard steroidcyclophosphamide treatment and bortezomib treatment[] Moreover the percentage of glomeruli with crescent or necrosis formation was reduced by both treatments The clinical efficacy of bortezomib for AAV hasnot been determined because only one case of an AAVpatient treated with bortezomib was reported In thatcase complete remission could not be achieved by acombination treatment with corticosteroid cyclophosphamide and rituximab therefore bortezomib mgm2week for weeks was added [] After the additionof bortezomib the patient achieved complete remissionand the doses of corticosteroid could be withdrawn []Thusthe proteasome may be associated with thedevelopment of AAV and inhibition of the proteasomemay be effective for inducing the remission of AAVOur study has several limitations The study population was small and limited to MPOAAV patients withrenal involvement and thus further studies are neededto compare patients with PR3AAV or nonrenal vasculitis In addition this was a retrospective crosssectionalstudy a larger prospective longitudinal study includingvasculitis patients with relapse would provide more definitive results Since the present study was performed atone facility it is necessary to verify the accuracy of theELISA test Moreover allinactivevasculitis patientswere treated with corticosteroids at the testing so treatments themselves may affected to decreased levels in inactive vasculitis Therefore further studies are needed tocompare AAV patients without treatments at the testingor to investigate other diseases patients treated withwithout corticosteroids Finally although we did demonstrate that serum 20Sproteasome levels were elevated inour patients with AAV the cause of this elevation wasnot identified In patients with multiple myeloma elevation of serum 20Sproteasome may be associated withoverexpression in tumor cells or abnormal cellular turnover [] On the other hand elevation of serum 20Sproteasome was observed in septic patients and therelation between elevated serum 20Sproteasome levelsand increased lymphocyte apoptosis was demonstratedin critically ill patients [] In patinets with RA andSLE it was speculated that the expression of inflammatory cytokines may have influenced the elevation of theserum 20Sproteasome [] Althoug elevated serum20Sproteasome in active AAV may be reflected an acutephase response there was no significant correlation between the serum 20Sproteasome levels and serum CRPlevels in the multiple regression analysis To clarify themechanisms of the serum 20Sproteasome elevation invasculitis patients further in vitro and ex vivo investigations are neededConclusionThe serum levels of 20Sproteasome in our patients withactive MPOAAV were significantly elevated compared tothe levels in the patients with inactive MPOAAV and thecontrols Moreover the serum levels of 20Sproteasomewere related to the BVAS results The serum level of 20Sproteasome may therefore be a useful marker for diseaseactivity in AAVSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s41927020001374Additional file Supplementary file S1 The relationships betweenthe serum 20S proteasome levels and the patients clinical symptomsAbbreviationsAAV Antineutrophil cytoplasmic antibodyassociated vasculitisANCA Antineutrophil cytoplasmic antibody BVAS Birmingham VasculitisActivity Score MPA Microscopic polyangiitis MPO MyeloperoxidaseAcknowledgementsPart of this study was reported at the 18th International Vasculitis and ANCAWorkshop Tokyo and it was published in Rheumatology suppl as an abstractAuthors contributionsHM and KH designed the study executed the experiments and participatedin the data management statistical analyses and reporting logicalinterpretation and presentation of the results MY KO and RT participated inthe data collection MT and HS took part in the logical interpretation andpresentation of the results KH and MK coanized the course of the workAll authors read and approved the final manuscriptFundingThis study was supported in part by a research grant to KH from MSD KKTokyo Japan The funds for this study were used only to purchase ELISAkitsAvailability of data and materialsAll of the raw datasets used and analyzed in this study are available uponreasonable request from the corresponding authorEthics approval and consent to participateAll procedures performed in this study involving human participants were inaccordance with the ethical standards of the institutional committee andwith the Declaration of Helsinki and its later amendments orcomparable ethical standards The study protocol was approved by the 0cMaruyama BMC Rheumatology Page of Zoeger A Blau M Egerer K Feist E Dahlmann B Circulating proteasomesare functional and have a subtype pattern distinct from 20S proteasomes inmajor blood cells Clin Chem Froment C UttenweilerJoseph S BousquetDubouch MP Matondo MBes JP Esmenjaud C Lacroix C Monsarrat B BurletSchiltz O Aquantitative proteomic approach using twodimensional gel electrophoresisand isotopecoded affinity tag labeling for studying human 20S proteasomeheterogeneity Proteomics Hideshima T Richardson P Chauhan D Palombella VJ Elliott PJ Adams JAnderson KC The proteasome inhibitor PS341 inhibits growth inducesapoptosis and overcomes drug resistance in human multiple myelomacells Cancer Res Boccadoro M Man G Cavenagh J Preclinical evaluation of theproteasome inhibitor bortezomib in cancer therapy Cancer Cell Int Fröhlich K Holle JU Aries PM Gross WL Moosig F Successful use ofbortezomib in a patient with systemic lupus erythematosus and multiplemyeloma Ann Rheum Dis Bontscho J Schreiber A Manz RA Schneider W Luft FC Kettritz RMyeloperoxidasespecific plasma cell depletion by bortezomib protectsfrom antineutrophil cytoplasmic autoantibodiesinducedglomerulonephritis J Am Soc Nephrol Novikov P Moiseev S Bulanov N Shchegoleva E Bortezomib in refractoryANCAassociated vasculitis a new option Ann Rheum Dis 2016751e9 Yousef AA Suliman GA Mabrouk MM The value of correlation of serum 20Sproteasome concentration and percentage of lymphocytic apoptosis incritically ill patients a prospective observational study Crit Care R215Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsEthics Committees of Tokyo Medical University Ibaraki Medical CenterWritten informed consent for inclusion in the study was obtained from allpatientsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki Japan 2Department of Intensive CareMedicine Tokyo Medical University Ibaraki Medical Center Ami IbarakiJapan 3Department of Nephrology Tokyo Medical University ShinjukuTokyo JapanReceived April Accepted May ReferencesArrigo AP Tanaka K Goldberg AL Welch WJ Identity of the 19S prosomep with the large multifunctional protease complex of mammaliancells the proteasome Nature Hershko A Ciechanover A The ubiquitin system Annu Rev Biochem Murakami Y Matsufuji S Kameji T Hayashi S Igarashi K Tamura T Tanaka KIchihara A Ornithine decarboxylase is degraded by the 26S proteasomewithout ubiquitination Nature Hershko A Roles of ubiquitinmediated proteolysis in cell cycle control CurrOpin Cell Biol Kho CJ Huggins GS Endege WO Hsieh CM Lee ME Haber E Degradationof E2A proteins through a ubiquitinconjugating enzyme UbcE2A J BiolChem Stoltze L Nussbaum AK Sijts A Emmerich NP Kloetzel PM Schild H Thefunction of the proteasome system in MHC class I antigen processingImmunol Today Baz A Henry L Caravano R Scherrer K Bureau JP Changes in the subunitdistribution of prosomes MCPproteasomes during the differentiation ofhuman leukemic cells Int J Cancer Pasquini LA Marta CB Adamo AM Pasquini JM Soto EF Relationshipbetween the ubiquitindependent pathway and apoptosis in different cellsof the central nervous system effect of thyroid hormones Neurochem ResJung T Grune T Structure of the proteasome Prog Mol Biol Transl Sci Groll M Ditzel L Lowe Löwe J Stock D Bochtler M Bartunik HD Huber RStructure of 20S proteasome from yeast at a resolution Nature Boes B Hengel H Ruppert T Multhaup G Koszinowski UH Kloetzel PMInterferon gamma stimulation modulates the proteolytic activity andcleavage site preference of 20S mouse proteasomes J Exp Med Jakob C Egerer K Liebisch P Türkmen S Zavrski I Kuckelkorn U Heider UKaiser M Fleissner C Sterz J Kleeberg L Feist E Burmester GR Kloetzel PMSezer O Circulating proteasome levels are an independent prognosticfactor for survival in multiple myeloma Blood Egerer K Kuckelkorn U Rudolph PE Rückert JC Dörner T Burmester GRKloetzel PM Feist E Circulating proteasomes are markers of cell damageand immunologic activity in autoimmune diseases J Rheumatol Watts R Lane S Hanslik T Hauser T Hellmich B Koldingsnes W Mahr ASegelmark M CohenTervaert JW Scott D Development and validation of aconsensus methodology for the classification of the ANCAassociatedvasculitides and polyarteritis nodosa for epidemiological studies AnnRheum Dis Berden AE Ferrario F Hagen EC Jayne DR Jennette JC Joh K Neumann INoël LH Pusey CD Waldherr R Bruijn JA Bajema IM Histopathologicclassification of ANCAassociated glomerulonephritis J Am Soc Nephrol 0c" | Thyroid_Cancer |
"Lung adenocarcinoma LAD is a prevalent type of bronchogenic malignant tumor and one of themost critical factors related to human death Long noncoding RNAs lncRNAs are involved in many complexbiological processes and have been emerged as extremely important regulators of various cancers LINC02418 anovel lncRNA hasnt been mentioned in previous studies on cancer development Therefore its important todefine the potential function of LINC02418 in LADMethods Gene expression was examined by RTqPCR or western blot CCK8 colony formation TUNEL andtranswell assays were utilized to study the role of LINC02418 in LAD The interaction of miR46773p withLINC02418 or KNL1 was verified through luciferase reporter RIP and RNA pulldown assaysResults High expression of LINC02418 was observed in LAD specimens and cells Downregulation of LINC02418obstructed the proliferation and motility of LAD cells Moreover LINC02418 negatively modulated miR46773pexpression and miR46773p overexpression could repress cell proliferation and migration Moreover kinetochorescaffold KNL1 expression was negatively modulated by miR46773p but positively regulated by LINC02418Furthermore miR46773p could bind with LINC02418 or KNL1 Finally KNL1 overexpression reversed theinhibitory function of LINC02418 deficiency in the malignant behaviors of LAD cellsConclusions LINC02418 contributes to the malignancy in LAD via miR46773pKNL1 signaling providing aprobable therapeutic direction for LADKeywords LINC02418 miR46773p KNL1 LAD Correspondence 171428256qqcom junqingxu126com Tao Wang and Ruiren Zhai are cofirst authors3Department of Radiology Xijing Hospital Fourth Military Medical UniversityXian Shaanxi China4Department of Radiology Shenzhen University General Hospital ShenzhenUniversity Clinical Medical Academy No1098 Xueyuan Avenue NanshanDistrict Shenzhen Guangdong ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cWang BMC Pulmonary Medicine Page of BackgroundAs a main subtype of nonsmall cell lung cancer NSCLC lung adenocarcinoma LAD is one of the leadingcauses of cancerrelated deaths around the world [ ]Previous studies have identified major pathways involvedin LAD development including the activationof the EGFR KRAS and ALK signals [] Althoughvarious molecular targeted therapies have been developed the prognosis of LAD patients is still disappointing[ ] Therefore defining the molecular mechanismsunderlying this fatal disease would be of considerablesignificance for LAD treatmentLong noncoding RNAs lncRNAs are a subtype ofnoncoding RNAs ncRNAs consisting of RNA over nucleotides in length that are not translated intoproteins [ ] Mounting evidence has elucidated a pivotal role of lncRNAs in cancer progression For examplelncRNA ANCR regulates EZH2 expression to inhibitbreast cancer progression [] LncRNA LINC00312 expedites cell migration and vasculogenic mimicry in LADby binding with YBX1 [] LncRNASNHG1 modulatesDNMT1 expression to accelerate the development ofgastric cancer [] A large number of studies haveunmasked that lncRNAs exert critical functions in diverse processes in lung cancer such as proliferation []migration [] and epithelialmesenchymaltransitionEMT [] Additionally the involvement of lncRNAsin LAD is also recognized For instance lncRNA DGCR5suppresses the expression of miR223p to promoteLAD progression [] Galectin3 activates TLR4NFκBsignaling pathway to facilitate the development of LADvia upregulatingexpression []LncRNA MIR31HG overexpression promotes cell proliferation in LAD and associates with poor prognosis []The oncogenic property of some common lncRNAs inLAD has been widely reported such as LINC00707 []MIR31HG [] OIP5AS1 [] MALAT1 [] etcHerein we intended to probe into the biological role ofa novellncRNA in LAD Using microarray analysisdifferentially expressed lncRNAs were identified Thetop ten upregulated lncRNAs in LAD samples werechosen for further analysis in LAD cells LINC02418 wasselected to be the research object in current studylncRNANEAT1MechanisticallymiRNAslncRNAs can interact with microRNAsto upregulate messenger RNAsmRNAs [ ] therefore forming a competing endogenous RNA ceRNA pathway Here bioinformatics analysis and mechanismbased experiments wereused to determine the miRNAs that could bind withLINC02418 Similarlythe target mRNAs of miR46773p were identified In this study wasdesigned to investigate whether lncRNA LINC02418could affect LAD development via regulating itsdownstream genesMethodsCell culture and transfectionHuman LAD cell lines A549 SPCA1 H1299 and PC and normal lung epithelial cells BEAS2B were boughtfrom the Cell Bank of the Chinese Academy of Sciencesand incubated with Dulbeccos modified Eagles mediumDMEM Invitrogen Life Technology Inc Carlsbad CAcontaining fetal bovine serum FBS with CO2 at °C in humid air All cell lines were authenticated viaSTR profiling before usingThesuppression of LINC02418 expression wasachieved by shLINC0241812 ShLINC024181 andshLINC024182 were obtained from GenePharmaShanghai China LINC02418 and KNL1 were overexpressed with pcDNA31 vectors Invitrogen CarlsbadUSA MiR46773p mimics were applied to elevatemiR46773p expression MiR46773p mimics and NCmimics were also bought from GenePharma ShanghaiChina The transfection of above plasmids was conducted by use of Lipofectamine® agent InvitrogenClinical samples collection and microarray analysisThree pairs of LAD and matched noncancerous tissuesamples were acquired from patients including twomale one female two patients years old one patient years old one patient at stage of III two patients atstage of IIIIV who received operation in the SecondAffiliated Hospital of Air Force Medical University Patients enrolled in this study signed the informed consents Ethic Committee of the Second Affiliated Hospitalof Air Force Medical University has approved samplecollection of this research Microarray analysis was implemented to profile the expression of lncRNAs in LADIn detail total RNA was isolated from three pairs of tissues and quantified utilizing NanoDrop ThermoWaltham MA USA followed by qualitychecking byuse of Agilent Bioanalyzer Agilent TechnologiesSanta Clara CA USA Subsequently the GeneChipIVT express kit Affymetrix Santa Clara CA USAwas utilized to label the qualified RNA samples and thenAffymetrix GeneChip Primeview Human cDNA microarray was used for hybridization in line with the manufacturers guides After that data were analyzed usingGeneChip Scanner AffymetrixRealtime quantitative polymerase chain reaction RTqPCRTotal RNA was extracted utilizing TRIzol InvitrogenThermo Fisher Scientific Inc and diluted to ngmlComplementary DNA cDNA synthesis was conductedvia applying Taqman Advanced miRNA cDNA SynthesisKit or Pyrobest DNA Polymerase and MMLV ReverseTranscriptase Thermo Fisher Scientific USA RTqPCR was then operated using One Step SYBR® Prime 0cWang BMC Pulmonary Medicine Page of Script¢ RTPCR Kit II Takara Biotechnology Co LtdDalian China based on the producers protocol Geneexpression relative to GAPDH or U6 was assessed usingthe 2ÎÎCt methodCell counting kit8 CCK8 assayCell proliferation was estimated utilizing the CCK8 kitBoster based on the manufacturersrequirementsBriefly cells à were supplemented into 96wellplates After cell adhesion each well received μlCCK8 solution and then cells were further incubatedfor h at °C Cell proliferation ability was monitored by detecting absorbance at nm utilizing microplateInstrumentsHopkinton MA USA at the indicated time points and hBioTekreaderEL340Colony formation assayCells were seeded in sixwell plates and grown in mediawith FBS Two weeks laterthe colonies werefixated using methanol and dyed using crystalviolet for half an hour Colonies with over cells werecounted manuallyTranswell assayFor invasion estimation à cells were added ontothe upper chambers BD Biosciences San Jose CAUSA with Matrigelcoating and incubated in DMEMDMEM supplementing with FBS was put into thebottom chambers Twentyfour hours later cotton swabswere used to scrape off cells in the upper chamber Themethanol and crystal violet were separately use tofasten and color the cells in the lower chamber For themigration assays transfected cells were seeded into theupper chambers with no Matrigelcoating while othersteps were similar to that in invasion assays Finally theinvaded or migrated cells were counted using aninverted biological microscope magnification Western blotCells were lysed by use of RIPA Beyotime ShangahiRocheChina containing protease inhibitor cocktailPleasanton CAand phenylmethylsulfonylfluorideRoche Protein samples were then subjected to sodiumdodecyl sulfate polyacrylamide gel electrophoresis SDSPAGEfollowed by transferring onto nitrocelluloseNC membranes SigmaAldrich After blocking via5skim milkthe membranes were cultured withprimary antibodies dilution against Bax Bcl2Ecadherin Ncadherin MRP2 MRP9 KNL1 CellSignaling Technology Danvers MA followed by incubation with secondary antibodies dilution foran hour at room temperature βactin or GAPDH wasthe loading control Thereafter signals were capturedwith the employment of the ECL chromogenic substrateTerminaldeoxynucleoitidyl transferase mediated nick endlabeling TUNEL assayAfter fixation and permeabilization cells were processedwith dUTPend labeling Clontech Mountain View CAand ²6diamidino2phenylindole DAPIin succession After that cells were observed and analyzed underfluorescent microscope Olympus Tokyo JapanLuciferase reporter assayA549 and SPCA1 cells à grown in a 96wellplates were cotransfected with ng of LINC02418WT or LINC02418Mutreporters Sangon BiotechShanghai China and miR46773p mimic or NC mimicsinto LAD cells by use of Lipofectamine InvitrogenCarlsbad California USA KNL1WT or KNL1Mut reporters Sangon Biotech Shanghai China and miR46773p mimics or NC mimics were also cotransfectedinto indicated LAD cells After transfection for daysthe luciferase activity normalized to Renilla luciferase activity was examined by luciferase reporter assay systemPromega Madison WIRNA immunoprecipitation RIP assayRIP assay was conducted utilizing the EZMagna RIP kitMillipore Billerica MA according to the manufacturers protocol A549 and SPCA1 cells at confluence were obtained and then lysed in complete RIPlysis buffer Cell extract was processed at °C for about h with RIP buffer which contained human Ago2 antibody or control IgG Millipore coated magnetic beadsAfter beads washed the RNA complexes were culturedwith Proteinase K to digest proteins RNA concentrationwas measured though employing a NanoDrop spectrophotometerThermo Scientific with the qualityassessed by a bioanalyser Agilent Santa Clara CAFinally the immunoprecipitated RNAs were purified andanalysed by RTqPCRRNA pulldown assayRNA pulldown assay was used to detect the probableinteraction among miR46773p LINC02418 and KNL1MiR46773p was biotinylated to be miR46773p biotinprobe by GenePharma Company Shanghai ChinaMiR46773p biotin probe and miR46773p nobiotinprobe were added into the lysates of A549 and SPCA1cells After h incubation Dynabeads M280 Streptavidin Invitrogen CA were put into above mixture Twohours later RNA in the pulled down complexes wasexamined using RTqPCR analysis after purification 0cWang BMC Pulmonary Medicine Page of Statistical analysisEach assay was implemented in triplicate and data wereexhibited as mean ± standard deviation SD Based onSPSS for Windows Version SPSS Inc Chicagostudents ttest was employed to compare differencesbetween two groups while oneway analysis of varianceANOVA was applied for the comparisons among noless than two groups P was deemed as statisticallysignificantResultsThe biological function of LINC02418 in LADTo identify the molecular mechanisms underlying LADdevelopment we first examined the differentiallyexpressed lncRNAs in LAD samples relative to pairednontumor ones The upregulated lncRNAs in LADtissues in comparison with adjacent normal tissueswere displayed using a heatmap Fig 1a Then thelinesFig 1bP top ten upregulated lncRNAs in LAD tissues were further analyzed in LAD cell lines via qRTPCR Resultsmanifested that compared to the normal BEAS2Bcells LINC02418 was expressed higher in four LADP However other celllncRNAs were not significantly differential expressedin the four kinds of LAD cells relative to control cellsP ns was no signifiFigure S1Acance Thus we chose LINC02418 as the researchobject in subsequent experiments A549 and SPCA1cells possessing highest LINC02418 level were used forlossof function assays To probe the biological role ofLINC02418 LINC02418 was knocked down in A549andshLINC0241812 with shNCtransfected cells as thescramble control The results showed that knockdownof LINC02418 resulted in an obvious decline inLINC02418 expression compared with control grouptransfection withSPCA1cellsbyFig The biological function of LINC02418 in LAD a Heatmap showing highly expressed LINC02418 in LAD b LINC02418 expression in LAD celllines and normal pulmonary epithelial cell line was examined by RTqPCR c The transfection efficiency of shLINC024181 and shLINC024182 inA549 and SPCA1 cells were measured by RTqPCR d CCK8 assay was performed to assess cell proliferation of A549 and SPCA1 cells after theknockdown of LINC02418 e Colony formation assay was carried out to detect the proliferation of A549 and SPCA1 cells in response toLINC02418 knockdown f Transwell assay was applied to access cell migration in A549 and SPCA1 cells after downregulation LINC02418 gWestern blot was used to examine the level of migrationrelated proteins after knocking down LINC02418 P 0cWang BMC Pulmonary Medicine Page of Fig 1c P Additionally CCK8 and colony forindicated that LINC02418 absencemation assaysinhibited the proliferation ofthese two LAD cellsFig 1de P TUNEL assay illustrated that cellapoptosis was promoted by LINC02418 depletionFigure S2A P As shown in Fig 1f P and S2B P the migration and invasion of shLINC0241812transfected cells were notably blockedin contrast to that in shNC group suggesting thatLINC02418 silencing weakened LAD cell migrationand invasion abilities Furthermore western blot analyses displayed that silencing LINC02418 suppressedthe expression of migrationrelated proteins MRP2and MRP9 which indicated that LINC02418 depletioncould inhibit cell migration Fig 1g Further whendownregulating LINC02418 the levels of Bax and Ecadherin were augmented while Bcl2 and Ncadherinexpression was declined Figure S2CIn totalLINC02418 is upregulated and LINC02418 knockdownsuppresses cell proliferation and motility in LADThe specific role of miR46773p in LADThereafter bioinformatics analyses were employed topredictthe miRNAs that could possibly bind withLINC02418 As a result miR46773p was then identified from starBase Fig 2a In order to investigatewhether LINC02418 could regulate miR46773p expression RTqPCR analysis was performed Interestingly anobservable increase in the expression of miR46773pwas observed in LAD cells with LINC02418 deficiencyP Additionally miR46773p wasFig 2bfound to be with a low expression trend in LAD celllines relative to BEAS2B cells Fig 2c P Subsequently we observed a significantly heightened level ofmiR46773p in miR46773p mimics group comparedwith NC mimics group Fig 2d P MoreovermiR46773p mimics suppressed cell proliferation in twoP Furthermore overexLAD cells Fig 2efpression of miR46773p notably impaired LAD cellP Moreover asmigration capability Fig 2gdisplayedupregulation2h miR46773pinFigFig The specific role of miR46773p in LAD a Potential binding targets for LINC02418 were predicted by bioinformatics analysis b Relativeexpression of miR46773p influenced by LINC02418 knockdown was detected via RTqPCR c Relative expression of miR46773p in LAD celllines and normal pulmonary epithelial cell line was examined by RTqPCR d The transfection efficiency of miR46773p mimics was checked byRTqPCR e CCK8 assay was carried out to test cell proliferation when A549 and SPCA1 cells were treated with miR46773p mimics f Colonyformation assay was used to detect cell proliferation in miR46773p mimicstransfected A549 and SPCA1 cells g Transwell assay was performedto examine the migration of A549 and SPCA1 cells treated with miR46773p mimics h Western blot was conducted to access the level ofmigrationrelated proteins in A549 and SPCA1 cells when overexpressing miR46773p P 0cWang BMC Pulmonary Medicine Page of decreased the expression of migrationrelated proteinsMRP2 and MRP9 Taken together miR46773p isdownregulated and serves a tumorrestraining part inLADKNL1 is a downstream target gene of miR46773pBased on research over the past decades miRNAs areknown to have a vital impact on cancer progression bydirectly modulating the expression of its target genes[] Herein candidates MAP 1LC3BIGSF3KNL1 MKL2 EFTUD2 CA12 MOCS1 and SLC31A1were identified as the potential targets of miR46773pfollowing analyses of the RNA22 and miRmap databasesFig 3a RTqPCR was then employed to assess the influence of miR46773p on the expression of these genesin A549 and SPCA1 cells Results indicated that onlyKNL1 expression was notably reduced by miR46773pmimics compared with that in NC mimics control groupin both the two LAD cells while the level of other genesnot Fig 3b P P Therefore KNL1 wasselected to carry out the following assays As illustratedin Fig 3c the level of KNL1 protein was also decreasedby miR46773p mimics Besides both mRNA andprotein expressions of KNL1 were also hampered in faceof LINC02418 knockdown Fig 3dInaddition LINC02418 miR46773p and KNL1 were allP Fig KNL1 is a downstream target gene of miR46773p a RNA22 and miRmap were used to research the potential target genes for miR46773p b The expression of the indicated mRNAs in miR46773p mimictransfected cells were tested using RTqPCR c The expression of KNL1protein was detected by western blot when upregulating miR46773p d The mRNA and protein levels of KNL1 were examined by RTqPCR andwestern blot analyses after downregulating LINC02418 e The enrichment of LINC02418 miR46773p and KNL1 in RISC complex was determinedby RIP assay f The binding site between miR46773p and LINC02418 was predicted by starBase left Luciferase reporter assay measured theluciferase activities of LINC02418WT and LINC02418Mut right g The binding site between miR46773p and KNL1 was predicted by starBaseupper Luciferase reporter assay measured the luciferase activities of KNL1WT and KNL1Mut h RNA pull down assay examined the enrichmentof LINC02418 and KNL1 in miR46773p biotin probe group miR46773p nobiotin probe served as negative control P P P 0cWang BMC Pulmonary Medicine Page of concentrated in antiAgo2 group instead of antiIgGgroup Fig 3e P Furthermore the binding sequences between miR46773p and LINC02418 were obtained from starBase Fig 3f left Then we found thatthe luciferase activity of pmirGLOLINC02418WT wasreduced by miR46773p mimics whereasthat ofpmirGLOLINC02418Mut showed no obvious alteration between miR46773p mimic group and NC mimicgroup Fig 3f right P Similarly starBase predicted the sequences of miR46773p and KNL1 wherethe binding occurred Fig 3g upper The luciferaseactivity of pmirGLOKNL1WT was also lowered by enhanced miR46773p while that of pmirGLOKNL1Mutwasnt affected Fig 3g lower P Moreover theresults of RNA pulldown assay testified that bothLINC02418 and KNL1 were enriched in miR46773pbiotin probe group Fig 3h P In sum KNL1 isthe downstream molecule of miR46773p in LADanalysesindicatedcotransfectionLINC02418 promotes the malignant phenotypes of LADcells through miR46773pKNL1 signalingSubsequently we planned to determine whetherLINC02418 influenced LAD developmentthroughmiR46773pKNL1 pathway RTqPCR and westernblotofpcDNA31KNL1recovered LINC02418 depletionlessened KNL1 expression in A549 cells Fig 4abP ns was no significance Then it was verified that KNL1 upregulation reversed the hinderingimpact of LINC02418 deficiency on A549 cell proliferation Fig 4cd P ns was no significanceAdditionally the stimulated cell apoptosis induced byLINC02418 silencing was rescued by KNL1 overexpresP ns was no significancesion Figure S3Ait was confirmed that overexpression ofSubsequentlyKNL1 offsetthe obstructivefunction caused byLINC02418 depletion on A549 cell motility Fig 4e andP ns was no significance AdditionallyS3BKNL1ofLINC02418 depletion on the levels of Bax Bcl2 Ecadherin and Ncadherin Figure S3C These findingssuggest that LINC02418 aggravates malignant behaviorsin LAD via miR46773pKNL1 pathwayupregulationneutralizedinfluencethattheDiscussionis the most commonLung adenocarcinoma LADsubtype of nonsmallcelllung cancer NSCLC accounting for the majority of diagnosed primary lungcancer cases and also with low 5year survival rate[ ] In recent few decades great progresses havebeen achieved in LAD treatment including antiPD1PDL1 therapy and targeted therapy [ ] In themeantime treatment strategies for LAD have also improved [] Nonethelessrate ofthe survivalpatients with LAD still remains poor Hence identifying effective targets is imperative for ing freshstrategies for LAD treatmentliterature has delineated thatRecently a great amount of work has uncovered thelncRNAs in multiple cancers and a growingveil oflncRNAs arebody ofimplicated in various malignanciessuch as LADbreast cancer and gastric cancer [ ] Nonetheless whether LINC02418 works in LAD has notbeen revealed Currently we found the notable upregulation of LINC02418 in LAD and the absence ofLINC02418 suppressed LAD cell proliferation andmotilitythemalignancy in LADimplying that LINC02418 promotesMiRNAs are also defined as a fraction of ncRNAswith the length of nucleotides [ ] Previous research suggests that miRNAs exert their functions in diverse cancers [ ] As an illustrationmiR4500 is downregulated and elicits an anticancerfunction through regulating HMGA2 expression incolorectal cancer [] MiR4315p targets UROC28to influence the expression of EMT markers in hepatocellular carcinoma [] MiR205 regulates E2F1 expression to promote the cisplatin sensitivity of gliomacells [] In LAD miRNAs also play important roleslike miR133 participates in LAD metastasis via targeting with FLOT2 [] MiR6293p inhibits SFTPCexpression to facilitate cell proliferation and is associated with poor survivalin LAD [] MiR608 andmiR4513 greatly enhance the prognosis of LADtreated with EGFRTKIs [] Present study showedthe decreased expression of miR46773p in LAD andrevealed that miR46773p overexpression suppressedcell proliferation and migration in LAD highlightingmiR46773p as a cancersuppressor in LADoftherenalcancerdevelopmentProteins translated from messenger RNAs mRNAsplay critical roles in cancer For instance EGFR enhances[]YWHAZ serves as an oncogenic gene in cervical cancer [] Also the oncogenic function of KNL1 hasbeen validated in cancer For example miR193b3psilencing promotes cell proliferation in gastric cancerthrough upregulating the expression of KNL1 []CeRNA hypothesis have been proposed and proven tobe transcripts crossregulated by competing certainmiRNAs [ ] To be specific lncRNA and mRNAcan competitively bind with the shared miRNA tomodulate cancer progression For instancelncRNAHOXDAS1 promotes liver cancer metastasis throughsponging miR130a3p and targeting SOX4 []LncRNA TUG1 facilitates the development of papillary[]LncRNAUCA1 exertsfunction inesophageal cancer through acting as the ceRNA ofthyroid cancer via miR145ZEB1 axisits oncogenic 0cWang BMC Pulmonary Medicine Page of Fig LINC02418 may promote malignancy in LAD by targeting miR46773pKNL1 axis ab The mRNA and protein expressions of KNL1 indifferently transfected groups were estimated by RTqPCR and western blot cd Cell proliferation in differently transfected groups was evaluatedby CCK8 and colony formation assays e Cell migration in differently transfected groups was detected by transwell assay P ns meansno significanceSOX4 [] Ourstudy revealed that miR46773pcould bind with LINC02418 and KNL1 and KNL1was the mediator downstream of LINC02418miR46773p signaling in LAD Finally rescue assays indicated that the inhibited LAD cell functions inducedby LINC02418 silencing were counteracted by KNL1overexpressionUpregulation of LINC02418 in LAD tissue sampleswas identified by a microarray analysis indicating theclinical potential of LINC02418 in LAD patients Thisstudy didnt elucidate the role of LINC02418 in clinicalinvestigatefeatures or prognosis Thus we willthe clinical value of LINC02418 in LAD in futureresearchIn LINC02418 contributes tomalignant phenotypes of LAD cells through sequestering miR46773p to boost KNL1 levelthrowinglight on the molecular mechanism of LINC02418 inLAD providing a novel target for LAD treatmentConclusionsLINC02418 facilitates malignant cell behaviorsinLAD via sponging miR46773p to upregulate KNL1expression 0cWang BMC Pulmonary Medicine Page of Supplementary informationSupplementary information accompanies this paper at httpsdoi101186s12890020012290Ethics approval and consent to participateThis study was approved by the Ethics Committee of the Second AffiliatedHospital of Air Force Medical University Patients involved in this work signedthe informed consents before sample collectionAdditional file Figure S1 A Expression pattern of lncRNAs inLAD cells and normal BEAS2B cell was tested by RTqPCR P P ns no significanceAdditional file Figure S2 A TUNEL assay measured cell apoptosisin LINC02418 downregulated cells B Transwell assay detected cellinvasion when knocking down LINC02418 C Western blot testedexpression of apoptosis and EMTrelated proteins in response toLINC02418 depletion P Additional file Figure S3 A TUNEL assay measured cell apoptosisin differently transfected groups B Transwell assay detected cellinvasion in differently transfected groups C Western blot testedexpression of apoptosis and EMTrelated proteins in differently transfected groups P ns no significanceAdditional file Supplementary Information file The originalunprocessed gel images for western blot data in Figs 1g 2h 3c d 4bS2C and S3CAbbreviationsANCR Angelman syndrome chromosome region ANOVA Analysis ofvariance CA12 Carbonic anhydrase CCK8 Cell counting kit8ceRNA Competing endogenous RNA DGCR5 DiGee syndrome criticalregion gene DMEM Dulbeccos modified Eagles medium DNMT1 DNAmethyltransferase E2F1 E2F transcription factor EFTUD2 Elongationfactor Tu GTP binding domain containing EGFR Epidermal growth factorreceptor EMT Epithelialmesenchymal transition EZH2 Enhancer of zeste polycomb repressive complex subunit FBS Fetal bovine serumGAS5 Growth arrest specific HMGA2 High mobility group AThook HOTAIR HOX transcript antisense RNA HOXDAS1 HOXD antisense growthassociated long noncoding RNA IGSF3 Immunoglobulin superfamilymember KNL1 Kinetochore scaffold LAD Lung adenocarcinomaLINC02418 Long intergenic nonprotein coding RNA lncRNAs Longnoncoding RNAs MALAT1 Metastasis associated lung adenocarcinomatranscript MAP LC3B Microtubule associated protein light chain betaMIR31HG MIR31 host gene miRNAs microRNAs MKL2 Myocardin like MOCS1 Molybdenum cofactor synthesis ns no significanceNEAT1 Nuclear paraspeckle assembly transcript NSCLC Nonsmall celllung cancer PVT1 Pvt1 oncogene RIP RNA immunoprecipitationRIPA Radioimmunoprecipitation assay RTqPCR Realtime quantitativepolymerase chain reaction SD Standard deviation SLC31A1 Solute carrierfamily member SNHG1 Small nucleolar RNA host gene SOX4 SRYbox transcription factor TIMP2 TIMP metallopeptidase inhibitor TLR4 Toll like receptor TUG1 Taurine upregulated TUNEL Terminaldeoxynucleoitidyl transferase mediated nick end labeling UCA1 Urothelialcancer associated YWHAZ Tyrosine 3monooxygenasetryptophan monooxygenase activation protein zeta ZEB1 Zinc finger Ebox bindinghomeobox AcknowledgementsWe appreciate all the people involved in this studyAuthors contributionsTW and RZ conceptualization project administration manuscript reviewdata analysis XL and KW investigation experiment record figures JXcorrespondence original manuscript writing All authors gave useful advicesThe authors read and approved the final manuscript002EFundingNoneAvailability of data and materialsRelevant data and materials have been presented within the manuscript andadditional filesConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thoracic Surgery The Second Affiliated Hospital of Air ForceMedical University Xian Shaanxi China 2Department of TumorCenter Sunshine Union Hospital Weifang Shandong China3Department of Radiology Xijing Hospital Fourth Military Medical UniversityXian Shaanxi China 4Department of Radiology Shenzhen UniversityGeneral Hospital Shenzhen University Clinical Medical Academy No1098Xueyuan Avenue Nanshan District Shenzhen Guangdong ChinaReceived June Accepted July ReferencesChou J Wang B Zheng T Li X Zheng L Hu J Zhang Y Xing Y Xi T MALAT1 induced migration and invasion of human breast cancer cells bycompetitively binding miR1 with cdc42 Biochem Biophys Res Commun Wei X Zhang K Qin H Zhu J Qin Q Yu Y Wang H GMDS knockdownimpairs cell proliferation and survival in human lung adenocarcinoma BMCCancer Janku F Stewart DJ Kurzrock R Targeted therapy in nonsmallcell lungcanceris it becoming a reality Nat Rev Clin Oncol Ihle NT Byers LA Kim ES Saintigny P Lee JJ Blumenschein GR Tsao A LiuS Larsen JE Wang J Effect of KRAS oncogene substitutions on proteinbehavior implications for signaling and clinical outcome J Natl Cancer InstPao W Girard N New driver mutations in nonsmallcell lung cancer LancetOncol Mensztern D Campo MJ Dahlberg SE Doebele RC Garon E Gerber DEGoldberg SB Hammerman PS Heist RS Hensing T Molecularlytargeted therapies in nonsmallcell lung cancer annual update JThorac Oncol Suppl 1S1Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin Zheng Y Liu L Shukla GC A comprehensive review of webbased noncoding RNA resources for cancer research Cancer Lett Gomes CC de Sousa SF Calin GA Gomez RS The emerging role of longnoncoding RNAs in oral cancer Oral Surg Oral Med Oral Pathol Oral Radiol Zhao L Wu D Sang M Xu Y Liu Z Wu Q Stachydrine amelioratesisoproterenolinduced cardiac hypertrophy and fibrosis by suppressinginflammation and oxidative stress through inhibiting NFkappaB and JAKSTAT signaling pathways in rats Int Immunopharmacol Peng Z Wang J Shan B Li B Peng W Dong Y Shi W Zhao W He D DuanM The long noncoding RNA LINC00312 induces lungadenocarcinoma migration and vasculogenic mimicry through directlybinding YBX1 Mol Cancer Hu Y Ma Z He Y Liu W Su Y Tang Z LncRNASNHG1 contributes to gastriccancer cell proliferation by regulating DNMT1 Biochem Biophys ResCommun Jing H Qu X Liu L Xia H A Novel Long Noncoding RNA lncRNALL22NC03N64E91 Promotes the Proliferation of Lung Cancer Cells and is aPotential Prognostic Molecular Biomarker for Lung Cancer Med Sci Monit Hao Y Yang X Zhang D Luo J Chen R Long noncoding RNA LINC01186regulated by TGFbetaSMAD3 inhibits migration and invasion throughepithelialMesenchymaltransition in lung cancer Gene Pan C Yao G Liu B Ma T Xia Y Wei K Wang J Xu | Thyroid_Cancer |
Scars Burns HealingVolume 1011772059513120940499 reuse guidelinessagepubcomjournalspermissions The Authors journalssagepubcomhomesbhKeloids are pathological scars that grow over time and extend beyond the initial site of injury after impaired wound healing These scars frequently recur and rarely regress They are aesthetically disfiguring can cause pain itching discomfort as well as psychological stress often affecting quality of life Many treatment modalities including surgical and nonsurgical have been explored and have been reported to be beneficial however none have been absolutely satisfactory or optimal for the treatment of all keloid subtypes to date This poses a major challenge to clinicians Often a combinational therapeutic approach appears to offer the best results with higher patient satisfaction compared to monotherapy The aetiopathogenesis of keloids is not fully elucidated however with recent advances in molecular biology and genetics insight is being gained on the complex process of scar formation and hence new therapeutic and management options for keloids In this paper we explore the literature and summarise the general concepts surrounding keloid development and review both current corticosteroids surgical excision siliconebased products pressure therapy radiotherapy cryotherapy laser therapy imiquimod and 5fluorouracil and emerging stem cell therapy mitomycin C verapamil interferons bleomycin botulinum toxin type A and angiotensinconverting enzyme inhibitors treatments Increased knowledge and understanding in this area may potentially lead to the discovery and development of novel therapeutic options that are more efficacious for all keloid typesKeywordsKeloids scar recurrence wound healing treatment managementLay SummaryKeloids are problematic scars that are difficult to treat and manage The aetiopathogenesis of keloids is not clear however recent advances in molecular biology and genetics are beginning to shed light on the underlying mechanisms implicated in keloid scar formation which will hopefully lead to the development of treatment options for all keloid types This review summarises current and emerging therapiesIntroductionWound healing is an intricate and complex series of processes comprising overlapping phases of inflammation granulation tissue formation and tissue remodelling and results in tissue structure integrity and damage being restored1 Abnormal wound healing can give rise to keloids which are benign dermal fibroproliferative nodular lesions that tend to recur after excision Keloid scars Faculty of Medical Sciences The University of the West Indies Cave Hill Campus Bridgetown Barbados West Indies Pine Medical Centre 3rd Avenue Belleville St Michael Barbados West IndiesCorresponding authorNkemcho Ojeh Faculty of Medical Sciences The University of the West Indies Cave Hill Campus PO Box St Michael Bridgetown BB Barbados West Indies Email nkemchoojehcavehilluwieduCreative Commons Non Commercial CC BYNC This is distributed under the terms of the Creative Commons AttributionNonCommercial License creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0c Scars Burns Healinginfluence in keloid aetiology9 Although no one specific gene has been associated with the development of keloids a number of genes and gene loci have been identified610 Genomewide association studies and admixture mapping studies have identified singlenucleotide polymorphisms across certain loci genetically linked to keloid development including the NEDD4 gene which encodes E3 ubiquitin ligase enzyme and the myosin genes MY01E and MYO7A10 Studies have also reported the involvement of several human leucocyte antigen HLA alleles p53 bcl2 and fas genes1014 Furthermore rare genetic disorders have been reported to present with spontaneous keloids including Dubowitz syndrome Bethlem myopathy RubinsteinTaybi syndrome Noonan syndrome Geominne syndrome and others1017 These lines of evidence suggest that genetic factors play a role on keloid predispositionPathophysiology of keloidsKeloid pathology is complex involving both genetic and environmental factors Keloids form as a result of abnormal wound healing and excessive dermal fibrosis Development of keloids has been linked to overproliferation and reduced apoptosis of dermal fibroblasts overproduction of collagen fibres and other extracellular matrix ECM components as well as abnormal ECM production and remodelling1 Various cytokines growth factors and proteolytic enzymes have been implicated in the formation of keloids including transforming growth factor TGF epidermal growth factor EGF vascular endothelial growth factor VEGF plateletderived growth factor PDGF connective tissue growth factor CTGF tumour necrosis factorα TNFα insulinlike growth factor1 IGF1 fibroblast growth factor FGF interleukin6 IL6 and matrix metalloproteinases MMPs31418 Furthermore signalling pathways such as Tolllike receptor signalling SMAD signalling and fibronectin have been reported to be associated with keloid development1819Histopathology of keloidsHistologically keloids comprise an abundance of unordered dermal collagen and vasculature with high inflammatorycell infiltrate and overactive mesenchymal cells1415 In addition to collagen elastin fibronectin and proteoglycans are deposited in excess amounts in keloid scars9 Collagen creates frequent crosslinks in ordinary wounds whereas collagen is irregularly anised in keloids forming nodules in the dermis20 During normal Figure Earlobe keloids as a consequence of ear piercingarise from skin trauma or inflammation and may develop years after the initial insult and rarely regress2 The scar tissue extends beyond the original wound site and can be disfiguring and cause psychosocial issues impairing quality of life3 In addition patients may present with symptoms such as burning pain pruritus movement limitation and hyperaesthesia2AetiologyThe aetiology of keloids is still poorly understood The most common regions of the skin for keloids include upper arms skin overlying joints chest shoulders and headneck regions particularly the ear lobes Figure The anatomical location of a keloid appears to alter its morphological characteristics Some keloids can develop spontaneously however most occur years after local trauma and other events including inflammation surgery burns elective cosmesis foreign body reactions acne insect bites vaccinations or mechanical force45Epidemiology and keloid geneticsThe incidence of keloids is highest among darkerpigmented persons of African Asian and Hispanic descent and is estimated to be in the range of Males and females have an equal risk of developing keloids6 although incidence is slightly increased in females likely attributable to them having more cosmetic procedures like ear piercing7 Persons aged years are also at a higher risk of developing keloids Additional risk factors include having blood type A hyperIgE and hormonal peaks during pregnancy or puberty8Familial keloid case studies and twin studies support the notion that genetic factors have an 0cOjeh for the treatment of keloids2627 Intralesional TAC injections have been shown to reduce scar volume and height improve scar pliability and diminish associated scar pain and itching8 as well as prevent recurrence3 Corticosteroids have antiinflammatory and antimitotic properties1 Several other mechanisms have been reported by which corticosteroids reduce keloid scar including inhibition of fibroblast growth attenuation of procollagen and glycosaminoglycan synthesis reduction of endothelial budding and enhancement of collagen and fibroblast degeneration2829 Corticos teroids inhibit TGF1 expression and induce apoptosis in fibroblasts inhibit VEGF and alphaglobulins which are involved in the wound healing process28 VEGF which promotes angiogenesis was reported to be highly expressed in keloid fibroblasts compared to controls but exogenous addition of the glucocorticoid dexamethasone suppressed its expression in vitro32 Furthermore VEGF expression was overexpressed in keloid tissue which later reduced following intralesional TAC injections in vivo33TAC is typically administered at intervals of weeks until pruritic and painassociated symptoms diminish and the scar flattens34 The dose of TAC is in the range of mgmL depending on the size and anatomical location of the lesion and the age of the patient34 TAC is used either alone as a monotherapy or in combination with other treatment modalities13 The response rates to corticosteroid injections vary clinically with regression rates in the range of reported after one year and recurrence rates in the range of reported after five years3 Combined therapy comprising surgical excision followed by TAC treatment also varied with reported recurrence rates in the range of Previous clinical studies where TAC was used alone reported efficacy and good clinical outcome with this treatment including reduced keloid height length width related pruritus and erythema and improved pliability3637 A recent randomised parallelgroup study that compared the role of intralesional TAC fractional CO2 laser or intralesional verapamil in the treatment of keloid in patients showed reduction in scar height vascularity and pliability in all three groups using the Vancouver Scar Scale score however pigmentation was not completely resolved with any of the treatments The response was fastest with TAC followed by verapamil and laser and this was statistically significant38 However intralesional TAC in combination with other treatment modalities such as 5fluorouracil 5FU pulsed dye laser PDL surgery interferon IFNα2b verapamil and Figure Current and emerging treatment strategieswound healing the early wound immature collagen type III can be modified into mature collagen type I In keloid tissue it mostly comprises disanised collagen types I and III made up of palestaining hypocellular collagen clusters lacking nodules or surplus myofibroblasts21 Furthermore recent research has provided four distinct findings only present in keloid specimens presence of keloidal hyalinised collagen presence of a tonguelike advancing edge underneath normalappearing epidermis and papillary dermis a horizontal cellular fibrous band in the upper reticular dermis and a prominent fascialike band22Treatment of keloidsCurrently various forms of treatment for keloids exist however no single treatment has proven to be the most effective This review will explore and discuss current and emerging treatment modalities Figure Some of the ongoing or completed clinical trials of keloid therapy registered on clinicaltrialsgov and accessed on May are summarised in Table Studies with the status terminated suspended or withdrawn were excludedCurrent treatmentsCorticosteroidsSeveral corticosteroids can be used for the treatment of scars including triamcinolone acetonide TAC hydrocortisone acetate dexamethasone and methyl prednisolone24 However since TAC has been the most widely used corticosteroid 0c Scars Burns HealingTCNVI esahPTCNlebacilppa toN vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoC yaMnodessecca vogslairTacinlil Cnoypareht doek fo slairt lliacinilC elbaTeltit ydutSreifitnediTCNlebacilppa toN ybdewoll iednotecaenoonicmairt llanoiseartnliilsdoeK fo tnemtaerTeht n iof resal OC lanoitcarFidoeKli sdoretS lanoiseartnl iI svyparehTdoretSdetsissA resaL lanoitcarFTCNlebacilppa toNTCNlebacilppa toNTCNVI esahP TRS ypareht noitadar liaicifrepuS iEnmativ enocilis enositrocordyh yparehtyhcarbetaresod ihgh tnavudahtij wnoisicxe lacigruS cihportrepyh idoeKlidoeKlidoeKl TRS yparehTnoitadaRi l laicifrepuS fonoitauavEevitcepsorteRA ilsdoeK fo tnemtaerT rof yparehtyhcarBetaResoDhgHi isracSdoeKdnal dnaytili llbareoTeht gnitauavEydutSevitarapmoCdezimodnaRA TCNlebacilppa toN llamsaphcir teetap suogootuAllidoeKl llllamsaPhciR teetaP suogootuAyb racSdoeK fo tnemtaerTli TCB®tiKnegeRhti wdenatboiTCBtiKnegeRhti wdenatbOi l ieg tcartxenono ro draugracS noitolamredeM xirtaciCracScihportrepyh ildna sdoeK fo tnemtaerTeht rof separehTi l acipoTowT foycaciffE sracScihportrepyHTCNI esahP muiclac fonoitcenj i laruomutartnIidoeKlrefsnartortceeybdewol llof edirohclTCNIII esahP dnaenoonicmairt llanoiseartnlIidoeKl ienodnefriP l ilsdoeK fo tnemtaerTeht rof noitaroportceEmuiclaCl acipoTdnaenoonicmairTl lanoiseartnl I foycaciffEienodnefrip lacipotnoitcenji UFidoeKllygooisyhpohtaPdna tnemtaerT gnirracSdoeKli iIOALSDAL sracSdoeK fo tnemtaerT roflTCNII esahP I esahP inks gnviltneavuqeiil dereyalib fargilpAsresalidoeKl iil tenragmunmuamuirttymumydoenideslupgno l dna OC lanoitcarFidoeKl fo tnemtaerTn ii sresaL tenraGmunmuAmuirttymumydoeNli fonoitneverPdna tnemtaerTeht rof farg ilpA foydutS toli PAisdoeKl ilsdoeKdesicxE foecnerruceR desluPgnoLdnaedixoDnobraCi l anoitcarFgnisUydutSA deunitnoC cihportrepyhTCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl eht rof eciveDdesaB saGnogrAnahti WyparehtoyrC lanoiseartnlI sracScihportrepyHdnadoeK fo tnemtaerTli TCNIII esahPxirtaciC fonoitacil ppa lacipoT sracs cihportrepyHcihportrepyhidoekl ilsracS sdoeKdna sracScihportrepyH fo tnemtaerTeht n i IXRTACCI sracScihportrepyHdnadoeK fo tnemtaerTli TCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl rof yparehtoyrC lanoiseartnl I foesUeht fonoitauavEevitcepsorPl 0cOjeh TCNVI esahPresal PTKmneht foycaciffE idoekl cihportrepyheahcun siliadoekl xirtacic xirtacic racs lacigrus racS reifitnedi vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoCdeunitnoC elbaTeltit ydutSTCNlebacilppa toNresal eyddesluP cihportrepyh idoeKl desluPmnagnisU tnemtaerT racSnohtdWesluP fo tceffEi TCNlebacilppa toN thgil AVUderutcafunamnamreG idoeklsracs amredorelcSsnoitidnoC rali imSdnaamredorelcS fo tnemtaerT rof thgLAVUi resaLeyD ecivedgnitti me gnisorbif rehtosnoitidnocTCNlebacilppa toN ecivederusserPTCNVI esahPl egenocilis lacipoT cihportrepyh idoeKlilERUSSERP sdoeK raE fo tnemtaerTeht n i eciveDerusserPidoeKl fo tnemtaerTeht no l eGenocili iSgnyrdfleS tnerapsnarT fo tceffEsracssracS l ianmodbAcihportrepyHTCNlebacilppa toNypareht noitadaRiidoeKl yrtsigeRnoitadaRdoeKlii TCNIII esahPnosahtemateB fonoitacil ppa lacipoT sracs cihportrepyHsretehtaC suoneV lartneC retfA sracSTCNI esahPII esahPTCN I esahPII esahP dna sll dicacidisuf dnaetareavl isdoeklidocitrococuglnoitadarri iBVU amredorelcs idoeKl inkS fo tnemtaerTeht n i yparehT thgL BVUil B teovartlUi leraunnaamounargl sracs siliadoekl enca desilacolxirtaM l amreDderetlAhti w snoitidnoC lecFVS suogootua fo snoitcenjI axal situc racs inkSCSDAxirtacic idoekl idevireDesopdA suogootuAhtil waxaL situCdna sracS foyparehT sll eCmetS lamyhcneseMTCNlebacilppa toN gnitti meAVUderutcafunamnamreG amredorelcs idoeKlsnoitidnoC ralimetsys thgil lamounarg sracs enca leraunna imSdnaamredorelcS rof thgLAVUi ilteovartlu VU FVS sll ecnoitcarf raucsavl lamorts FVS etahpsohp lynatit muissatop PTK sll ecmets l amyhcnesemdeviredesopdai hserF fo tnemtaerTeht rof resaLPTKmn l evoNa foydutS to liP CSDA licaruoroulf UFsracS lacigruS 0c Scars Burns HealingA variety of methods can be used for the surgical removal of keloids depending on the size of the keloid anatomical location skin type and age of patient52 These include linear closure and flap coverage excision with grafting Wplasty and Zplasty53 To reduce risk of keloid recurrence the surgeon performing the excision should establish tensionfree wound closure As a general rule closure of the wound should be accomplished with minimal tension and sutures leaving everted wound borders Zplasties threelayered sutures subcutaneousfascial tensile reduction sutures or local ï¬ap surgery can be employed on a casebycase basis5455 The final outcome of the scar is often positively correlated with the experience of the operating surgeon and technique utilised as well as the patients active participation in their wound care52Siliconebased productsSince the 1980s siliconebased products have been used in the treatment of keloids and hypertrophic scars with silicone gel or silicone gel sheeting considered as firstline therapy for minor keloids and hypertrophic scars2635 There are various other forms of silicone including creams sprays gel cushion and liquid24 The precise mechanism of action of silicone products is not fully understood but it is proposed that they enhance hydration and create an occlusive environment53 which influences fibroblast regulation and decreases collagen synthesis56 Silicone gel sheeting has been shown to have minimal side effects including local irritation which can be resolved quickly24 Studies have shown that the beneficial effects of silicone gel sheets include pain reduction tenderness and pruritus and flattening the keloid57 The silicone gel sheeting is recommended to be worn from two weeks after primary wound treatment for h for months58Studies have demonstrated an improvement of up to in keloid scars after using silicone gel sheeting35 and a decrease in the incidence rates of keloids and hypertrophic scars after surgery59 In addition controlled studies have reported the clinical effectiveness of silicone gel and silicone gel sheeting in the prevention and treatment of keloids3558 However a recent metaanalysis review60 and Cochrane review61 found that even though studies published data in support of the efficacy of silicone gel sheeting in the treatment and prevention of keloid and hypertrophic scarring they provided weak evidence and were of poor quality60 Therefore given Figure Auricular keloid surgical excision used as monotherapy Auricular keloid before a and after b surgical excisonsurgery all yielded significant improvements compared to treatment with TAC monotherapy339Intralesional steroid injections can cause several adverse side effects such as telangiectasis atrophy steroid acne pigmentary changes necrosis ulcerations and systemic side effects3 There is also significant pain associated with intradermal corticosteroid injections that can be reduced when local anaesthetic lidocaine is administered to control the pain44 Furthermore the side effects have been reported to diminish when intralesional TAC is used in combination with 5FU45Surgical excisionSurgical excision is a traditional method of removing keloids Figure However excision creates a new wound and can result in a similar or larger keloid47 Therefore surgical excision is not recommended as a monotherapy as it results in high recurrence rates in the range of For better postoperative surgical outcomes surgical excision is often combined with other forms of treatment including radiotherapy intralesional corticosteroid injections IFN injection bleomycin cryotherapy pressure therapy and silicone gel or sheeting82649 Successful use of dermal substitutes and epidermal skin grafting with keloid excision has also been reported50 A recent case series study in which patients with anterior chest wall keloids were given a treatment protocol consisting of complete excision Zplasty postoperative adjuvant radiotherapy and postsurgical wound selfmanagement reported excellent outcomes with a recurrence rate of only The use of steroid tape and injections helped to resolve the recurrence of keloids51 0cOjeh the lack of substantial evidence welldesigned clinical trials and studies are required to gain a better understanding of the effectiveness of siliconebased products in preventing and treating keloidsPressure therapyPressure therapy has been used to treat and manage keloids and hypertrophic scars for decades3563 It has been routinely employed as firstline treatment in the treatment of hypertrophic scarring resulting from burns64 The underlying mechanisms of action of compression techniques remain unclear however several hypotheses exist some of which include increased pressure to the scar surface reduces perfusion and decreased oxygen to the location of injury reduces collagen synthesis It is also thought that pressure increases apoptosis reduces scar hydration stabilising mast cells and decreases angiogenesis165 The application of pressure can be achieved using a variety of materials such as adhesive plaster moulds pressure earrings and customfitted splints16 which have improved scar cosmesis and rates of keloid recurrence66 A continuous pressure of mmHg preferably at the lower range soon after wound reepithelialisation for h per day for months is recommended166869 The efficacy of pressure therapy depends mainly on the anatomical location of the scar with trunk and limb areas being more appropriate sites for pressure therapy In addition a pressure garment is predominantly used for auricular keloids where pressure clips are commonly utilised after surgery6670 As an adjuvant therapy this form of pressure garment has also been successfully used to prevent the recurrence of keloids6667 In contrast a metaanalysis review that analysed the effectiveness of pressure garment therapy for the prevention of abnormal scarring after burn injury was unable to demonstrate any beneficial effects of pressure garment therapy on prevention or treatment of abnormal scarring59 Notwithstanding success rates of pressure therapy are contingent upon patient compliance69 which can sometimes be low due to discomfort Overall pressure therapy is tolerated better and is devoid of the pain often associated with intralesional therapies and hence can be considered as a good adjuvant therapy for keloid scarsRadiotherapyIn the treatment of keloids using superficial Xray irradiation was first described71 Since then it has been used less frequently as a monotherapy and more widely as an effective adjunct treatment after surgical excision72 with success rates in the range of and recurrence rates of about Radioactive skin patches have also been used in combination with other treatment modalities for keloids7879 Radiotherapy is most commonly used h after surgical excision7480 and acts by suppressing angiogenesis and inhibiting ï¬broblast activity1 Decreased fibroblast proliferation induced cell senescence and apoptosis leading to reduction in collagen production and suppression of keloid development have also been reported81Different radiotherapy modalities have been used after surgical excision including electron beam radiotherapy brachytherapy superï¬cial and orthovoltage radiotherapy with varying degrees of success84 Mankowski et a0 al conducted a literature review of studies to compare the clinical outcome of different forms of radiation treatment used for the management of keloids77 The metaanalysis demonstrated that radiation used as monotherapy yielded higher rates of recurrence compared to combinational therapy with postsurgery excision Comparison between the different radiationbased treatments revealed that the lowest rate of recurrence was observed with brachytherapy followed jointly by Xray and electron beam The authors also reported that the rate of recurrence was dependent on anatomical site of the keloid with chest keloids having the highest recurrence rate73The adverse effects of radiotherapy often linked with dose of radiation used can be grouped into acute skin reactions and late complications Acute reactions arise as early as seven days after keloid treatment and include oedema necrosis ulceration desquamation erythema and pigmentary changes with the latter two being the most common Late complications which include changes in pigment atrophy telangiectasis and alopecia may present several weeks after radiotherapy Emollient and steroid ointment used after radiotherapy can help alleviate the side effects19 A recommended radiation dose Gy over several sessions can also minimise adverse effects1980 Radiotherapy carries a risk of malignancy8586 Therefore caution should be used in radiationvulnerable sites such as the head neck thyroid and breast and in patients aged years2 Protecting fragile ans and selecting the most appropriate sitedependent dose protocol can help minimise further complications of radiotherapy87 0c Scars Burns HealingCryotherapyCryotherapy is a lowtemperature treatment that causes vascular damage resulting in tissue necrosis88 It has been used to treat keloids as a monotherapy or in combination with other treatment methods such as intralesional steroid injections89 Various delivery methods used for cryotherapy include spray and contact probes or the intralesionalneedle cryoprobe method Compared to contact and spray methods intralesionalneedle cryoprobe was found to be the most effective method in treating keloid scars90 Positive outcomes were observed in a number of studies that used liquid nitrogen and cryotherapy to treat keloids with success rates in the range of External cryotherapy has been associated with several side effects including hypopigmentation blistering pain delayed healing and infection9093 Moreover larger keloids have been shown to need multiple cryotherapy sessions9093 To minimise side effects intralesional cryotherapy was introduced and there are now a number of nitrogenbased cryodevices that have been described for the treatment of keloid scars with two commercially available a liquid nitrogenbased device88 and an argon gasbased device94 The intralesional cryotherapy was designed to overcome the hypopigmentation seen mostly in darkskinned individuals with external cryotherapy It works by destroying the core of the keloid sparing the surface epithelial cells including melanocytes9596 As a result it enhances volume decrease while minimising the risk of hypopigmentation and other surface reactions90 A recent comprehensive review based on the preferred reporting items for systematic reviews and metaanalysis was performed to investigate the efficacy of intralesional cryotherapy on keloid scars90 The review of eight studies that met the inclusion criteria revealed that average scar volume decreased in the range of but complete eradication of the scar on average was lacking Recurrence of keloid scars was in the range of The authors also reported that patients complaints of pain and pruritus was considerably reduced however hypopigmentation was seen mostly in Fitzpatrick skin type patients after treatment90Laser therapyLaser therapy for keloid treatment was introduced in the 1980s97 Since then different systems have been used for the treatment of keloid and hypertrophic scars4898 These lasers target skin chromophores like haemoglobin and melanin based on the principle of selective photothermolysis99 Lasers can be classified as ablative and nonablative The most common ablative lasers include the 2940nm erbiumdoped yttrium aluminium garnet ErYAG laser and the 10600nm carbon dioxide CO2 laser These emit a laser beam that is absorbed by water in the skin leading to local tissue destruction and reduction of lesion volume3 Common examples of nonablative lasers include 585nm or 595nm PDLs 1064nm neodymiumdopedyttriumaluminiumgarnet NdYAG neodymiumdopedvanadate NdVan laser and nm Qswitched NdYAG laser with low fluence100 These lasers induce thermal injury to the scars microvasculature leading to thrombosis and ischaemia which result in collagen denaturation and collagen fibre realignment101532nm laser Laser therapy requires several treatments at intervals of weeks depending on scar type and type of laser used98104 with possible side effects including itching pigmentary changes blister formation and postoperative purpura98 The use of the nonfractional vascular nm PDL in the treatment of keloid and hypertrophic scars has been welldocumented105 and has response rates in the range of PDL monotherapy has been shown to be effective108110 as well as CO2 laser monotherapy38111112 In a clinical study where patients with moderate to severe keloids were treated with highenergy pulsed CO2 laser the treatment was efficacious and welltolerated with minimal side effects112 In other studies where CO2 laser ablation was compared with other forms of treatment CO2 laser was as efficient as the other forms38111 It must be noted however that these studies are small and randomised controlled studies are lacking98Laser therapy such as PDL CO2 and NdYAG have been associated with a high rate of recurrence at months111113 However optimal results can be achieved with combination treatment especially with intralesional TAC injections116 Kumar and coworkers conducted a cohort study on patients with keloids previously treated with an NdYAG laser and reported complete scar resolution and flattening in seven patients only when intralesional TAC was used after laser therapy41 Moreover combined therapy with PDL and TAC119 and PDL TAC and 5FU36 were shown to produce better clinical results In a recent study that evaluated and compared the efficacy of combination therapy of 0cOjeh scars statistically fractional CO2 laser and intralesional TAC injection or TAC injection alone in keloid and hypertrophic significant improvements were reported in overall scar quality with the combined treatment options comto TAC monotherapy120 Moreover pared combined CO2 laser and IFNα2b injections given to patients with auricular keloids resulted in no recurrence in of patients three years after treatment121 Laser therapy can also be combined with other laser treatment topical corticosteroids and cyanoacrylate glue98 and have shown promising results however larger controlled clinical studies are needed to further evaluate their efficacy and safetyRecently lasers are also being explored as tools for assisted drug delivery Kraeva et a0al proposed an alternative technique of corticosteroid administration of laserassisted drug delivery of topical TAC This was shown to be effective when used on a keloid on the posterior scalp of a patient after each CO2 laser session122 More efficient intraepidermal drug delivery options are also being investigated Singhal et a0al developed TACcontaining polymeric microps that were prepared using a cryomilling technique for freezing fracture After ablation with a fractional ErYAG laser these microps can be deposited in cutaneous micropores and provide highdose intraepidermal reservoir systems with minimal transdermal permeation leading to sustained and targeted local drug delivery123It must be noted that one of the biggest limitations in studies available at present is the lack of histological definition between keloid and hypertrophic scars so conclusions are not valid on efficacyImiquimod creamImiquimod cream is approved for the treatment of basal cell carcinoma actinic keratoses and genital warts21 As an immuneresponse modifier it stimulates the production of proinflammatory cytokines such as TNFα interleukins and IFNs by activated Tcells124 thereby changing the expression of genes associated with apoptosis125 and reducing collagen production16 Studies have reported conflicting findings regarding efficacy of imiquimod cream postoperatively following keloid excision likely due to keloid location Many studie | Thyroid_Cancer |
Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young at diagnosis patients who received curative resection for breastcancer The signiï¬cance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identiï¬ed variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI P and OS HR CI P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identiï¬ed as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI and CI respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identiï¬ed for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to beneï¬t prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and Chinascontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkins lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classiï¬cation system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassiï¬ed as luminal Alike ER PR¥ HER2 andKi6715luminal Blike ER andor PR HER2HER2enriched ER PR HER2 or triplenegative ERPR HER2 subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have deï¬niteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insuï¬cient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was deï¬ned as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was deï¬ned asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the KaplanMeier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asigniï¬cance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the rms package of R The discriminationofthe nomogram models was estimated by the Harrellsconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank à statistics with the Xtileprogram Pearsons chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the ï¬ndings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsuï¬cientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patientsFIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical signiï¬cancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The models explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI and CI respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS patients intermediateriskgroup TPS patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS patientsintermediaterisk group TPS patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the ï¬rsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentiï¬ed as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE KaplanMeier curves for A diseasefree survival and B overallsurvival stratiï¬ed by HBsAg status in very young patients with breast canceridentiï¬ed as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE KaplanMeier curves for A diseasefree survival and B overall survival stratiï¬ed by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may beneï¬t patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insuï¬cientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population conï¬nes the universality of our resultsFurthermore the information was insuï¬cient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE KaplanMeier curves for A diseasefree survival and B overall survival stratiï¬ed by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the ï¬rst evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to conï¬rm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the ï¬nal versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al Very young women years with operable breastGcancer 101093annoncmdf039at presentation Ann Oncolfeaturesof disease Anders CK Hsu DS Broadwater G Acharya CR Foekens JA Zhang Y et alYoung age at diagnosis correlates with worse prognosis and deï¬nes a subsetof breast cancers with shared patterns of gene expression J Clin Oncol 101200JCO2007142471 Narod SA Breast cancer in young women Nat Rev Clin Oncol 101038nrclinonc2012102 Trepo C Chan HL Lok A Hepatitis B virus infection Lancet 101016S0140673614602208 de Martel C Gees D Bray F Ferlay J Cliï¬ord GM Global burden of cancerattributable to infections in a worldwide incidence analysis LancetGlobal Health 8e180 101016S2214109X19304887 Lu FM Li T Liu S Zhuang H Epidemiology and prevention of hepatitisJ Viral Hepatitis 17Suppl B virus infection in China 101111j13652893201001266x Liu J Zhang S Wang Q Shen H Zhang M Zhang Y et al Seroepidemiologyof hepatitis B virus infection in million men aged years in ruralChina a populationbased crosssectional study Lancet Infect Dis 101016S1473309915002182 Zhang Y Fang W Fan L Gao X Guo Y Huang W et al HepatitisB surface antigen prevalence among rural women of childbearingage in Hainan Province China a crosssectional study Virol J 1011861743422X1025 Cui Y Jia J Update on epidemiology of hepatitis B and C in China JGastroenterol Hepatol 28Suppl 101111jgh12220 Chan SL Wong VW Qin S Chan HL Infection and cancer the case ofHepatitis B J Clin Oncol 101200JCO2015615724 Fwu CW Chien YC You SL Nelson KE Kirk GD Kuo HS et al Hepatitis Bvirus infection and risk of intrahepatic cholangiocarcinoma and nonHodgkinlymphoma a cohort study of parous women in Taiwan Hepatology 101002hep24150 Kamiza AB Su FH Wang WC Sung FC Chang SN Yeh CC Chronichepatitis infection is associated with extrahepatic cancer developmenta nationwide populationbased study in Taiwan BMC Cancer 101186s1288501629185 Nath A Agarwal R Malhotra P Varma S Prevalence of hepatitis B virusinfection in nonHodgkin lymphoma a systematic review and metaanalysisInternal Med J 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell 107314APJCP201516135285lung cancer Asian Paciï¬c J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in Vickers AJ Elkin EB Decision curve analysis a novel method for evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res 10115810780432CCR040713Conï¬ict of Interest The authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c' | Thyroid_Cancer |
Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group ARED PBM Group BINFRARED PBM Group CPLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring º Analyses of body contouring will be performed by non invasive methods º The waist circumference measurement will not discriminate the factors underlying the volume modifications º The habits of the participants such as diet and exercise routines may affect the results º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical TrialsReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individuals self esteem4 Studies have shown that aesthetic treatments significantly increase a patients quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6 There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10 The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18 Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions their frequency times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25 It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described below biggersmaller Ïn2 accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participants abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group ARED ± nm Group BINFRARED ± nm Group CPLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant powerone LED mWAperture diameterone LED cmPower density at aperture mWcm2Beam spot size at targetone LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContactweek for a month a total of sessionsInfrared LEDContinuousContactweek for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire The WHO Quality of Life WHOQOL BREF as well as the Body Shape Questionnaire BSQ34 Self Image Scale will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on a protected by password computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths nm and near the infrared regions nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18 Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39 If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individuals self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on a protected by password computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients Landecho MF Tuero C Valent V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts Kouris A Platsidaki E Christodoulou C et a0al Patients self esteem before and after chemical peeling procedure J Cosmet Laser Ther Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci Thornfeldt CR Thaxton PM Horn | Thyroid_Cancer |
genes of papillary thyroidcarcinoma by integrated bioinformatics analysisGang Xue11Department of Otorhinolaryngology Head and Neck Surgery The First Afï¬liated Hospital of Hebei North University Zhangjiakou China 2Department of Histology andEmbryology Hebei North University Zhangjiakou ChinaJingFang Wu2 Da Pei1 DongMei Wang2 Jing Zhang2 and WenJing Zhang2Xu Lin2Correspondence JingFang Wu wjfxg163comBackground Papillary thyroid carcinoma PTC is one of the fastestgrowing malignant tumor types of thyroid cancer Therefore identifying the interaction of genes in PTC is crucialfor elucidating its pathogenesis and finding more specific molecular biomarkersMethods Four pairs of PTC tissues and adjacent tissues were sequenced using RNASeqand differentially expressed genes were screened P005 logFC1 The enrichment analysis indicated that the vast majority of differentially expressed genes DEGs mayplay a positive role in the development of cancer Then the significant modules were analyzed using Cytoscape software in the proteinprotein interaction network Survival analysisTNM analysis and immune infiltration analysis of key genes were analyzed And the expression of ADORA1 APOE and LPAR5 genes were verified by qPCR in PTC compared withmatching adjacent tissuesResults Twentyfive genes were identified as hub genes with nodes greater than Theexpression of genes were verified by the GEPIA database and the overall survival anddiseasefree survival analyses were conducted with KaplanMeier plotter We found onlythree genes were confirmed with our validation and were statistically significant in PTCnamely ADORA1 APOE and LPAR5 Further analysis found that the mRNA levels andmethylation degree of these three genes were significantly correlated with the TNM staging of PTC And these three genes were related to PTC immune infiltration Verification ofthe expression of these three genes by RTqPCR and Western blot further confirmed thereliability of our resultsConclusion Our study identified three genes that may play key regulatory roles in the development metastasis and immune infiltration of papillary thyroid carcinomaThese authors contributedequally to this work and shouldbe considered as coï¬rstauthorsReceived May Revised August Accepted August Accepted Manuscript online August Version of Record published August IntroductionThyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the Western world [] Thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma PTC follicular thyroid carcinoma FTC and anaplastic thyroid carcinoma ATC according to the histological subtype [] Clinicalresults vary across these subtypesThe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to PTC which accounts for of all thyroid carcinomas [] Inaddition patients with PTC suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately of cases may progress to a potentially fatal recurrentailment [] Due to these reasons uncovering the causes of PTC and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555tasksWith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics [] Currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of PTC An examination of differentially expressedgenes DEGs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma As a form of molecular marker mRNA containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages [] Some studiesutilized public databases such as The Cancer Genome Atlas TCGA and the Gene Expression Omnibus GEO toidentify significant biomarkers of papillary thyroid carcinoma However these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the DEGsIn the present study we analyzed the DEGs in PTC tissues versus the matched adjacent tissues by RNASeq andbioinformatics methods to obtain the DEGs Then we screened out the key modules and extracted the key genes inthose modules by constructing DEGs interaction network Then the possible role of differentially expressed geneswas analyzed using GO annotation and KEGG pathway enrichment analysis The expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases Finally wefound that the three genes ADORA1 APOE and LPAR5 were highly expressed in PTC and were associated withPTC methylation TNM staging and immune infiltrationMethodsTissue samplesThirty pairs of PTC and adjacent tissues were collected from January to July at the First Affiliated Hospitalof Hebei North University This experiment was approved by the Ethical Committee of the First Affiliated Hospitaland all patients provided informed consent All tissues were frozen in liquid nitrogen after surgical resectionRNA library construction and sequencingTotal RNA was isolated from four adjacent normal and cancerous thyroid samples utilizing TRIzol reagent QiagenValencia CA USA as indicated by the manufacturers guidelines RNAs of PTC tissues and paracancerous tissuessample numbers 1C 1P 2C 2P 3C 3P 4C 4P the number represents different samples the C indicates a cancersample and the P represents a matched paracancerous tissue sample were used Six libraries were built utilizingan Illumina standard kit as indicated by the manufacturers protocol All sequencing was carried out on an IlluminaHiseq LC Bio ChinaDifferentially expressed genes screeningThe level of expression of mRNAs was evaluated using StringTie by calculating FPKM [] The DEGs between PTCand paracancerous tissue were screened with log2 fold change1 and P005 was regarded as statistically significant the analyses were conducted using the R package Ballgown []Functional enrichment analysis and pathway analysisTo reveal the functional roles of the DEGs the Annotation Visualization and Integrated Discovery function annotation tool DAVID httpdavidabccncifcrfgovhomejsp was used to perform Gene Ontology GO enrichmentanalysis and Kyoto Encyclopedia of Genes and Genomes KEGG pathway enrichment analysis P values less than were considered as cutoff criteriaPPI network construction and identiï¬cation of hub genesPPI networks were constructed successively using STRING database tringdb [] The interactions ofDEGs with a combined score were set as significant and Cytoscape software version was utilized tovisualize and analyze the results of the STRING database To find key hub genes in this PPI network the significantmodule was analyzed by using the plugin MCODE of Cytoscape software The criteria for selection were set to thedefault The key genes were chosen with degrees ¥ Subsequently genes in that module were used to analyse theirfunctional roles with FunRich software The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Table PCR primersGene symbolADORA1ActinAPOELPAR5bp base pair F forward primer R reverse primerPrimer sequenceF5cid3CCACAGACCTACTTCCACACC3cid3R5cid3TACCGGAGAGGGATCTTGACC3cid3F5cid3CACTCTTCCAGCCTTCCTTCC3cid3R5cid3AGGTCTTTGCGGATGTCCAC3cid3F5cid3 GTTGCTGGTCACATTCCTGG 3cid3R5cid3 GCAGGTAATCCCAAAAGCGACcid3F5cid3 CACTTGGTGGTCTACAGCTTG3cid3R5cid3 GCGTAGTAGGAGAGACGAACG3cid3Data validation and analysisTo verify the accuracy of our RNAseq results we used the Gene Expression Profiling Interactive Analysis databaseto verify the expression of key genes in PTC and adjacent tissues The overall survival and diseasefree survivalanalyses were performed by KaplanMeier plots for these PTCrelated hub genes Genetic alterations of hub genesin PTC and their correlations with other genes were analyzed utilizing the cBioPortal for Cancer Genomics Hub genesrelated to clinicopathological features were analyzed using the online database UALCAN httpualcanpathuabedu[] The correlation of ADORA1 APOE and LPAR5 expression with the immune infiltration level in PTC and theexpression of these three genes in different kinds of cancers was performed using the Tumor Immune EstimationResource database []For qRTPCR analysis total RNA was isolated from normal and cancerous papillary thyroid samples utilizingTRIzol reagent Qiagen Valencia CA USA cDNA was synthesized with RNA reverse transcription kit TIANGENBIOTECH Beijing China qRTPCR was performed with an ABI RealTime PCR System Applied Biosystems Life Technologies USA The expression of the genes of interest was normalized to actin The primers forADORA1 APOE LPAR5 and actin are shown in Table For Western blot RIPA buffer was used to extract protein from four pairs of tissue from PTC patients and theprotein concentrations were measured via BCA methods Briefly the SDSPAGE gel was used for electrophoresis andPDVF membrane was used for transmembrane transfer PDVF membrane was blocked and then incubated with primary antiADORA1 antibodies dilution Bioss bs6649R APOE dilution Bioss bs4892R LPAR5antibodies dilution Bioss bs15366R and actin dilution Bioss bs0061R at ¦C overnight followed by incubation with secondary antibodies Zhongshanjinqiao dilution at ¦C for h The signal wasdetected using ECL methodStatistical analysisAll the data were analyzed by R and SPSS SPSS Inc USA KaplanMeier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma The statistical difference was set at P ResultsDifferentially expressed genes screening based on RNASeqTo screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed RNASeq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes After RNASeq we acquire million reads for each sample The fold changesbetween PTC cancer tissues and matched paracancerous samples were calculated Setting the cutoff criterion as Pvalue and a fold change there were upregulated and downregulated genes These DEGswere considered to be candidate genes for subsequent study Figure 1A showed the expression of the top genes inPTC versus matched paracancerous tissuesFunctional enrichment analysis and pathway analysisConsidering that there were many falsepositive genes among these DEGs we verified our results one by onethrough the TCGA database We found that only genes in our data were consistent with the gene expression of the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Identiï¬cation of DEGs by RNAseqThe heat map A and PPI network of the DEGs B C The volcano plots of the DEGs D The most significant module was selectedby MCODE in Cytoscape Red represents the upregulated genes and blue represents the downregulated genesFigure GO and KEGG pathway enrichment analysis of DEGs through RNASeqA Bubble plot of Gene Ontology enrichment analysis of DEGs B Bubble plot of Kyoto Encyclopaedia of Genes and Genomespathway enrichment analysis of DEGsTCGA database To investigate the potential function of these DEGs in PTC genes functional enrichment was conducted by using GO and KEGG pathway analyses For the biological process category the DEGs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development Thecellular component category results showed PTCrelated DEGs were enriched in collagencontaining extracellularmatrix synaptic membrane cellcell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane DEGs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity Figure 2AAs Figure 2B showed the KEGG pathway results showed DEGs were enriched in cytokinecytokine receptorinteraction MAPK signaling pathway proteoglycans in cancer Rap1 signaling pathway axon guidance Cushing The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure GO enrichment analysis and KEGG analysis for the key genesA Top elements involved in biological processes B Top elements involved in molecular function C Top elementsinvolved in cellular components D Top pathways related to the key genes through KEGG analysissyndrome parathyroid hormone synthesis secretion and action AGERAGE signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ECMreceptor interaction arrhythmogenic right ventricular cardiomyopathyARVC endocrine resistance renin secretion Type II diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesPPI network construction and module analysisPPI networks were constructed successively by the database by loading the PTC related DGEs into the STRINGdatabase Figure 1BC Using Cytoscape we analyzed the most significant module in the PPI network Figure 1DThe PPI network consisted of nodes and edges Following the use of MCODE in Cytoscape the significantmodule was selected The top hub genes ADCY8 ADORA1 ADRA2C APOE C5AR1 CCL13 CCL20 CDH2CHGB CXCL12 EVA1A FAM20A FN1 GNAI1 GPC3 GRM4 LPAR5 MELTF or MFI2 MFGE8 NMU OPRM1SERPINA1 SSTR3 TIMP1 and TNC were evaluated by degree in the PPI network Figure 1D The resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationGprotein coupled receptor activity cell adhesion molecule activity and GPCR ligand binding Figure Data analysis and validationAfter the key genes were selected the expression of key genes in PTC and its adjacent tissues were verified by theGEPIA database Figure ADORA1 APOE EVA1A LPAR5 MFGE8 OPRM1 SERPINA1 SSTR3 and TIMP1were positively related to the overall survival analysis of PTC patients while C5AR1 and GNAI1 were negativelyrelated Figure ADCY8 ADORA1 CHGB FN1 LPAR5 NMU and TNC showed positive associations withdiseasefree survival analysis of PTC patients but not APOE Figure Next we analyzed the alterations of the key genes by using the cBioPortal database Figure The key geneswere changed in of queried samples Figure 7B Figure 7A showed the frequency of alterations of eachPTC related key gene SSTR3 FN1 and ADORA1 were altered the most and respectively Figure 7Dshowed the network of the genes and their altered neighbouring genes in PTC patients out of a total of Among these genes only ADORA1 APOE and LPAR5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of PTC patients The qPCR experiments and Western blotdata verified that these three survivalrelated genes were all overexpressed in PTC Figure Then based on UAL The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Validation of the key DEGs in the GEPIA databaseADORA1 APOE CCL13 CDH2 CXCL12 EVA1A FAM20A FN1 GNAI1 LPAR5 MFGE8 NMU SERPINA1 TIMP1 and TNC areoverexpressed in PTC tissues compared with paracancerous tissue while GNAI and GPC3 are downregulated The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Overall survival analysis of key genes in PTC using KaplanMeier plotsExpression levels of ADORA1 APOE C5AR1 EVA1A FAM20A GNAI1 LPAR5 MFGE8 OPRM1 SERPINA1 SSTR3 and TIMP1are related to the overall survival of patients with PTCCAN the clinical features and degree of methylation of these three genes were analyzed The transcription levels ofADORA1 APOE and LPAR5 were significantly higher in PTC patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status Figure In addition ADOR1 and LPAR5 exhibiteda hypomethylation state in the cancer group but APOE showed a hypermethylation state in PTC samples Figure10ATo further clarify the role of these genes we conducted an analysis of immune infiltration The ADOR1 expression level was positively corelated with infiltrating levels of B cells r0111 P151e2 CD8 T cells r0246P396e neutrophils r0162 P331e and DCs r0232 P232e The expression of APOE was The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Diseasefree survival analysis of key genes in PTC using KaplanMeier plotsExpression levels of ADCY8 ADORA1 APOE CHGB FN1 LPAR5 NMU and TNC are significantly related to the diseasefreesurvival of patients with PTCpositively associated with B cells r0228 P439e CD8 T cells partialcor P930e neutrophils r0197 P114e and DCs partialcor P358e LPAR5 expression level was positively related to B cells r0259 P815e CD4 T cells r0238 P103e macrophages r0175P105e neutrophils r027 P142e and DCs r0256 P104e and negatively related to Purity r P294e and CD8 T cells r P618e Figure 10B These findings stronglysuggested that LPAR5 ADOR1 and APOE may play specific roles in immune infiltration in PTC especially those ofDCs Finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure The key genes expression and mutation analysis in PTC by the cBioPortal for Cancer GenomicsA The genetic alterations of key genes of PTC samples Queried genes are altered in of queried patientssamplesB The expression heatmap of key genes C The alteration frequency of key genes in PTC D Network of key genesmutations and their frequently altered neighboring genes in PTC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure The mRNA and protein expressions of ADORA1 APOE and LPAR5 in PTC tissuesAC Validation of expression levels of ADORA1 APOE and LPAR5 by RTqPCR in cases of PTC and matched adjacent tissuesD ADORA1 APOE and LPAR5 protein levels are increased in four cases of PTC and matched adjacent tissues as measured byWestern Blot P0001DiscussionPTC is a common cancer with great heterogeneity in morphological features and prognosis [] Although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features [] Activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of PTC To better clarify the molecular mechanism of PTC occurrence development and metastasis we identified key genes related to PTC progression through comprehensive bioinformaticsmethods and we screened three of the PTC prognosisrelated genes for a comprehensive analysisIn the present study we identified differentially expressed genes by RNASeq with GO enrichment analysis showing that the DEGs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cellcell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity And KEGG pathway results showed DEGs were enrichedin cytokinecytokine receptor interaction MAPK signaling pathway proteoglycans in cancer Rap1 signaling pathway axon guidance Cushing syndrome parathyroid hormone synthesis secretion and action AGERAGE signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ECMreceptor interaction arrhythmogenic right ventricularcardiomyopathy ARVC endocrine resistance renin secretion Type II diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesTo further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a PPI regulatory network A total of DEGs with nodes greater than were screened out in the networkThe key genes were ADCY8 ADORA1 ADRA2C APOE C5AR1 CCL13 CCL20 CDH2 CHGB CXCL12 EVA1AFAM20A FN1 GNAI1 GPC3 GRM4 LPAR5 MELTF MFGE8 NMU OPRM1 SERPINA1 SSTR3 TIMP1 andTNC Biological process and molecular function analyses of these key DEGs indicated that they were significantly The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Relative expression of ADORA1 APOE and LPAR5 in normal thyroid tissues and PTC tissues individual cancerstages and nodal metastasis status respectively UALCANP0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentTo verify the credibility of the experiments and data the DEGs screened were verified by the GEPIA databaseAmong the selected genes genes showed expression differences consistent with our RNASeq data Among the The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure Methylation level and immune inï¬ltration level of ADORA1 APOE and LPAR5A Relative methylation level of ADORA1 APOE and LPAR5 based on normal thyroid tissues and PTC tissues individual cancerstages and nodal metastasis status respectively UALCAN B The correlation between the three genes and TIICs TIMER TIICstumor infiltrating immune cells genes ADORA1 APOE CCL13 CDH2 EVA1A FAM20A FN1 LPAR5 MFGE8 NMU SERPINA1 TIMP1and TNC levels were overexpressed in PTC tissues while GPC3 and GNAI1 were downregulatedADORA1 belongs to the Gprotein coupled receptor family and protects human tissues and cells under physiological conditions [] Lin et al suggested that ADORA1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells [] Similarly Jayakar indicated that knockdown of APOE expression can reduce the level of MMPs by regulating the AP1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma [] Bioinformatics predictions were that APOE mRNAshows a significant increase in PTC [] CCL13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that CCL13 has a role in promoting the proliferation of tumorforming volumein nude mice [] CDH2 is overexpressed in various cancers Some research results indicate that overexpression ofCDH2 can increase the invasive ability and induce EMT in lung cancer cells [] Qiu et al confirmed CDH2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings [] EVA1A acts as a regulatorof programmed cell death and Shen et al indicates that EVA1A can inhibit the proliferation of GBM cells by inducing autophagy and apoptosis via inactivating the mTORRPS6KB1 signaling pathway [] FAM20A may play a keyrole in haematopoiesis There are few reports on the relationship between FAM20A and cancer and our experimentfound that FAM20A is more highly expressed in papillary thyroid carcinoma than in other cancers FN1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology [] Some researchersindicated that FN1 participates in regulating many types of cancer progression such as gastric cancer [] skin squamous cell carcinoma [] and papillary thyroid carcinoma [] It has been shown that LPAR5 is related tothe pathogenesis of pancreatic cancer [] Consistent with our study Zhang et al believes that LPAR5 may be involved in the development of papillary thyroid carcinoma [] According to previous reports MFGE8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555Figure The expression of ADORA1 APOE and LPAR5 in thyroid cancer tissues and normal thyroid tissuesThe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the TIMER database P005P001 P001 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201555101042BSR20201555[] MFGE8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect[] Consistent with our data Zhang et al indicate that NMU is one of the DEGs of papillary thyroid carcinoma[] Recently a researcher has shown that abnormal expression of NMU is associated with a variety of cancers []For SERPINA1 there are currently six s pointing out that SERPINA1 may be a key gene for PTC consistentwith our results [] Clinical studies have shown that high expression of TIMP1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers [] TNC is a component of theextracellular matrix ECM and is closely related to the malignant biological behavior of cancer In particular TNCoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer [] GPC3 belongs to the glypicans family It has been reported that overexpression of GPC3can promote the metastasis of hepatocellular carcinoma [] but we found that it is expressed at low levels in PTCSimilar to GPC3 some scholars believe that GNAI1 is a tumorpromoting gene and reported upregulated GNAI1mRNA in human glioma which is inconsistent with our data []Only the ADORA1 APOE and LPAR5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of PTC patients Considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes We found thatthe mRNA and methylation levels of these three genes were significantly correlated with TNM staging In additionADORA1 APOE and LPAR5 were all related to immune infiltration especially to dendritic cells Finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissuesHowever our research has certain limitations First only four pairs of cancer and adjacent tissues were analyzedusing RNAseq in this experiment so further research requires a larger sample size Second further experiments areneeded to validate the specific mechanisms of these key genesData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis study was supported by grants from the Hebei Provincial Department of Finance Specialist Capacity Building and SpecialistLeadership Program [grant number ] the Hebei Provincial Natural Science Foundation Project [grant number H201840505]and the Hebei North University Basic Research Business Expenses Project [grant number JYT2019015]Author ContributionXu Lin conducted the bioinformatics analysis Xu Lin and Gang Xue contributed as first authors Xu Lin wrote the manuscriptJingFang Wu and Gang Xue critically revised the Gang Xue and Da Pei obtained clinical specimens and the others contributed to verification of the RNAseq resultsAbbreviationsATC anaplastic thyroid carcinoma ECM extracellular matrix FTC follicular thyroid carcinoma PTC papillary thyroid carcinomaReferences Kitahara CM and Sosa JA The changing incidence of thyroid cancer Nat Rev Endocrinol 101038nrendo2016110 AschebrookKilfoy B Ward MH Sabra MM and Devesa SS Thyroid cancer incidence patterns in the United States by histologic type Thyroid 101089thy20100021 Pourseiraï¬ S Shishehgar M Ashraf MJ and Faramarzi M Papillary Carcinoma of Thyroid with Nasal Cavity Metastases A Case Report IranJ Med Sci Ullmann TM Gray KD Moore MD Zarnegar R and Fahey III TJ Current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers Gland Surg 1021037gs20170908 Jin X Deng B Ye K et al Comprehensive expression proï¬les and bioinformatics analysis reveal special circular RNA expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy Mol Med Rep 103892mmr201910671 Rapisuwon S Vietsch EE and Wellstein A Circulating biomarkers to monitor cancer progression and treatment Comput Struct BiotechnolJ 101016jcsbj201605004 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20201555101042BSR20201555 Pertea M Pertea GM Antonescu CM et al StringTie enables improved reconstruction of a transcriptome from RNAseq reads NatBiotechnol 101038nbt3122 Frazee AC Pertea G Jaffe AE et al Ballgown bridges the gap between transcriptome assembly and expression analysis Nat Biotechnol 101038nbt3172 Von Mering C Huynen M Jaeggi D et al STRING a database of predicted functional associations between prot | Thyroid_Cancer |
Genomics Score Based onGenomeWide Network Analysis forPrediction of Survival in GastricCancer A Novel PrognosticSignatureZepang Sun Hao Chen Zhen Han Weicai Huang Yanfeng Hu Mingli Zhao Tian LinJiang Yu Hao Liu Yuming Jiang and Guoxin LiDepartment of General Surgery Nanfang Hospital Southern Medical University Guangzhou ChinaGCPurpose Gastric canceris a product of multiple genetic abnormalitiesincluding genetic and epigenetic modiï¬cations This study aimed to integrate variousbiomolecules such as miRNAs mRNA and DNA methylation into a genomewidenetwork and develop a nomogram for predicting the overall survival OS of GCMaterials and Methods A total of GC cases as a training cohort with a random of examples included as a validation cohort were screened from The Cancer GenomeAtlas database A genomewide network was constructed based on a combination ofunivariate Cox regression and least absolute shrinkage and selection operator analysesand a nomogram was established to predict and 5year OS in the trainingcohort The nomogram was then assessed in terms of calibration discriminationand clinical usefulness in the validation cohort Afterward in order to conï¬rm thesuperiority of the whole gene network model and further reduce the biomarkers for theimprovement of clinical usefulness we also constructed eight other models accordingto the different combinations of miRNAs mRNA and DNA methylation sites and madecorresponding comparisons Finally Gene Ontology GO and Kyoto Encyclopedia ofGenes and Genomes KEGG analyses were also performed to describe the function ofthis genomewide networkResults A multivariate analysis revealed a novel prognostic factor a genomics scoreGS comprising seven miRNAs eight mRNA and DNA methylation sites In thevalidation cohort comparing to patients with low GS highGS patients HR P were significantly associated with increased allcause mortality Furthermoreafter stratiï¬cation of the TNM stage I II III and IV there were significant differencesrevealed in the survival rates between the highGS and lowGS groups as wellP The and 5year Cindex of whole genomicsbased nomogram were and respectively The other models have comparable or relativelypoor comprehensive performance while they had fewer biomarkers Besides thatDAVID further revealed multiple molecules and pathways related to the genomewidenetwork such as cytomembranes cell cycle and adipocytokine signalingEdited byXin Maizie ZhouStanford University United StatesReviewed byHailin TangSun Yatsen University Cancer CenterSYSUCC ChinaJie TianInstitute of Automation CAS ChinaCorrespondenceYuming Jiangjiangymbest163comGuoxin Ligzliguoxin163com These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in GeneticsReceived April Accepted July Published August CitationSun Z Chen H Han Z Huang WHu Y Zhao M Lin T Yu J Liu HJiang Y and Li G GenomicsScore Based on GenomeWideNetwork Analysis for Predictionof Survival in Gastric Cancer A NovelPrognostic SignatureFront Genet 103389fgene202000835Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreConclusion We successfully developed a GS based on genomewide network whichmay represent a novel prognostic factor for GC A combination of GS and TNMstaging provides additional precision in stratifying patients with different OS prognosesconstituting a more comprehensive subtyping system This could potentially play animportant role in future clinical practiceKeywords gastric cancer genomewide network miRNA mRNA DNA methylation nomogramBACKGROUNDGastric cancer GC is one of the most common malignanthuman tumors and the third leading cause of cancerrelatedmortalities worldwide Reports estimate that nearly one millionnew cases and deaths occur each year across the worldTorre Despite the rapid research advancement GCrelated impacts on human life remain high around the globeAccording to the global cancer burden data hundreds of billionsof dollars in economic losses are incurred each year due to GCAt the same time stomach cancer has been reported to cause million disabilityadjusted life years with of these resultingfrom years of life lost and from years lived with disabilityGlobal Burden of Disease Cancer Collaboration Despite major breakthroughs in GC prevention diagnosisand treatment therapies reported over the past decade prognosisremains a challenge at diï¬erent TNM stages Jiang 2018aSun 2019ab Notably patients with similar clinicalfeatures and at the same tumor stage who receive uniformtreatment have exhibited varying clinical outcomes Bang Jiang 2018b Such evidence indicates the existingchallenges to traditional TNM staging Serra possiblydue to a lack of molecular tools for eï¬ectively predicting theprognosis and the therapeutic eï¬ect of GC patients Thereforemore rigorous and reliable systems that accurately reï¬ect theheterogeneity of diï¬erent patients and guide the development oftreatment approaches are urgently needed Duarte Serra Tumors are a product of multiple genetic mutations includinggenetic gene expression and epigenetic DNA methylation andhistone modiï¬cation modiï¬cations as well as deregulationsof tumorsuppressor genes and protooncogenes Anna Choi In addition changes in a set of geneticmaterials have been closely associated with cancer outcomesAnna Choi Therefore to eï¬ectivelypredict the prognosis of tumors such as GC a single biomarkeris insuï¬cient necessitating the need for a gene networkA variety of mRNAs have been associated with GC prognosisCamargo with microRNAsmiRNAs alsoimplicated in tumor prediction in the recent years Li Ueda Camargo These smallnoncoding RNAs comprising nucleotides primarily functionto regulate protein translation by inhibiting the expressionoffrom geneticsepigenetics is currently receiving considerable research attentionDNA methylation isthe most common epigenetic eventassociated with cancer development and progression Anna Consequently numerous studies have implicated DNAtarget messenger RNAs mRNAs Apartmethylation in the diagnosis and the prognosis of various tumorsincluding GC Camargo Choi Althoughthese studies have revealed several biomarkers that have proved tobe prognostic predictors in GC only a handful have been adoptedin clinical therapies or are used to build predictive models forthe disease Anna Duarte Camargo Choi Serra Previous studies have identiï¬ed and recommended numerousbiomarkers for GC However since malignant tumors ofteninvolve multiple layers and diï¬erent levels of genetic changesincluding the genome transcriptome and proteome or evenepigenetic content selecting reasonable candidate factors fromtens of thousands of biomarkers and comprehensively analyzingthem as an independent feature is imperative to eï¬ectivelydevelop a suitable prognostic target Therefore genetic networkscontaining a panel of abnormal factors from diï¬erent regulatorylevels represent the best chance for achieving prognostic valueThe whole genomewide network analysis is reported inseveral other cancers such as colorectal cancer breast cancer andlung cancer Hou Zhang and it showsgreat value in diï¬erentiating the prognosis of these patientsTherefore it is feasible and advantageous to apply genomewidenetwork analysis to GCIn the current study we performed a series of sophisticatedstatistical analyses and identiï¬ed genetic molecules that werehighly correlated with the prognosis of GC Speciï¬cally wescreened The Cancer Genome Atlas TCGA a genome projectwith types of cancer including gene expression and DNAmethylation as well as other biological information Furthermorewe extended these independent prognostic factors to theomics concept Then a genomewide network was constructedInterestingly the genomics score GS obtained herein couldsupplement TNM staging and enhance the prognostic value ofdiï¬erent patients Moreover we developed multiple prognosticmodels then validated and compared them to ascertain theirstrengths and weaknesses Finally we performed pathwayenrichment and gene oncology annotation analyses to elucidatethe function of this gene networkMATERIALS AND METHODSData Acquisition and PreprocessingLevel data were downloaded from the TCGA databaseusing TCGAAssembler Module Ain January whichwas then pretreated with Module B The dataset comprised ofclinical variables from patients including age sex stageFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreprimary site grade treatment and survival as well as associatedgenomewide data In addition the expression levels of miRNAs mRNA and DNA methylation sitesIllumina methylation were obtained from and patients respectively Afterward an intersection with a totalof samples was eventually retained Furthermore patientswith missing active followup data were excluded from theanalysis leaving patients in the ï¬nal cohort Figure Moreover genomewide level data whose expression levelsfor miRNAs mRNA and DNA methylation sites were missingin more than of all samples were excluded from the ï¬nalanalysis Finally GC patients with miRNAs mRNA and DNA methylation sites were chosen forfurther analysisGenomeWide Network AnalysisGene expression and DNA methylation data were normalizedusing R package before subsequent processing Univariate andleast absolute shrinkage and selection operator LASSO Coxregression models were combined and used to identify themost useful prognostic factors in miRNAs mRNA and DNAmethylation sites associated with survival Firstly univariate Coxregression was performed on each candidate miRNA mRNA andDNA methylation site to elucidate its role in patient survivalthen signatures with P value less than were retained forsubsequent analysis Thereafter the LASSO Cox regression modelwas applied to select and shrink the data SupplementaryFigure S3 Tibshirani Finally a GS based on a genomewide network comprising seven miRNAs eight mRNAs and DNA methylation sites was constructed for predicting survivalA summary of the whole screening process is displayed inSupplementary Figure S1Development and Comparison ofIndividualized Prediction ModelsThe TCGA cohort with cases was used as the trainingset with a random cases from the total cohort includedas a validation group The random number is Firstlywe developed the original GS based on biomarkers sevenmiRNAs eight mRNAs and DNA methylation sites Thenconsidering the complexity of the original GS and diï¬cultclinical applicationin order to obtain a more concise andFIGURE A Venn diagram displays the patients screening processFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scoreeï¬ective GS we also constructed eight other models accordingto the diï¬erent combinations of miRNAs mRNA and DNAmethylation and made corresponding comparisons Finally atotal of nine GS models based on the genomewide networkfrom LASSO were adopted to screen for the most appropriatemarkers These included the following models genomics sevenmiRNAs eight mRNA and DNA methylation sites miRNAsseven miRNAs mRNA eight mRNA methylation DNAmethylation sites miRNAs methylation seven miRNAsand DNA methylation sites miRNAs mRNA sevenmiRNAs and eight mRNA mRNA methylation eightmRNA and DNA methylation sites Coxmodel twomiRNAs six mRNA and nine DNA methylation sites andCoxmodel one miRNA one mRNA and seven DNAmethylation sites Among them markers from Coxmodel were separately detected from miRNAs mRNA or DNAmethylation sites using multivariate Cox regression analysis afterLASSO Supplementary Tables S2S4 On the other handmarkers from Coxmodel depended on signatures from amultivariate Cox regression analysis combining the genomewide network and the clinical characteristics SupplementaryTable S5 Thereafter we constructed several nomograms byincorporating significant P GS variables and otherclinical features following multivariate Cox regression Iasonos and a clinical nomogram was built as a blank controlThe equations used for calculating the GS of these models arelisted in Supplementary Table S6To calculate the discrimination and the stability of diï¬erentCox regression models we applied Cstatistics and calibrationAdditionally we performed an analysis oftimedependentreceiver operator characteristics ROC based on the and 5year survival endpoints to assess the prognostic accuracyof the diï¬erent nomograms Furthermore we evaluated thepotential net beneï¬t of diï¬erent predictive models using decisioncurve analysis DCA DCA compares the clinical usefulness ofdiï¬erent indicators by calculating the potential net beneï¬t of eachdecision strategy at each threshold probability Thus DCA wasa significant novel approach for comparing the old and the newmodels Vickers and Elkin Screening for Potential miRNA TargetGenesWe predicted the potential target genes of the seven miRNAsfrom LASSO by screening the miRTarBase miRDB andTargetScan databases Common genes from each miRNA acrossthe three databases were then used for subsequent studies Morethan of the miRNAs showed negative regulation to targetgenes Consequently the expression data from TCGA were usedto perform a batch of correlation analysis of each miRNAwith corresponding target genes and the three genes with thelargest absolute negative correlation were retained as the mostlikely targets Additionally at least three potential target genesfrom miRTarBase which is coexpressed with miRNAs wereconsidered as equally important and were subjected to Cytoscapeversion for identiï¬cation of miRNAtarget genes coexpression network analysis Supplementary Figure S2Functional Enrichment AnalysisThe potential target genes that were negatively correlated withmiRNAs in TCGA as well as the coding sequences for mRNAand DNA methylation sites were used for functional enrichmentanalysis using the Kyoto Encyclopedia of Genes and GenomesKEGG pathway and Gene Ontology GO using DAVID Supplementary Figure S2 Functional enrichment analysisindicates why the gene network produces images on the survivalof GC from a molecular mechanism Visualization was then doneusing the ggplot2 package implemented in RStatistical AnalysisThe patients were divided into lowrisk and highrisk groupsby the median GS as the cutoï¬ point Survival estimates wereobtained according to the KaplanMeier method and comparedusing the logrank test Variables that reached signiï¬cance withP were entered into the multivariable analyses usingthe Cox proportional hazards model with an entry stepwiseapproach to identify covariates associated with increased allcause mortality and then hazard ratio with conï¬denceintervals CIs of each variable was achieved All the statisticalsigniï¬cance values were set as twosided P LASSOCox regression was performed through the glmnet packageTimedependent ROC analysis at diï¬erent followup times wasimplemented using the timeROC package of R project in orderto further expound the performance of diï¬erent GS modelsand DCA was used to compare their clinical use by rmdapackage Finally nomogram based on the Cox regression modelwas constructed using the rms package Cindex and calibrationto calculate the discrimination and the stability of these modelswere performed using cstatistics and Bootstrap sample Harrellsconcordance index Cindex indicated a better prognostic modelif its value was closer to and the calibration diagram showedthat the better the prediction if the closer the correction line wasto the diagonal All statistical methods are applied to both thetraining group and the validation group Statistical analyses wereperformed using SPSS statistical software version and Rsoftware version RESULTSPatient CharacteristicsAmong the GC patients analyzed in this study were male whereas were female The average ageof the entire study population was ± years In termsof pathological stage cases were identiï¬ed as stageI were stage II were stage III and were at stage IV With regards to treatment patients received surgery cases of R0 surgery R1 and nineR2 whereas were subjected to ï¬uorouracilbasedchemotherapy The genomic nomogram classiï¬ed samplesinto low GS GS ¤ and into high GS GS groups based on the median cutoï¬ Figure A detaileddescription of tumor location pathology grade and Laurenclassiï¬cation is outlined in Supplementary Table S1 while aheat map of the genomic scores layered by clinicopathologicalFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Distribution of patient cases and density based on genomics score GS in the total The Cancer Genome Atlas cohort AB Scatter plots of genomicsscores regarding the classiï¬cation of low and high GS CD and the bold line represents the medianfactors is illustrated in Supplementary Figure S4 The medianmean CI survival time for OS was days in the total cohort days in the highGS group and mean days in the lowGSgroup Supplementary Figure S5 Toward the last followupa total of deaths and censoring were recorded Theestimated cumulative and 5year OS in the total cohortwere and respectively although these ratesincrease to and respectivelyin the lowGS group Conversely the and 5year OS decreased to and respectively in the highGS group Thebaseline information of the validation cohort is also listed inSupplementary Table S1 and Supplementary Figure S6Survival AnalysisWe identiï¬ed a basic genomewide network comprisingseven miRNAs eight mRNAs and DNA methylationsites as the prognostic factor for OSfrom hundreds ofthousands of univariate Cox regression and LASSO analysesThis network was then classiï¬ed as other models in thetraining and the validation groups Among the featuresidentiï¬ed poor prognosis was significantly associated witha high expression of seven miRNAs hsamir100 hsamir hsamir136 hsamir193b hsamir22 hsamir653and hsamir6808 six mRNAs NRP18829 RNF144A9781ZNF227570 DUSP11843 CPNE8144402 MAGED19500and LOC9145091450 and seven DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics Scorecg22813794cg26014401cg25361506cg07020967 cg08859156 cg12485556 cg15861578 cg15861578cg25161386 and cg22740006 Conversely poor prognosis wasstrongly associated with a low expression of SOX148403 and DNA methylation sites including cg02223323 cg00481239cg14791193 cg15486740 cg20100408 cg20350671 cg22395807cg24361571andcg26856948 Table Univariate analysis performed onclinical characteristics revealed a significant association betweenage pathological stage TNM and surgery with OS Table On the other hand results from multivariable Cox regressionshowed that age pathological stage and GS were significantlyassociated with allcause mortality in GC Table and Figure Furthermore stratiï¬cation of the pathological stage I II IIIand IV revealed significant diï¬erences in survival rates betweenthe highGS and the lowGS groups Figure A similarresult was found when the data were stratiï¬ed by demographicvariables sex and age clinical characteristics primary sitegrade and Lauren classiï¬cation as well as treatments surgeryand chemotherapy Supplementary Figures S7 S8 Onthe other hand categorizing GS into high or low groupsusing the median value across diï¬erent modelsindicatedthe genomics nomogram had the highest HR valuethatInterestingly HR was almost equal to miRNAs methylationmRNA methylation Coxmodel and Coxmodel nomograms which contained fewer gene features Moreoverthe HR value showed a marked decrease in miRNAs mRNAmethylation and miRNAs mRNA nomograms which includedthe least characteristics Table Nomograms Based on GenomeWideNetworkA genomics nomogram was ï¬rst constructed based on thegenomewide network comprising gene features Figure To obtain a more concise and eï¬ective nomogram we also builta Coxmodel gene features and Coxmodel nine genefeatures nomograms Supplementary Figures S9 S10 Nexta clinical nomogram based on stage and age was built as acontrol Supplementary Figure S11 Thereafter we performedinternal and external validation to evaluate the feasibility of allnomograms using a threegrouped random bootstrap samplingFigure and Supplementary Figures S9S11 We observedgood predictive performance in the ï¬rst three nomograms butnot in the simple clinical modelValidation of the Nomograms Using ROCand DCATo ensure a good comparison across diï¬erent GS nomogramswe performed a timedependent ROC at and years offollowup as well as DCA In the validation group genomicsnomogram revealed the best comprehensive performance with and 5year area under the curve AUC values of and respectively Table and Coxmodel miRNAs methylation and mRNA methylation nomogramshad a comparable performance with and 5year AUCvalues of and respectivelybut it had fewer biomarkers Table Although the Coxmodel nomograms had the least biomarkers including miRNA mRNAand DNA methylation sites it had a relatively poor performancewith and 5year AUC values of and respectively Besides that the miRNA mRNA methylationmiRNAs methylation and miRNAs mRNA nomogramsrecorded and 5year AUC values of and respectively Finally we found thatcompared to miRNA and and mRNAnomogram and methylation nomogramhad higher and 5year AUC values of and Nevertheless all of them showed better performance thanthe clinical nomogram which recorded and 5year AUCvalues of and respectively Figures 6AB andSupplementary Figure S12 The Cindex based on diï¬erentnomograms exhibited a similar eï¬ect Supplementary Table S8Additionally DCA showed that the genomics Coxmodel mRNA methylation and methylation nomograms had asignificant net beneï¬t compared to other GS models and theclinical nomogram Figures 6CDPotential miRNA Target GenesA total of hsamir22 hsamir100 hsamir136 hsamir193b hsamir653 hsamir1304 and hsamir6808 potential target genes were identiï¬ed from themiRTarBase miRDB and TargetScan databases SupplementaryFigure S14 We then performed a correlation analysis betweeneach target gene and miRNAs and ï¬nally generated a miRNApotential target gene plot Supplementary Figure S15A as wellas a miRNAtarget gene coexpression network SupplementaryFigure S15B using CytoscapeFunctional Analysis of GenomeWideNetworkWe imported the potentialtarget genes mRNA andDNA methylation sitecoding sequences identiï¬ed above intoDAVID for KEGG and GO analyses and identiï¬ed biologicalprocesses molecular functions as well as cellular componentsFigures 7AC Their corresponding KEGG pathways were alsoplotted Figure 7DDISCUSSIONGC can be divided into two types or four main categoriesaccording to the Lauren and World Health anizationWHO classiï¬cations Lauren Nagtegaal although neither of these classiï¬cations is based on molecularmarkers In the last decade however three novel molecularbased classiï¬cation systems have been suggested for GC TheSingaporeDuke Group was the ï¬rst to describe a classiï¬cationwith two intrinsic genomic subtypes GINT and GDIF whichhad diï¬erent gene expression Tan Serra The subtypes have diï¬erent levels of resistance to variouschemotherapy drugs and show limited prognostic value LaterTCGA used molecular evaluation to propose a new classiï¬cationwith four subtypes EBV MSI GS and CIN The identiï¬cation ofthese subtypes has provided a roadmap for patient stratiï¬cationFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Univariable and multivariable analyses of the genomics score and the clinicopathological characteristics for overall survival in the training group and thevalidation groupVariablesTraining group n Validation group n Univariable analysisMultivariable analysisUnivariable analysisMultivariable analysisHR CIP valueHR CIP valueHR CIP valueHR CIP valueAge at diagnosis tears¥Pathological stageIIIIIIIVSurgeryR0R1R2UnknownGenomics scoreaLowHighT stagingT1T2T3T4N stagingN0N1N2N3M stagingM0M1SexFemaleMalePrimary siteCardiaFundusbodyAntrumUnknownPathology gradeIIIIIIIVUnknownLauren classiï¬cationIntestinal typeDiffused typeUnknownChemotherapyYesNoNANANANA NA NANANA NA NANANA NANANANANANANANANANANANANANANA NANANANANAaBased on biomarkers seven miRNAs eight mRNAs and DNA methylation sitesFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE miRNAs mRNA and DNA methylation whose expression levels showed a significant association with overall survival in least absolute shrinkage andselection operatorMolecular probeID reference geneCoefï¬cientmiRNAsmRNAcg02223323cg00481239cg07020967cg08859156cg12485556cg14791193cg15861578cg15486740cg20100408cg20350671cg22395807cg24361571cg25361506cg25622155cg25161386cg22740006cg22813794cg26014401cg26856948hsamir100hsamir1304hsamir136hsamir193bhsamir22hsamir653hsamir6808NRP18829RNF144A9781ZNF227570SOX148403DUSP11843CPNE8144402MAGED19500LOC9145091450MAP7D2SHC4EID1TMEM117RPS4XPREPC1orf144ZC3H10ACOT13TTRAPHLADPB1IL1RAPL1ATXN10MIR3652Unconï¬rmedUnconï¬rmedNUFIP2PCLRFN4STYXL1MDH2Unconï¬rmedGOLGA3HR CISEz valuep valueas well as targeted therapeutic trials Cancer Genome AtlasResearch However initial data on disease outcomes fromthis cohort did not show diï¬erences in survival among thefour groups A series of positive studies on prognosis basedon TCGA classiï¬cation was also reported Sohn In addition the Asian Cancer Research group divided GCinto four subtypes MSI EMT MSSTP53 and MSS TP53based on gene expression data and found significantly diï¬erentsurvival outcomes across them Cristescu Serra Despite the significant milestones of these studiesthey are all mainly based on the analysis of gene expressionmRNA Besides that a study proposed a ï¬vemiRNAmodel while it had a Cindex of only Zhang In the current study we included methylation data andperformed functional enrichment analysis making our workstronger The aforementioned classiï¬cations are also complicatedand need further optimization to increase clinical applicabilityFurthermore they focused on typing and ï¬nding new targetswhereas our study reports on prognostic analysisSome of the biomarkers we identiï¬ed herein including hsamir22 hsamir100 hsamir136 hsamir193b hsamir1304NRP1 DUSP1 and MAP7D2 cg02223323 have previouslybeen reported in GC Grandclement and B Chen Mu Zuo Zheng Chen Kurata and Lin Liu KT Song Teng Liu Pan Wang Others such as CPNE8MAGED1 RNF144A SOX14 ACOT13 cg15486740 EID1cg00481239 RPS4X cg08859156 and TTRAP cg15486740have been identiï¬ed in various tumors other than GC Kamio Zeng Zhou Kuang Stanisavljevic Liu X Tosic Nagasawa Yang The remainingbiomarkers including hsamir653 hsamir6808 LOC91450Frontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE KaplanMeier curve of overall survival in all patients then stratiï¬ed by genomics score GS pathological stage and age Survival analysis in the lowand highGS groups was further divided based on stages stages IIVcg20100408 LRFN4cg14791193 GOLGA3ZNF22 C1orf144cg26856948HLADPB1cg22740006 MDH2cg22813794 MIR3652 cg24361571 NUFIP2 cg25161386PREPcg22813794 TMEM117cg07020967 ZC3H10 ZC3H10 IL1RAPL1 cg20350671 PCcg22740006 SHC4 cg00481239 and ATXN10 cg22395807have not been previously reportedcg12485556STYXL1Currently focus has been directed on identifying prognosticmiRNAs for GC Particularly one miRNA can regulate multipletargets while multiple miRNAs can regulate a single mRNATherefore a single miRNA may play an opposite role incancer progression by regulating diï¬erent target genes Forexample Mir22 and Mir100 were found to be tumorsuppressors in various cancers including GC Chen Zuo Similarly a high expression of Mir136 wasfound to promote proliferation and invasion in GC cell linesby inhibiting PTEN expression Chen while acontrasting result was reported when HOXC10 was targetedZheng Similarly Mir193b reportedly inducedGC proliferation or apoptosis by mediating diï¬erent mRNAexpressions Mu Song whereas a highMir1304 expression in GC was reported as a negative predictorfor prognosis of lung and thyroid cancers Kurata and Lin Liu Pan However the function of Mir653and Mir6808 has not been previously reported In the currentstudy we found an association between a high expression of allmiRNAs and poor survival Diï¬erent outcomes may be observedin our study relative to previous reports owing to the hugeFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreTABLE Comparison of different genomics score models based on the median value for overall survival in the training group and the validation groupVariablesTraining group n Validation group n Hazard ratio CIP valueHazard ratio CIP valueGenomics nomogramAgePathological stageGenomics scoreaClinical nomogramAgePathological stagemiRNAs nomogramAgePathological stageGenomics scorebmRNA nomogramAgePathological stageGenomics scorecMethylation nomogramAgePathological stageGenomics scoredmiRNAs methylation nomogramAgePathological stageGenomics scoreemiRNAs mRNA nomogramAgePathological stageGenomics scorefmRNA methylation nomogramAgePathological stageGenomics scoregCoxmodel nomogramAgePathological stageGenomics scorehCoxmodel nomogramAgePathological stageGenomics scoreiaBased on biomarkers seven miRNAs eight mRNA and DNA methylation sites bBased on seven miRNAs cBased on eight mRNA dBased on DNA methylationsites eBased on seven miRNAs DNA methylation sites fBased on seven miRNAs eight mRNA gBased on eight mRNA DNA methylation sites hBased ontwo miRNAs six mRNA nine DNA methylation sites iBased on one miRNAs one mRNA seven DNA methylation sitesnumber of corresponding miRNA target genes herein and lackof evidence on their role in GC developmentMessenger RNAs have been reported to play an essentialrole in GC cancer For example high NRP1 expression andhypermethylation were associated with poor GC prognosisWang whereas another study indicated thatit could be an antitumor target Grandclement and B In addition high DUSP1 expression levels were foundto promote progression drug resistance and poor prognosisof GC Teng On the other hand SOX14showed opposite prognostic values in cervical cancer andleukemia with antitumor and carcinogenic eï¬ects respectivelyStanisavljevic Tosic Studies have alsoimplicated CPNE8 MAGED1 and RNF144A in ovarian andbreast cancers Zeng Nagasawa Yang However LOC91450 and ZNF22 have not beenreported in cancerAccumulating evidence indicates that DNA methylation playsa significant role in cancer progression However only a handfulof studies have described the relationship between levels ofFrontiers in Genetics wwwfrontiersinAugust Volume 0cSun et alGenomics ScoreFIGURE Genomics nomogram to predict the probability of and 5year overall survival OS in the training cohort A and the validation cohort B todetermine how many points for each variable to the probability of OS locate the variable on its axis draw a line straight upward to the point axis repeat this processfor each variable sum up the points achieved for each of the risk factors locate the ï¬nal sum on the total point axis and draw a line straight down to ï¬nd thepatients probability of OSsinglesite methylation and GC prognosis Particularly highexpressions of MAP7D2 ACOT13 EID1 RPS4X and TTRAPhave been associated with poor prognosis in gastric lung andpancreatic cancers as well as hepatic carcinoma respectivelywhile a high TTRAP expression reportedly inhibits the growthof osteosarcoma Kamio Zhou Kuang Liu KT Liu X Notably therelationship between methylation levels and corresponding geneexpression proï¬les is unknown necessitating further researchFurthermore the remaining DNA methylation sites and theircorresponding genes have not been reported Lastly no studyhas described the prognostic signiï¬cance using a genomewide networkLast but not l | Thyroid_Cancer |
In a novel coronavirus SARSCoV2 was found to cause a highly contagious disease characterized by pneumonia The disease COVID19 quickly spread around the globe escalating to a global pandemic In this review we discuss the virological immunological and imaging approaches harnessed for COVID19 diagnosis and research COVID19 shares many clinical characteristics with other respiratory illnessesAccurate and early detection of the infection is pivotal to controlling the outbreak as this enables case identification isolation and contact tracing We summarize the available literature on current laboratory and pointofcare diagnostics highlight their strengths and limitations and describe the emerging diagnostic approaches on the horizonWe also discuss the various research techniques that are being used to evaluate host immunity in laboratoryconfirmed patients Additionally pathological imaging of tissue samples from affected patients has a critical role in guiding investigations on this disease Conventional techniques such as immunohistochemistry and immunofluorescence have been frequently used to characterize the immune microenvironment in COVID19 We also outline the emerging imaging techniques such as the RNAscope which might also aid in our understanding of the significance of COVID19specific biomarkers such as the angiotensinconverting enzyme ACE2 cellular receptorOverall great progress has been made in COVID19 research in a short period Extensive global collation of our current knowledge of SARSCoV2 will provide insights into novel treatment modalities such as monoclonal antibodies and support the development of a SARSCoV2 vaccineKeywordsCOVID19 immunology pathology diagnostics specific T cellsIntroductionIn December a novel respiratory disease named coronavirus disease COVID19 was detected by physicians in Wuhan China The disease was found to be caused by the severe acute respiratory syndrome SARSCoV2 RNA virus12 Within a matter of weeks COVID19 had spread rapidly and escalated to a global pandemic At the time of writing June million cases had been reported and patients had succumbed to the disease worldwide3 Indeed patients with COVID19 are at high risk of developing a severe and critical disease4 Therefore rapid and accurate diagnostic tests are urgently needed to effectively isolate identify and treat infected individuals and to contain the spread of the virus Failure to do so will inevitably lead to spikes in cases and the resultant overcrowding and collapse of healthcare services5 Moreover research into this novel virus is also critical to understand its pathogenesis and its interaction with the human immune system Insights from such research will guide the design of public health policies and protocols to 1Lee Kong Chian School of Medicine Nanyang Technological University Singapore Singapore2Yong Loo Lin School of Medicine National University of Singapore Singapore Singapore3Institute of Molecular Cell Biology IMCB Agency of Science Technology and Research ASTAR Singapore Singapore4Department of Anatomical Pathology Singapore General Hospital Singapore SingaporeThese authors contributed equallyReceived June and in revised form July Accepted for publication July Corresponding AuthorJoe Poh Sheng Yeong Institute of Molecular Cell Biology IMCB Agency of Science Technology and Research ASTAR College Road Academia Level Diagnostics Tower Singapore Singapore Email yeongpsimcbastaredusg 0c SLAS Technology identify susceptible individuals and diagnostic prognostic and treatment approaches for patientsCurrent diagnostic approaches predominantly involve established virological procedures such as nucleic acid hybridization techniques reversetranscriptase PCR [RTPCR] and recombinase polymerase amplification RPA as well as immunologic approaches like antibody assays Each approach boasts unique strengths and weaknesses For instance while RTPCR demonstrates high sensitivity and specificity its capabilities have been severely limited for practical reasons during this current pandemic due to global shortages of skilled personnel reagents and equipment and a processing time of up to days By contrast immunologic tests such as antibody assays are rapid and require minimal equipment but they have limited utility in the context of acute diagnosis of SARSCoV2 infections This is because it can take several days to weeks following symptom onset for a patient to mount a detectable antibody response6Immunological tools in research include enzymelinked immunosorbent assays ELISAs flow cytometry and mass cytometry CyTOF Imaging techniques for pathological analyses include conventional approaches such as hematoxylineosin HE staining immunohistochemical IHC staining or transmission electron microscopy TEM and RNAscope Each of these methods is used to examine the pathophysiology underlying COVID19 from a different perspective each with their own advantages and disadvantages For example it has been established that the entry of SARSCoV2 intro cells depends on the binding of viral proteins with the human receptor angiotensinconverting enzyme ACE2 receptors7 Additionally evidence shows that the type II transmembrane protease TMPRSS2 is also essential for viral entry by priming the viral spike protein for binding to ACE28 Therefore considerable research efforts employing different techniques have been directed at mapping the distribution of ACE2 and TMPRSS2 in tissues and their relationship to the observed manifestations of disease Together the combination of these approaches has advanced our understanding of COVID19In this review we discuss the current approaches in COVID19 diagnosis and research with a focus on findings from virological and pathological imaging methods We also discuss immunological methods which are increasingly recognized as an integral component of the disease processDiagnosticsThe most common symptoms of COVID19 at initial presentation are nonspecific and include a high fever a new and persistent cough and fatigue910 Due to similarities between the clinical characteristics of COVID19 and many other respiratory illnesses the accurate and early detection of infection is pivotal for outbreak control Any delays in diagnosis are increasingly measured in lives lostAccording to the World Health anization WHO the immediate goal for research into COVID19 diagnostics is the development of RNA assays antibody and antigen assays and pointofcare detection11 The intermediateterm priority would be their integration into multiplex diagnostic platforms while the longterm goal would be the investigation of prognostic markersIn this section we summarize the current and emerging diagnostic tools for SARSCoV2 through the lens of immunologyLabBased TestsRTPCR Molecular Testing The detection of viral nucleic acids by RTPCR is the primary method used to confirm a suspected case of COVID19 RTPCR and other nucleic acid hybridization techniques are an integral part of virology and are applied in a broad range of settings including screening diagnosis informing medical and therapeutic decisions and assessing cure rates from therapy12 Chinese officials released the genomic sequence of SARSCoV2 to public databases early in the course of the outbreak13 and the WHO has since published seven protocols for RTPCRbased diagnostics Because of the high sensitivity and specificity of RTPCR it is regarded as the gold standard for virus detection14 There are two essential steps in the process viral RNA extraction and PCR amplification and probebased detection Multiple largescale highthroughput instruments are available for automating both steps such as the Roche Cobas system which has an advertised throughput of tests per hours15However RTPCRbased testing is costly and timeconsuming requiring up to days using centralized laboratory equipment and skilled personnel furthermore global supply chain challenges have led to significant shortages of essential reagents Lastly falsenegative results due to low sample volumes variable sampling techniques and sampling locations sample degradation during transportation andor improper nucleic acid extraction are a concern16 In addition the differences in detectable viral material in different sampling locations eg nasopharyngeal vs bronchoalveolar lavage fluid [BALF] vs rectal samples might also explain the falsepositive RTPCR results on repeat testing in recovered COVID19 patients Indeed one postmortem case study revealed residual virus in lung tissue despite consecutive negative results on PCR testing from nasopharyngeal swabs19 Separately Winichakoon et al outlined a case of repeatedly negative nasopharyngeal and oropharyngeal swabs in a clinically deteriorating patient where only a BALF PCR test returned positive20Given the high expression of ACE2 on alveolar epithelial cells and negative expression on nasal oral and nasopharynx 0cTan et al cells21 it would be prudent to perform bronchoalveolar lavage on patients to rule out falsenegative results from swabs of upper respiratory tract samples20LabBased Immunological Assays In contrast to RTPCR techniques that detect viral nucleic acids serological and immunological assays aim to detect antibodies against SARSCoV2 or antigenic proteins in infected individuals Neutralization assays are considered the gold standard for assessing neutralizing protective antibodies22 however these assays require specialized biosafety level BSL3 facilities and still take several days to complete Another type of labbased antibody assay is the traditional ELISA which detects all binding antibodies The four principal types of ELISA are direct indirect competitive and sandwich ELISA the indirect ELISA is the most common method for determining antibody concentrations ELISAs have good concordance with neutralization assays for the detection of antibody responses in SARSCoV223 Unfortunately both methods require skilled operators and are limited by low throughput due to the absence of fully automated systemsSerological diagnostics offer several advantages Re quirements for specimen quality are comparatively less stringent than for nucleic acid tests as the antibodies are uniformly distributed in the serum24 Consequently sampling location concerns do not apply here Furthermore good correlation between IgG ELISAs performed on both conventional serum samples and plasma samples have been reported25 of which the latter may be conveniently obtained from residual blood submitted for other routine laboratory testsOne pitfall of antibody assays is their limited utility early in the course of any infection Sparse data are available with regard to the antibody responses produced by patients with COVID19 It seems that SARSCoV2 IgM is detectable at a median of days after symptom onset while IgG is detectable after days26 with the seroconversion rate approaching by day An Italian research group noted that the performance of a commercial VivaDiag COVID19 IgMIgG test was very poor with a sensitivity of only and a negative predictive value of in a cohort of suspected COVID19 patients in the emergency room setting27 As such we believe that for now RTPCR testing is likely more appropriate for diagnosing acute COVID19Notably a longitudinal study examining the IgGIgM profiles of patients found that seroconversion for IgG and IgM occurred in no specific chronological order with a median of days after symptom onset28 all patients achieved seroconversion by day Consequently the detection of both IgG and IgM simultaneously rather than one antibody alone would be idealAnother concern surrounding serologic diagnostics is the production of falsepositive results from crossreactivity due to the high prevalence of the four endemic human coronaviruses in the human population In SARSCoV2 the spike S protein which includes two regions S1 and S2 and the nucleocapsid N protein NP are the major immunogens29 and therefore most diagnostics rely on the detection of antibodies specific for these antigens One work suggests that of the possible targets the S1 subunit antigen is more specific than either the whole S antigen or the N antigen for detecting SARSCoV2 antibodies with no crossreactivity to other coronaviruses except for SARSCoV23 Given that only SARSCoV infections were recorded worldwide30 the risk of false positives from this crossreactivity is miniscule However NP ELISAs are more sensitive than S1 in detecting antibodies in those with a mild infection23 Importantly as in SARSCoV most of the neutralizing antibodies are directed against the S protein31 of which S1 contains a receptorbinding domain RBD responsible for making contact with ACE2 to facilitate viral entry7 Thus theoretically only diagnostics that detect S1specific antibodies are suitable to infer immunity to COVID19 this fact is corroborated by evidence that antiS RBD but not antiNP IgG levels correlated with neutralizing antibody titers in sera from a cohort of recovered patients32 The number of commercial antibody assays is growing detecting either antiNP antibodies antiS1S antibodies or both there is also large variation in their claimed sensitivities and specificities33 Based on the available evidence an ideal serological assay would be a combined test that simultaneously detects both antibodies to NP and S1 antigens assessment of antiNP antibodies has good sensitivity and would be best suited for supporting the diagnosis of infection while the additional antiS1 antibody assay would allow for the determination of immunityRapid TestsPointofCare RTPCR Tests A small number of commercial pointofcare tests utilizing RTPCR have been developed These typically involve the same methodology as conventional RTPCR but implemented with automated and portable benchtopsized instruments that can be operated closer to patient care settings than a centralized laboratory A prominent example is Cepheids Xpert Xpress SARSCoV2 run on the Gene Xpert platform This apparatus can provide a result within min Others include the MesaBioTech Accula Test and MicrosensDx RapiPrep COVID19 Despite displaying good sensitivity and specificity these instruments are generally limited by a very low throughput of only one to four tests per run per machine34 and as such are only suited to small laboratories or clinics 0c SLAS Technology Figure Loopmediated isothermal amplification LAMP A LAMP begins when the forward inner primer FIP binds to the A2C region while the forward primer A1 binds to A1C which displaces the FIP complementary strand B The backward inner primer BIP binds B2C while the backward primer B3 binds B3C and displaces the BIP complementary strand C A complementary sequence that initiates loop formation is produced D Loop structures are formed that allow for LAMP with the use of loop primersFigure CRISPR technique Viral RNA is converted to dsDNA using RTRPA recombinase polymerase amplification A The CAS12a nuclease enzyme is activated upon complex binding to the target sequence resulting in cleavage of the target sequence and the fluorescent RNA reporter B T7 transcription converts DNA to complementary RNA Cas13 nuclease enzyme activity is activated upon complex binding to the target sequence resulting in a similar cleavage of the target sequence and the fluorescent RNA reporterImmunological AssaysRapid Antibody Assays Compared with labbased antibody assays rapid assays such as lateral flow immunoassays LFIAs Fig and chemiluminescent immunoassays CLIAs Fig offer the benefits of rapid diagnostic testing at a low cost These assays do not require specialized equipment or expertise35 and are thus excellent candidates for pointofcare testing or deployment on a large scale This an area of intense interest with governments worldwide aiming to order millions of tests to inform policy makers about attack rates in their populations36 LFIAs are predominantly singleuse kits designed for pointofcare use while CLIAs are fully automated analyzers that permit very high testing throughputUnfortunately these tests do not quantify the antibody titers and the performance of LFIAs has been called into question one evaluation of nine commercial LFIAs reported a sensitivity ranging from only to versus RTPCR and to versus ELISA37 Meanwhile the performance of CLIAs is superior with good sensitivity and specificity levels similar to those of ELISA38 Otherwise these tests share the same advantages and drawbacks as the 0cTan et al Table Summary of Diagnostic Approaches for COVID19CategoryType of TestTypical Test Result TimeCharacteristicsExamplesVirologicmolecular RTPCRDaysGold standard high sensitivity WHO RTPCR protocolstests Pointofcare RTPCR minLAMP CRISPR hImmunologic testsLFIA for antibodies minantigensand specificity high throughput but lab basedRapid good sensitivity and specificity pointofcare testing but low throughputRapid good sensitivity and specificity pointofcare testing but low throughputRapid pointofcare testing but not quantitative poor sensitivityCepheid Xpert Xpress SARSCoV2Sherlock Biosciences SHERLOCKVivaDiag COVID19 IgMIgG rapid testCorisbio COVID19 Ag RespiStripEpitope Diagnostics KT1033 EDI Novel Coronavirus COVID19 ELISA kitRoche Elecsys AntiSARSCoV2 Traditional ELISA hGood sensitivity and specificity but lab based not automatedCLIA minRapid good sensitivity and specificity high throughput but lab basedNeutralization assayDaysGold standard high sensitivity Not commercially and specificity able to quantify neutralizing antibodies but requires BSL3 lab facilityavailablelabbased antibody assays discussed above The characteristics and unique advantages and disadvantages of these different methodologies are outlined in Table Antigen Assays An alternative approach to immunological assays is to directly detect SARSCoV2 viral antigens Several commercial pointofcare antigen tests are available but their performance remains to be evaluated These tests may be suitable for making an early diagnosis and are deployable as pointofcare assays However they face the same sampling limitations as RTPCR and are hypothetically hampered by limited sensitivity due to the omission of an amplification process unlike nucleic acid testing For example one multicenter study evaluating the Corisbio COVID19 Ag RespiStrip a lateral flow assay for the SARSCoV2 NP reported a test sensitivity of only Rapid NonPCR Molecular Testing Nucleic acid testing using nonPCR methods is an emerging approach for rapid diagnostics and several assays have received Food and Drug Administration FDA emergency use authorization which facilitates the distribution of unapproved medical products or the offlabel use of approved medical products when certain criteria are met These methods share high sensitivity and specificity on par with RTPCR but with the principal advantages of more rapid testing at a lower cost40LAMP Fig is one such novel isothermal nucleic acid amplification method that does not require a thermal cycler One example is the ID NOW COVID19 test from Abbott Diagnostics which can deliver results in just min43 and uses a lightweight portable instrument allowing onsite testing of swab samples However it has a limited throughput of only one sample per runThe CRISPR enzymes Cas12 and Cas13 have also been adapted for rapid nucleic acid sensing Fig The DETECTR assay by Mammoth Biosciences45 as well as the SHERLOCK assay by Sherlock Biosciences46 potentially offers sensitivity and specificity comparable to those of RTPCR but can be completed in h However these approaches are still in the early stages of commercialization and current applications are available only as test kits to be run in labs while pointofcare versions exist as proofofconcept demonstrations47 Nonetheless their inherent characteristics hold great potential for diagnosis in the futurePrognostication of DiseaseProfiling of Genetic Susceptibility Work is in progress to ascertain the possible genetic basis for the apparent variations in COVID19 susceptibility and disease severity Cao et al compared expression quantitative trait loci eQTL for ACE2 in different populations finding significantly greater eQTL variants associated with higher ACE2 expression in 0c SLAS Technology Figure Lateral flow immunoassay LFIA A Serum sample deposited on the sample pad B AntiSARSCoV2 antibodies in the sample will bind to the target antigen with a labeled tag C Immobilized antihuman IgM antibodies will capture the SARSCoV2 antibodyantigen complex D Control antibodies are captured by immobilized antibodies in the control lineSerum Prognostic Markers Another application of immunological methods would be to measure markers that enable prognostication in COVID19 Higher titers of antibodies against SARSCoV2 have been associated with more severe disease2350 similar to previous studies in Middle East respiratory syndrome MERSCoV51 ELISA has been used to provide a quantitative measurement of serum and plasma IgM and IgG antibodies By monitoring the kinetics of IgM and IgG antibodies specific to the N and S proteins on SARSCoV2 it was found that intensive care unit ICU patients had a significantly lower level of SIgG within weeks of symptom onset but a higher level of NIgG antibodies compared with nonICU patients52 This finding highlights the possible utility of SIgG and NIgG as a prognostic tool for COVID19 patientsThe Ddimer level which consists of crosslinked fibrin degradation products that reflect ongoing blood clot formation and breakdown activity in the body is another proposed prognostic marker Modern commercial assays for Ddimers are based on monoclonal antibodies employing either ELISA or microlatex agglutination assays53 Reports have emerged that elevated Ddimer levels suggestive of a hypercoagulable state are associated with drastically worse outcomes A Chinese group reported that Ddimer levels of ¥ µgmL on admission were associated with a times increased mortality relative to Ddimer levels of µgmL in a cohort of COVID19 patients54 This finding of Ddimer levels as a negative prognostic marker was also noted in other studies conducted in China455 and the Netherlands56Similarly interleukin IL6 a key component of the cytokine release syndrome is another marker measured by ELISA and has been described to independently predict adverse outcomes in COVID195758 Tumor necrosis factor alpha TNFα another important proinflammatory cytokine has also been found to be strongly correlated with Figure Chemiluminescence enzyme immunoassay CLIA SARSCoV2 antigens will capture IgM and IgG antibodies from the sample serum Secondary antibodies that are conjugated with horseradish peroxidase HRP bind to the captured primary IgM and IgG antibodies and react with a chemiluminescent substrate to generate a strong chemiluminescent signal that is measured in terms of relative light units RLUEast Asian populations but reported no direct evidence supporting the existence of S proteinbindingresistant ACE2 mutants48 out of identified protein altering variants Separately Stawiski et al analyzed ACE2 polymorphisms within a much larger population dataset spanning more than population groups across the world and performed structural predictions to identify variants that might confer protection or rather increase susceptibility to SARSCoV2 S protein binding49 Out of a total of identified proteinaltering ACE2 variants variants were predicted to increase susceptibility while variants were speculated to confer protection however the degree of changes in receptorvirus binding interactions for each structural variant was not quantified These findings represent significant developments in our understanding of population risk profiles for COVID19 and future coronavirus infections 0cTan et al endan damage and mortality even after adjusting for disease severity scores59 Gao et al examined both IL6 and Ddimer levels they proposed a panel comprising tandem testing of these two markers which produced a sensitivity of and specificity of in early prediction of severe COVID1958 Elevated troponin levels a marker of myocardial injury measured with ELISA immunoassays also strongly predict progression to death in patients with severe illness60 These results suggest that multiplex cytokine and serum marker profiling will be a powerful tool in stratifying patients that may guide clinical decisions and resource allocationSummary In sum rapid progress has been made in diagnostics for COVID19 Yet the race against time continues for researchers and biotechnology firms to develop rapid costeffective and reliable test kits that can be deployed on a large scale At the time of writing labbased RTPCR testing has been the dominant diagnostic approach but alternative molecular approaches like isothermal amplification and CRISPR which have clear advantages are on the horizon Immunological tests such as CLIA and LFIA will become increasingly important because of the urgent need for pointofcare diagnostics for mass testing of infected asymptomatic individuals and their close contacts and will be valuable in complementing molecular approaches for confirming infection Furthermore immunological assays will be in great demand by policy makers worldwide for the assessment of immunity to COVID19 However the performance of these serological tests varies significantly particularly their degree of sensitivity and specificity we believe that caution must be taken in the interpretation of these tests Detailed evaluation of the reliability of serological tests will be a key area for future research Lastly given the importance of techniques like ELISA in prognosticating COVID19 immunological methods will undoubtedly occupy a crucial role in achieving all levels of the WHOs short medium and longterm diagnostic goalsCOVID19 Research ToolsImmunological ApproachesCOVID19 infection has a poor prognosis in individuals with comorbidities and abnormal immune functions Although research surrounding COVID19 is still in its infancy several studies have revealed lymphopenia and the cytokine storm as underlying mechanisms correlating to disease progression Here we discuss the various immunological techniques involved in assessing host immunity in COVID19 patientsELISA As discussed ELISA has also been used to detect the inflammatory cytokines implicated in the cytokine storm seen in patients with severe respiratory failure due to COVID19 One study found that the immune dysregulation in patients with severe respiratory failure was due to a significantly increased production of IL6 and defective lymphoid function because of an IL6mediated decrease in HLADR expression on CD14 monocytes Interestingly interferongamma IFNγ levels were below the detection level in these patients suggesting that T helper Th cells are unlikely to be major players in the overinflammatory response of severe patients61 A similar observation was made in a separate study whereby inflammatory cytokines that mediate major immune responses such as TNFα and IL1β were not significantly elevated in ICU patients62 These findings demonstrate that the immunophenotype of patients with COVID19 can vary depending on presently unclear host immune factors and the severity of their condition This relationship between disease severity and cytokine storm has also been highlighted in other studies that found a significantly elevated plasma concentration of granulocyte colonystimulating factor GCSF IP10 CCL2 and CCL3 in ICU patients compared with nonICU patients63ELISA is also being used as a companion diagnostic tool for therapeutic purposes In a study that explored the use of convalescent plasma therapy from donors as a form of treatment in severe COVID19 ELISA was used to assess the neutralizing activity of the RBDspecific IgM and IgG antibodies found in the donor convalescent plasma64 After the transfusion was complete ELISA was also used to detect IgG IgM and neutralizing antibody titers in the sera of patients to assess the response to treatment65EnzymeLinked Immunosorbent Spot Enzymelinked immunosorbent spot ELISPOT is a sensitive immunoassay that quantitatively measures cytokinesecreting cells at the singlecell level providing insight into immunerelated cellular activities66 Hence it is a promising tool for characterizing specific Tcell immunity in COVID19 patients By IFNγ ELISPOT analysis it was revealed that convalescent COVID19 patients had significantly increased levels of IFNγsecreting T cells when compared with healthy donors A significant correlation between neutralizing antibody titers and NPspecific T cells was identified in these patients suggesting that a combination of humoral and cellular immunity is integral to clearing SARSCoV2 Interestingly it was noted that in convalescent patients weeks IFNγsecreting Tcell numbers had postdischarge decreased suggesting that they may not be maintained for a prolonged period of time even in recovered patients67ELISPOT is also serving a vital role in vaccine development through the detection of potential Tcell epitopes in the S protein RBD of SARSCoV268 One study was able to harness ELISPOT assays to identify three Tcell epitopes that induced a strong adaptive immune response 0c SLAS Technology postimmunization demonstrating the promise of ELISPOT assays in the area of vaccine development32 Recently ELISPOT has also been applied to assess the immunogenicity of newly developed vaccines One such study successfully utilized an IFNγ ELISPOT assay to evaluate Tcell responses to a new SARSCoV2 vaccine in murine splenocytes and rhesus macaque peripheral blood mononuclear cells PBMCs The promising findings from this animal study informed the start of a phase I clinical trial with the same vaccine highlighting the usefulness of ELISPOT in assessing immune responses to new vaccines and promoting vaccine development69Flow Cytometry Unlike ELISA and ELISPOT flow cytometry determines the number of cytokinesecreting cells and has the capacity to immunophenotype based on surface and intracellular markers70 In relation to the current pandemic this technique enables the detection sorting and analysis of multiple subpopulations of immune cells specific to COVID Using flow cytometry researchers detected a cytotoxic immune environment in patient blood samples despite a reduction in the overall lymphocyte population71 As part of the SARSCoV2 antiviral response peripheral lymphocytes retain the capacity to activate and differentiate into subpopulations such as antibodysecreting cells CD3CD19CD27hiCD38hi follicular T cells CD4CXCR5ICOSPD1 CD4 Th cells CD38HLADRCD4 cytotoxic T Tc cells CD38HLADRCD8 and regulatory T Treg cells CD3CD4CD25CD127 These Tc cells harbor large amounts of cytotoxic granules while CD4 Th cells skewed toward a proinflammatory Th1 and Th17 phenotype727375 The overall hyperinflammation and cytotoxic environment supports the notion that a cytokine storm could be liable for the multisystemic insults in patients with severe COVID19Elicitation of antiviral Tcell responses specific to SARSCoV2 is of utmost importance to establishing viral control Many studies have demonstrated robust antiviral responses however there is no known set of markers reported to identify SARSCoV2specific T cells Collectively most groups have characterized SARSCoV2specific T cells based on HLADR CD38 CD69 CD25 CD44 and Ki67 expression T | Thyroid_Cancer |
"EPMA celebrated its 10th anniversary at the 5th World Congress in Pilsen Czech Republic The history of theInternational Professional Network dedicated to Predictive Preventive and Personalised Medicine PPPM 3PM is rich inachievements Facing the coronavirus COVID19 pandemic it is getting evident globally that the predictive approach targetedprevention and personalisation of medical services is the optimal paradigm in healthcare demonstrating the high potential to savelives and to benefit the society as a whole The EPMA World Congress Supplement highlights advances in 3P medicineIntroductionEuropean Association for Predictive Preventive and PersonalisedMedicine has been created in In the historical 1stEPMA World Congress took place in Bonn GermanyIn the EPMA celebrated its 10th anniversary at the 5thWorld Congress in Pilsen Czech Republic The decadeoldprofessional history of the EPMA is rich in achievementsHerewith we briefly highlight some of themGeographic distribution of the 3PMrelevant expertise underthe EPMAumbrella started with approximately countries in currently the EPMA is represented in countries University Hospital in Pilsen Medical Faculty in Pilsen CharlesUniversity Prague Czech RepublicEuropean Medical Association Brussels BelgiumEuropean Association for Predictive Preventive and PersonalisedMedicine EPMA Brussels BelgiumThe historical 1st EPMA World Congress in former Bundestag BonnGermany September Declarations The authors declare that they have no competing interest Permissions by responsible ethic commissions for correspondingcontributions have been received and thoroughly check prior topublishing the EPMA World Congress Supplement Research involving human participants andor animals was performedin accordance with international regualtions All the patient investigations conformed to the principles outlined in theDeclaration of Helsinki Informed consent was obtained from all individual participants included in the corresponding study Olga GolubnitschajaOlgaGolubnitschajaukbonndePredictive Preventive and Personalised 3P Medicine Departmentof Radiation Oncology University Hospital Bonn RheinischeFriedrichWilhelmsUniversitt Bonn VenusbergCampus Bonn Germany 0cworldwide who actively promote 3PM concepts in biomedicalsciences and practical medicine strongly benefiting patients andhealthcare systemsThe first issue of the EPMA Journal Springer Nature wasreleased in March In the journal received its firstIF in it reached Nowadays the EPMA J is ahighly recognised international forum for 3P medicine operating in a hybrid subscription access modus ScopusCiteScore of the EPMA J is wwwscopuscomsourceid19700201201originsourceInfozonerefpointranktabs1 thereby Scopus ranks the EPMA Jamongst the top in the category Health Policy due tohighly requested and wellcited strategic papers created bymultiprofessional groups of EPMA experts such as General report recommendations in predictive preventive and personalised medicine white paper ofthe European association for predictive preventive andpersonalised medicine 1011861878 Medicine in the early twentyfirst century paradigm andanticipation EPMA position paper 101186s1316701600724SCImago topranks the EPMA J in all three categoriesnamely Health policy Medical Biochemistry and Drugdiscoverywwwscimagojrcomjournalsearchphpq19700201201tipsidIn Springer Nature awarded the belowmentioned the status of an with a potential to change thew o r l d i n t h e c a t e g o r y M e d i c i n e a n d P u b l i cHealth wwwspringernaturecomgpresearcherscampaignschangetheworldmedicinepublichealthPregnancy Associated Breast Cancer The Risky StatusQuo and New Concepts of Predictive Medicine EPMA J s1316701801297Advances in Predictive Preventive and PersonalisedMedicine is a very successful EPMASpringer Nature bookseries which educates both professionals and the general population in 3P medicine Since book volumes havebeen released dedicated to a whole spectrum of PPPM relatedaspects such as digital health information technology framework application of artificial intelligence in healthcare drugdelivery systems liquid biopsy and multilevel diagnosticsamongst othersHorizon is the main European Scientific Programmewhich EPMA experts have contributed to with 3PMrelatedprotocols as well as with the topexpertise provided byRepresentatives and Members of the association involved inthe evaluation panelsEPMA JournalEPMA AWARD for EXCELLENCE in BIOMEDICALSCIENCES was created in and the 1st EPMA awardwas given to Prof Dr Josef Flammer University of Basel forphenotyping of the Flammer Syndrome which the EPMAinternational jury panel valued as being of great clinical utilityYoung professionals in PPPM Award was created bythe EPMA in Atthe international workshopslinked to the biannual EPMA World Congresses thepresentations made by young professionals get evaluated by an international jury panel The best presentations and smart 3PM concepts receive awards thateffectively promote the careers of young professionalsin innovative biomedical fieldsEPMA World Congress in Pilsen Czech Republicattracted 3PM experts from countries The congress wasdedicated to innovation in a broad spectrum of biomedicalfields with a specific focus on the concepts of predictive diagnostics targeted prevention and personalisation of medical services in Cancer Metabolic DisordersCardiovascular Disease Neurological Neurodegenerativeand Neuropsychiatric Disorders Inflammatory DisordersDentistry Biobanking and Screening ProgrammesMultiomics Microbiome Immune Pre andProbiotics and Innovative Technologies among othersFurther there were several new topics presented at the congress among others these were Implementation of 3PMConcepts in Plastic Surgery Application of ArtificialIntelligence in Medicine 3PM strategies and MedicalUse of Cannabis The latter topic was discussed in the EUParliament in and the EPMA position has been elucidated by the EPMA Representatives for more information seethe below linkhttpwwwepmaneteulatestevents2019epmapositiononmedicaluseofcannabispresentedattheeuparliamentOral and poster presentations provided valuable information regarding pilot projects towards personalisedhealthcare eg awarded by ICPerMedindividualisedpatient profiles multilevel biomarker panels patientstratification creation and application of innovative ITtools ethical issues doctorpatient collaboration optimalthe modern hospitalstructure and anisation ofambitioned to practically implementthe paradigmchange from reactive to predictive preventive andpersonalised medicineWorld First 3P Medical Unitthe historically first worldwide unitIn March dedicated to Predictive Preventive and Personalised3P Medicine led by SecretaryGeneral of the EPMAProf Dr Olga Golubnitschaja was created in Germanyatthe Department of Radiation Oncology UniversityHospital Rheinische FriedrichWilhelmsUniversittBonn 0cEPMA Journal3PM vision and strategiesPPPM for Twentyfirst Century Biosensing PainlessPersonalised PointofCare Monitoring with Wearableand Implantable DevicesAndrews RACorresponding author Nanotechnology Smart Systems NASA Ames Research Center Moffett Field CA USA email rjarusselljandrewsKeywords Artificial intelligence Biosensors Blood pressuremonitoring BraincomputerBrainmachine interfaceContinuous monitoring Diabetes ElectrocardiogramElectroencephalogram Epilepsy Fall detection Gait disorders Glucose monitoring Implantable sensors Ingestible sensors Internet of things Iontophoresis Interstitial fluidNanosensors Neurotechnology Pressure monitoring Salivamonitoring Seizure detection Smart contact lenses Smartmouthguards Smart patches Smart skin Smart watchesSmartphone apps Skin patches Sweat monitoring Tear monitoring Temperature monitoring Tissuedevice interfaceWearable sensors Wireless monitoringIntroductionMany people do not realize they already have adopted wearable devices for medical monitoringsmartwatches Typicalstories of smartwatches providing lifesaving diagnostic information include the following A smartwatch alarming allnight regarding abnormal heartrate alerted the wearer to seekmedical attention for what proved to be atrial fibrillation [] A hikerlost as nightfall approachedstumbled and fellon difficult terrain Unbeknownst to the hiker the fall triggered his smartwatch to automatically call the emergencyphone number in the USA thereby avoiding what couldhave been a tragic outcome Smartphones with accelerometerand GPS capabilities have apps for people with epilepsy whomay require emergency medical assistance []Medical monitoring has not always been so painless persistent and unobtrusive Atrial fibrillation required attachingelectrodes to the skin with a conductive gel in turn connectedto a devicepossibly portable but certainly obtrusiveMonitoring of blood glucose by diabetic patients required repeated fingerstickspainful intermittent and obtrusivePhases of Biofluid MonitoringDiagnostic techniques for biofluids eg blood urine salivaand cerebrospinal fluid CSF have evolved over the pastseveral decades Fig [] The first phaseextendingfrom the twentieth century to the presententailsobtaining a sample from the patient an invasive procedurefor blood and CSF and sending it to a laboratory for analysisResults are not available for hours to days for samples obtained from outpatients and minutes to hours for inpatientsFig The four technological waves of biochemical monitoring reference []with permissionThe second phase began about two decades ago with pointofcare POC monitoring where the laboratory comes to thepatient ie to the recently obtained sample rather thantransporting the sample to the laboratoryThe third phase more recently available consists of wearabledevices This is the epitome of POC monitoring since the patientand the device are inseparable Smartwatches can do this forpulse and blood pressure patches applied to the skin for continuous blood glucose monitoring The patches eg for glucosemonitoring typically monitor the analyte concentration in interstitial fluid ISF which closely parallels blood glucose []The line between the third and fourth phaseswearable andimplantable devicesis blurred Part of this is due to expansion of the fluids monitoring from blood or ISF to sweatsaliva and tears Most would call a mouthguard to monitor salivaa wearable devicebut what about a smart contact lens tomonitor tears Truly implantable devices eg inserted subcutaneously by a minor surgical procedure can monitor analytes suchas glucose for months potentially longer rather than the days toa week or so of most patches []Power to the PatientDigitizing Biofluid MonitoringDuring the sampletolab and POC phases urine was the idealbiofluidnoninvasively obtained and relatively easilytransported Blood required an invasive procedure a needlestickSweat and tears were not easily obtained in a manner guaranteeing uniformity and saliva could vary greatly depending on timeof sampling eg after a drink or a mealWearable devices have transformed those problems into oneconsisting of a tissuedevice interface TDI challenge 0cEPMA JournalContinuous biofluid monitoring is a reality sampling urineblood or other biofluids continuously was not practical previously outside a hospital setting with an indwelling catheterfor urine or blood or even CSF A second problem in phasesone and two was obtaining continuous diagnostic informationfrom the biofluidThe smartphone and smartwatches plus machine learning andartificial intelligence AI have allowed not only continuousbiofluid monitoring but also continuous realtime interpretation of that monitoring information in a precise and personalized mannerdigital biomarkers [] This can answer thequestionWhat does this biofluid monitoring value mean for this particular patient at this precise momentOnce answered that information can guide realtime precisepersonalized treatment eg continuous feedbackguided orclosedloop insulin release in diabetes The patient if desiredcan have control over when the biofluid monitoring information is gathered or processed or transmitted eg to adatabank The patient can remove the patch or the smartwatchor turn off the smartphone containing the app transferring thedataBlood Sweat Tears and SalivaAlthough the primary target has been a wearable monitor ofblood glucose for diabetic patients other biological signalsthat can be measured through the skin include chemicals beyond glucosepotassium chloride lactate electrical electrocardiogram ECG electroencephalogram EEG electromyogram EMG and physical temperature pressure lightsound [] Additionally non or minimally invasive monitoring has included measures ranging from respiratory rate tojoint movement to gait [ ] This review is primarilylimited to the TDI for biofluidsWearable skin patches depend on knowledge of thestructure of human skin [ ] Smart skin exhibitsmany technological advances as illustrated in Fig [] Skin patches usually monitor ISF concentrations of thechemical of interest relatively straightforward for ISF glucoseas a surrogate for blood glucose Sweat however poses adifferent problem since sweat is not continuously availablefor monitoring In the typical skin patch for sweat the patchincorporates an electrode to deliver a cholinergic agent such ascarbacholinto the skin for stimulation of sweationtophoresis [ ]Fig Recent research trends in smart skin from four viewpoints First the structures of smart skins are advancing from stretchable toultrathin to breathable sensors resulting in enhancement of biocompatibility and reduced burden of sensor attachment Second multimodality is expanding from electrical to physical to chemical sensors Third more advanced functions such as stimulation drug deliveryand displays are being incorporated in addition to sensing functions Fourth novel materials such as selfhealing conductors intrinsicallystretchable semiconductors and photoactive materials are being developed reference [] with permission 0cEPMA JournalMonitoring tears is challenging the rate of tearing is notuniform the device must be acceptable to the patientTearbased biofluid sensors include smart contact lenses anddevices placed in the lower eyelid Fig 3A [ ]Fig 3A Tearbased biosensors a Contact lens sensor previously under development by Google and Novartis to measure tear glucose concentrationPrototype platform contained integrated electronics for sensor response processing and wireless transmission b Multifunctional wearablelens for monitoring both glucose in tears and intraocular pressure using enzymesmart sensor system incorporated onto a contactfunctionalized graphenesilver nanowire hybrid nanostructures c A wireless glucose sensor incorporated into a contactlens platformwith wireless power transfer circuitry and display pixels for a fully integrated and transparent platform that does not hinder vision dWearable contact lens tear glucose biosensor applied to an artificial eye with schematic representation of smartphonebased quantification of glucose levels through reflection of incident light by the photonic microstructure within the lens The smart contact lens systemintegrated with a glucose sensitive hydrogel monitors changing glucose concentrations in vitro without complicated fabrication proceduresand allows rapid response time for continuous measurements e NovioSense electrochemical tear glucose sensor A small springlikesensing device is designed to be placed within the conjunctival fornix for continuous access to tear glucose reference [] withpermission Saliva is readily available but suffers from analyte variabilityeg temperature and concentration resulting from the presence of liquids of varying temperatures over time in the oralcavity hot vs cold drinks [] Patient acceptance of a devicein the oral cavitygiven that some saliva biofluid sensors aremouthguards or otherwise bulkyobtrusiveis another issueFig 3B [] 0cEPMA Journal Fig 3B Salivabased biosensors a Mouthguardbased wearable salivary uric acid biosensing platform with integrated wireless electronics andanalysis of salivary uric acid concentrations b Mouthguardbased sensor for glucose monitoring in saliva with onbody applicationand analysis of increasing glucose concentrations c Onbody depiction and crosssectional configuration of radio frequency trilayertoothmounted sensor for wireless monitoring of food consumption This dielectric sensor fabricated with biocompatible materials iscapable of being mounted onto tooth enamelto detect foods and fluids during ingestion when functionalized with analytesensitivelayers Projected uses were for detection of sugars alcohol salinity pH and temperature d Operational principles and electronicsconfiguration of a wireless usercomfortable sensing platform for longrange oral monitoring of sodium intake during hypertensionmanagement reference [] with permissionSometimes It Takes Guts to MonitorConfirmation of ingestion of prescribed medications particularly in unreliable patients eg dementia is another biosensing challenge One solution is the smart pilla capsulecontaining a microsensor that is swallowed monitoringwhether the medication is present in the stomach [] Thesmart pill communicates with a skin patch which not onlydocuments that the pill was swallowed and when but also ifdesired blood pressure pH and temperatureFor continuous monitoring a sensor can be stationed in thegut most likely the stomach Such monitoring could includemedication ingestion pH controlled drug delivery and imaging of the gut lining An ingestible sensor that is selfpowered by stomach acid in contact with zinc and copper electrodes on the sensor surface is being developed[] Another ingestible capsule under development usesa microneedle that inserts into the stomach wall to deliver a drug eg insulin [] 0cEPMA JournalWear Your Heart on Your Sleeve Wear Your Brain onYour HatThe topic of brain biomonitoringfrom EEG to nextgeneration brainmachine interfaces BMIsis beyond the scope of this but has been recentlyreviewed [] An area of concern regarding brainbiomonitoring is directtoconsumer DTC marketingof devices that are of undocumented value or possiblerisk [] Brain biomonitoring information obtained through DTC marketing raises questions of bothpersonal privacy and ultimate use of such data bymarketers Increasing DTC availability of brain electrical stimulation eg via a skullcap notably transcranial direct current alternating current and randomnoise stimulation techniques raises questions of safety [] Ethical considerations regarding DTC brainbiomonitoring and biostimulating remain unresolved[]Conclusions and Expert RecommendationsThe field of wearable and implantable biosensors is evolvingso rapidly that no review truly reflects the stateoftheartAdvances in the TDI and AI promise that such devices willnot only enhance diagnostic capabilities but also provide awealth of information for improved treatmentsSpecific recommendationsIncorporating the latest technology into biosensorsfromnanotechniques to microfluidicsis essential Asmartphone from ten years ago would be unacceptablein the consumer marketplace outdated diagnostic techniques in medicine are similarly unwarranted Similarly the latest AI is necessary to analyze the hugeamounts of data that wearable and implantable biosensorsprovide The consumerpatient must be involved in device development from the outset What may appear wonderful inthe lab or the boardroom may prove a failure in the marketplace and social media Consumerpatient acceptanceCPA is crucial for widespread adoption Flexibility is key Some patients may prefer a patch forcontinuous glucose monitoring others a smart contactlens and others an implanted device requiring a minorprocedure for implantation but not frequent replacementWhat works in a highincome country such as Belgiummay not work in a lowincome country such as BurkinaFaso Legislation and safeguards regarding the huge amounts ofpersonal medical data generated by wearable and implantable biosensors is essential since data collection and storage systems can be hacked This is especially crucial withregard to biomodulating devices eg cardiac pacemakers brain stimulation and controlled drug deliverysystems Given the vulnerability to hacking wearable and implantable biosensors require the same caution as other widespread threats to population health eg toxins both liquid and aerosol biological warfare agents and radiationReferences Weichert W My watch kept on alarming all night aboutmy heart rate Oxford Med Case Rep Seizario detecting seizures and falls Available seizariohealthhappycom [Accessed Apr ] Heikenfeld J Jajack A Feldman B Granger SWGaitonde S Begtrup G et al Accessing analytes inbiofluids for peripheral biochemical monitoring NatBiotechnol Guk K Han G Lim J Jeong K Kang T Lim EK et alEvolution of wearable devices with realtime diseasemonitoring for personalized healthcare Nanomaterials Khan S Ali S Bermak A Recent developments in printable flexible and wearable sensing electronics forhealthcare applications Sensors Kim J Campbell AS EstebanFernandez de Avila BWang J Wearable biosensors for healthcare monitoringNat Biotechnol Someya T Amagai M Toward a new generation ofsmart skins Nat Biotechnol Waltz E Sweet sensation Nat Biotechnol McCarthy A The biomarker future is digital ClinicalOMICS 2020JanFeb24 Massaroni C Nicolo A Lo Presti D Sacchetti MSilvestri S Schena E Contactbased methods for measuring respiratory rate Sensors Faisal AI Majumder S Mondal T Cowan D Naseh SDeen MJ Monitoring methods of human body jointsstateoftheart and research challenges Sensors McDonnell S Ingestible sensors powered by stomachacid Tech Briefs 2018Aug45Jarchum I To the stomach and beyond Nat Biotechnol Frank JA Antonini MJ Anikeeva P Nextgenerationinterfaces for studying neural function Nat BiotechnolIenca M Haselager P Emanuel EJ Brain leaks and consumer neurotechnology Nat Biotechnol 0c Wexler A Separating neuroethics from neurohype NatBiotechnol Jarchum I The ethics of neurotechnology NatBiotechnol The Navarra Genomes Project NAGEN Benefits for Predictive Preventive and PersonalizedMedicinePasalodos S1 Salgado J1 Miranda M1 Maillo A1Matalonga L2 Beltr¡n S2 Carmona R3 P©rezFlorido J3Etayo G4 Lasheras G4 Bernad T4 G³mezCabrero D1Angel Gonz¡lez L5 Brennan P6 Gut I2 Dopazo J3Pinillos I4 Lasa I1 Alonso A11Navarrabiomed Complejo Hospitalario de NavarraUniversidad Pºblica de Navarra UPNA IdiSNAPamplona Spain2Centro Nacional de An¡lisis Gen³mico CNAGCRGCenter for Genomic Regulation Barcelona Institute ofScience and Technology BIST Barcelona Spain3rea de Bioinform¡tica Fundaci³n Progreso y Salud Nodode Gen³mica Funcional INBELIXIRes Bioinform¡ticade ER BiERCIBERER CDCA Hospital Virgen delRoco Sevilla Spain4Navarra de Servicios y Tecnologa NASERTIC Spain5AVANTIA Pyramide Asesores Spain6NENC NHS Genomic Medicine Centre Newcastle uponTyne UKCorresponding author Dr Angel Alonso GenomicMedicine Unit Navarrabiomed Complejo Hospitalario deNavarra Universidad Pºblica de Navarra UPNA IdiSNAEPMA JournalCIrunlarrea Pamplona Spain emailangelalonsosancheznavarraesKeywords predictive preventive personalized medicinegenomics next generation sequencing NGS whole genome sequencing WGS rare diseases eHealth bioinformatics big data ICPerMed multiomicsBackgroundIn the past few years extraordinary developments in the fieldof next generation sequencing NGS technologies such aswhole genome sequencing WGS have made it possible forclinicians to have access to a huge amount of biological information which could potentially explain complex genetic diagnoses genetic predisposition to severe diseases reproductiverisks and inappropriate responses to certain medicationsThese advances herald a new era of predictive preventive personalized medicine PPPM although incorporation into clinical practice has proved to be challenging [] NAGEN is a Spanish regional pilot study to implement recentadvances of cutting edge genomic research technology intoreal clinical practiceGoal materials and methodsNAGEN s main goal is the implementation of the wholegenome sequencing WGS derived information as a clinicaltool for the development of PPPM in the Public Health ServiceA scientific implementation approach was used to identify andcategorize both the local barriers and facilitators to acceleratethe incorporation of translational genomics intohealthcare see Fig Fig Local barriers for genomic medicine implementation in Navarra NAGEN project 0cEPMA JournalKey Actions for this implementation Subjects NAGEN is recruiting patients and theirrelatives affected with one condition from a list of nearly rare diseases RD Albeit rare joint RDs prevalence ishigh with a very high social impact wide multidisciplinary medical coverage and a high rate of identifiablegenetic causes These features make it possible to involve themedical community raise population awareness and offergood support to evidencebased medicine practice The rateof genome per inhabitants facilitates a wide participation from patients and health professionals Results and incidental findings Pertinent findingsexplaining the referral condition secondary findings onpersonal and reproductive risks of severe inherited diseases and pharmacogenomic variants determining drugsdose and toxicity are reported based on patients choiceproviding the necessary evidence of the effectiveness ofmedical interventions based on genomic medicine Newgenetic counselling interventions variant validation andreporting pathways have been put in place for the bestprovision of services Electronic health record EHR adaptation The existingEHR has been modified to host a newly designed recruitment tool which enables and guides the identification andimmediate referral of patients from any point in theNavarre health system network An additional development also makes it possible that clinically actionable genomic results are available for participants doctors withall other clinical information across the system Clinical research A number of new exciting genomicresults potentially providing new insights into the geneticbasis of RDs and additional information on populationgenomics are being produced by NAGEN offeringexceptional material to support new research It is a maingoal of the Project to ensure an adequate data harmonization which enables data sharing for research under anappropriate regulatory and legal framework Optimized use of preexisting public infrastructures Inorder to overcome the lack of local facilities NAGEN externalizes WGS sequencing services to CNAGCRG the Spanish world leader public centre for genomicanalysis Bioinformatic analysis also relies primarily onCNAGCRG through the RDConnect GenomePhenome Analysis Platform which was deployed for theproject to store analyse and interpret the genomic datamaking use of the phenotypes encoded with the HumanPhenotype Ontology HPO and the experts from theBioinformatics Platform of the Rare Diseases Spanishnetwork CIBERer through the Interactive VariantAnalysis IVA tool based on the genome browserGenome Maps but expertise in this field has graduallybeen transferred to the newly created local TranslationalBioinformatics Unit during the course of the ProjectICT New ICT solutions have been adopted for NAGEN allowing the storage and high performance managing of massive genomic data through an innovative partnership with NASERTIC a local company providing dataanalysis infrastructures such as the new IBM POWER processor which build on crossdisciplinary collaborationin research and development with the local industry ELSI While genetic data protection is widely regulated forclinical and research purposes within the NAGEN project the local Health Research Authority has specificallyresolved that the massive genomic information resultingfrom WGS will also be part of the patients medical recordand it will accordingly be protected and stored In order toenable the use of genomic data for research the constitutionof a Genomic Library has been proposed which wouldaccept specific research enquiries on anonymized genomicsequences upon pertinent EC approval This scenario requires a new regulatory legal framework which has alsobeen explored through a specific partnership with Avantia from the Pyramide group a local consulting companywith wide experience in data protectionResultsKey results to date Clinical and preclinical results Around patients have todate followed through the aboveoutlined pathway and of the families have now found the longawaited geneticcause for their previously unexplained condition and nowhave hope of an improvement of their clinical care based onthese findings Remarkably of these diagnoses wereattributed to genes previously unknown to cause a humandisease or causing different phenotypes than those previously described Fig Additionally of participants carriedgenetic predispositions to severe diseases had reproductive risks and had pharmacogenomic actionable variants influencing prescription Table Further candidategenomic variants potentially explaining patients diseaseshave been identified in an additional of participatingfamilies which provides an extended base for new collaborative research projects Interestingly about differentmedical specialities have referred patients to NAGEN indicating a desirable multidisciplinary involvementin this implementation initiative Healthcare workforce education and public empowerment Monographic NAGEN symposiums hospitalbriefings clinical sessions and face to face meetings havebeen anized ing the participation to all medicalprofessionals in the region Moreover the designatedspecialities physician champions especially commissioned to facilitate recruitment help with the clinical 0cinterpretation of genomic variants and to spread the wordreceived category and CME credits from a NAGEN tailored genomics education programme Public involvement has also been possible through a press conference which was widely covered by national general andmedical press and social media conferences at theScience Week and Rare Diseases Day a specificwebsite wwwnagen1000navarraes and communicationsto national and international congresses Sustainability After deducting marginal costs due to theTranslational Bioinformatics Unit establishment and ICTinfrastructures the costeffectiveness analysis CEA recEPMA Journalognized a full running costs of ¬ per RD diagnosisprior to familial cascade genetic testing and including duoand trio studies costs when necessary compared with¬ average cost per diagnosis estimated for thestandard of care pathway [] Considering that costbenefit analysis CBA outperforms CEA for RDwe conducted a survey of all participants whichshowed that more than of them would be willing to pay more than ¬ for the genomic information they received after their participation inNAGEN regardless of whether their diagnosiswas ultimately achieved or notFig Pertinent clinical findings a Piechart showing the performance of genomic diagnoses achieved by NAGEN and of strong and mild candidates genomic variants b Table listing OMIM codes and diagnoses in red cases with no OMIM codificationTable Clinical Actionable Incidental FindingsClinical Actionable Incidental FindingsTypeConsentN of patients of casesDisease PredispositionReproductive RiskPharmacogenomicpatients and it was awarded as the Best Practice in PersonalisedMedicine by ICPerMed in Significantly it resulted in setting the new Genomic Medicine Unit of Navarrabiomed and itsNAGEN strategy which has now raised ¬ million for RDprojects on PM over the past years NAGEN is an exemplarpractice for the Spanish Senate Initiative for a National Strategyon Genomics and PM and has given rise to the launch ofthe Navarra Government Strategy on Personalised Medicine announced in November Conclusions and expected impactsGenomics has become a major contributor to multiomics andPPPM related approaches in management of major and fatal pathologies such as cancer diabetes and stroke [] NAGEN illustrates how translational research and innovation in thefield of genomics and PPPM is already delivering real benefits toAcknowledgements This study will always be in debt to all participa | Thyroid_Cancer |
evaluation of biochemical and hematological parameters in adults with Down syndromeDavid de Gonzalocalvo123 Isabel Barroeta45 Madalina Nicoleta Nan6 Jos Rives6 Diana Garzn45 Mara CarmonaIragui457 Bessy Benejam457 Laura Videla457 Susana fern¡ndez7 Miren Altuna45 Slvia Valldeneu45 Rafael Blesa45 Alberto Lle45 Francisco BlancoVaca689 Juan Fortea457 Mireia Tondo6Down syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting liveborn infants In addition to intellectual disability individuals with DS have other comorbidities and complex medical conditions The increase in the life expectancy of patients with DS requires expanding the knowledge about their clinical characteristics and related laboratory parameters Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism Therefore our main goal was to describe the biochemical and hematological findings in a DS cohort and to compare the values to those of a control population A total of DS individuals and control subjects were enrolled DS individuals had a higher frequency of several clinical conditions compared to control individuals and presented with significant differences with respect to the controls in both biochemical and hematological parameters We found age and sexrelated differences in several of the parameters A good understanding of the differences in our cohort might be of aid in the clinical followup of adults with DS especially considering that the lifespan of DS individuals may reach years of age in developed countriesAbbreviationsAD AF ALT AST B12 CKDEPI DS ESR FT4 eGFR GGT HbA1c Alzheimers disease Alkaline phosphatase Alanine aminotransferase Aspartate aminotransferase Vitamin B12 Chronic kidney disease epidemiology collaboration Down syndrome Erythrocyte sedimentation rate Free thyroxine Estimated glomerular filtration rate Gammaglutamyl transferase Glycated hemoglobin1Biomedical Research Institute Sant Pau IIB Sant Pau Barcelona Spain 2Institute of Biomedical Research of Barcelona IIBB Spanish National Research Council CSIC Barcelona Spain 3Translational Research in Respiratory Medicine University Hospital Arnau de Vilanova and Santa Maria IRBLleida Lleida Spain 4Sant Pau Memory Unit Department of Neurology Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau Universitat Aut²noma de Barcelona Barcelona Spain 5Center of Biomedical Investigation Network for Neurodegenerative Diseases CIBERNED Madrid Spain 6Department of Biochemistry Hospital de La Santa Creu i Sant Pau Biomedical Research Institute IIB Sant Pau CSant Quint Barcelona Spain 7Barcelona Down Medical Center Fundaci Catalana de Sndrome de Down Barcelona Spain 8Center of Biomedical Investigation Network for Diabetes and Metabolic Diseases CIBERDEM Madrid Spain 9Department of Biochemistry and Molecular Biology Universitat Aut²noma de Barcelona Barcelona Spain email mtondosantpaucatScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cHDLc LDLc MCH MCHC MCV MDRD4 MPV K RDW Na TG TSH Highdensity lipoprotein cholesterol Lowdensity lipoprotein cholesterol Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration Mean corpuscular volume Modification of diet in renal disease Mean platelet volume Potassium Red blood cell distribution width Sodium Triglycerides Thyroid stimulating hormoneDown syndrome DS is the most common worldwide cause of intellectual disability of genetic origin and the most common chromosomal disorder affecting liveborn infants with an estimated birth prevalence of per live births1 Despite the shorter life expectancy when compared to healthy subjects and adults with other causes of intellectual disability4 there has been a progressive increase in the life expectancy of patients with DS in recent decades currently reaching nearly years5 This fact has increased the need to expand the knowledge about the clinical characteristics of DS individuals and the health problems differentiating them from both pediatric and adult populations6 DS is associated with a distinct phenotype involving many body systems In addition to intellectual disability individuals with DS present with a high number of comorbidities and complex medical conditions whose frequencies are modified throughout the lifespan of the individuals7 The increase in life expectancy has led to a higher prevalence of agerelated pathologies including premature Alzheimers disease AD8Since optimal medical management is associated with improved quality of life and functioning among persons with DS910 medical professionals including pediatricians and other physicians should closely supervise this population throughout their lifespan and evaluate their laboratory results Previous investigations in DS cohorts have focused on select biochemical parameters such as uric acid and thyroid function biomarkers bone mineral density nutritional zinc status gonadal and endocrine function and glucose and lipid metabolism parameters11 However no previous work has described a comprehensive panel of biochemical and hematological parameters in a large cohort of DS patientsOur hypothesis is that a thorough analysis of the biochemical and hematological parameters will provide a basis to establish whether commonly observed alterations in DS individuals are intrinsic of the disease or have clinical implications similarly as for the general population Therefore our goals were to describe the biochemical and hematological findings in our DS cohort and to compare the values to those of a control populationMaterial and methodsStudy participants This was a singlecenter descriptive study of adults with DS recruited at Barcelona Down Medical Center Fundaci Catalana Sndrome de Down and Hospital de la Santa Creu i Sant Pau Barcelona in Catalonia Spain according to a populationbased health plan to screen for neurological comorbidities1718 The Down Medical Center provides medical care specifically for individuals with DS and possesses over medical records more than of the estimated Down syndrome population in Catalonia therefore it reflects the population with DS in our geographic area The period of patient recruitment for this study was February to June In adults with DS ¥ a0years a biochemical and hematological analysis was performed as part of their annual health plan visit A total of patients were enrolled in the study Six further patients were ultimately excluded for presenting with conditions unrelated to DS according to their medical records patients with hepatitis C patient with hepatitis B and patient with breast cancer resulting in a final total number of DS individuals included age range a0years A total of healthy control participants in the same age range a0years were enrolled in the study Volunteers were recruited from the SPIN Sant Pau Initiative on Neurodegeneration cohort santp aumem oryun itcomourresea rchspincohor t or social media SantPauMemory Further details on the clinical protocol of the SPIN cohort can be found elsewhere19Based on current guidelines1720 associated clinical conditions were obtained through a systematic review of the medical records including the following history of arterial hypertension dyslipidemia diabetes mellitus congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology psychiatric pathology epilepsy and Alzheimers disease Treatment data with a special focus on the treatment of hypothyroidism were also collectedBiochemical and hematological data Analyzed biochemical and hematological parameters were selected according to a defined laboratory blood profile as recommended in the guidelines for management of patients with DS1720Blood collection and processing were performed in accordance with the Standard Operating Procedures for Serum and Plasma Collection from the Early Detection Research Network EDRN Consensus Statement and Standard Operating Procedure Integration Working Group21 Blood samples were collected by venipuncture after an overnight fastWhole blood samples were collected in VACUTAINER tubes and fractionated by centrifugation at a0g for a0min at room temperature to obtain serum Serum was aliquoted into a0mL tubes and the following parameters were measured according to standard commercially available assays adapted to an Architect C4000 Abbott Diagnostics USA using automated procedures thyroid stimulating hormone TSH free thyroxine FT4 Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cAge yearsMalefemaleArterial hypertensionDyslipidemiaDiabetes mellitusCongenital heart diseaseGastrointestinal pathologyDermatological pathologyBone pathologyHypothyroidismTreatment for hypothyroidismHearing problemsOtolaryngology pathologyOphtalmological pathologyPsychiatric pathologyEpilepsyAlzheimers disease Controln Median P25P75n Down syndromenMedian P25P754n pvalue Table Characteristics of the Study Population Data are presented as frequencies percentages for categorical variables Continuous variables are presented as median interquartile range Differences between groups were analyzed using Wilcoxon ranksum test or Fishers exact testsodium Na potassium K glucose urea creatinine total bilirubin triglycerides TG total cholesterol aspartate aminotransferase AST alanine aminotransferase ALT alkaline phosphatase AF gammaglutamyl transferase GGT total proteins vitamin B12 and folate The estimated glomerular filtration rate eGFR was calculated according to the MDRD4 Modification of Diet in Renal Disease and CKDEPI Chronic Kidney Disease Epidemiology Collaboration formulasWhole blood samples in EDTAK3 were also obtained for determining blood cell count and indices The tubes were immediately inverted times to mix the anticoagulant additive with blood The blood was processed within a0h of extraction Using the impedance channel of the automated hematology analyzer Sysmex XE2100 Roche Diagnostics Kobe Japan the following parameters were determined red blood cell count RBC white blood cell count WBC platelet count hemoglobin hematocrit mean volume MCV mean corpuscular hemoglobin concentration MCHC mean corpuscular hemoglobin MCH red blood cell distribution width RDW and mean platelet volume MPV The erythrocyte sedimentation rate ESR was calculated with a VES cube Sysmex Analyzer Roche Diagnostics Kobe JapanValues were compared to normal reference ranges used in our laboratory established in a healthy population from our geographical area according to standardized guides22Statistical analysis Descriptive statistics were used to summarize the characteristics of the study population Data are presented as medians [25th percentile P2575th percentile P75] for continuous variables and as frequencies percentages for categorical variables Data normality was analyzed using the KolmogorovSmirnov test Continuous variables were compared between groups using the Wilcoxon ranksum test ANCOVA models adjusted for age and sex were used to compare continuous variables across the study groups Variables were logtransformed to achieve a normal distribution For clarity the original values are shown Categorical variables were compared between groups using Fishers exact test Spearmans rho coefficient was used to assess the correlation between continuous variables The statistical software package R wwwrproje ct was used for statistical analyses A Pvalue was considered statistically significantEthical aspects The study was approved by the Sant Pau Ethics Committee following the standards for medical research in humans recommended by the Declaration of Helsinki and in accordance with Spanish legislation for research in people with intellectual disabilities All participants or their legally authorized representatives gave written informed consent before enrolment in accordance with the guidelines of the local ethics committeeResultsStudy cohort characteristics We enrolled a total of individuals with DS males and females with a median age of years and control subjects males and females with a median age of years The clinical features of the DS and control populations are listed in Table a0 The frequency of the following clinical conditions was significantly higher in the DS group than in the control group history of congenital heart disease gastrointestinal pathology dermatologiScientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0ccal pathology bone pathology hypothyroidism hearing problems otolaryngology pathology ophthalmological pathology epilepsy and AD No differences were observed in the frequency of diabetes mellitus or psychiatric pathology for either group DS individuals presented with a lower frequency of arterial hypertension and dyslipidemia compared to the control group See Table a0 for further details on the cohort characteristicsBiochemical and hematological parameters in patients with Down syndrome We performed a detailed biochemical and hematological analysis of the DS cohort and compared the profiles obtained with our control population The reference values of the studied parameters the number and percentage of patients out of range and the median P25P75 of the whole study population are shown in Table a0 Seventythree percent of the studied hematological parameters and of the studied biochemical parameters were significantly different between the DS individuals and the control population The DS individuals presented with higher TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW values and lower TG total cholesterol folate eGFR MPV and WBC values These differences remained significant or close to signification after adjusting for confounding factors such as age and sex Statistical differences for RBC and MCHC were observed after adjustment An additional analysis to evaluate the impact of hypothyroidism treatment on TSH was performedNo differences were observed for TSH between both studied groups treated DS individuals vs untreated DS individuals Pvalue For categorical variables the percentage of DS individuals out of range for some parameters was also statistically significant compared to the control population Parameters with a higher percentage of values out of range in the DS group were TSH urea creatinine total proteins RBC MCV ESR MCH and WBC whereas those with a lower percentage of values out of range were K TG total cholesterol and ASTThe differences in the biochemical and hematological parameters and the number and percentage of patients out of range between DS individuals and the control population according to sex are displayed in Supplemental Tables a0 and For the female DS cohort parameters with significantly higher values were TSH urea creatinine AST hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol GGT eGFR RBC MPV and WBC For categorical variables parameters with significantly higher percentages of values out of range were TSH creatinine total proteins MCV ESR MCH and WBC whereas those with a significantly lower percentage of values out of range were total cholesterol and B12 Supplemental Table a0 For the male DS cohort parameters with significantly higher values were TSH hemoglobin hematocrit MCV ESR MCH and RDW whereas those with significantly lower values were TG total cholesterol eGFR MPV and WBC Regarding categorical variables parameters with significantly higher percentages of values out of range were TSH ESR and MCH whereas those with significantly lower percentages of values out of range were K TG total cholesterol and GGT Supplemental Table a0The differences in the biochemical and hematological parameters between males and females as well as the frequency and percentage of patients out of range in the control and DS groups are displayed in Supplemental Table a0 and Table a0 respectively For the control group parameters with significantly higher values in the male subgroup were K creatinine TG ALT hemoglobin hematocrit RBC and MCHC whereas those with significantly lower values were AF eGFR and ESR Among the categorical variables K had a significantly higher percentage of values out of range in the male subgroup and ESR had a significantly lower percentage of values out of range Supplemental Table a0 For the DS cohort parameters with significantly higher values in the male subgroup were creatinine total bilirubin TG ALT GGT hemoglobin hematocrit RBC MCHC and WBC whereas those with significantly lower values were folate MCV ESR RDW platelet count and MPV Regarding categorical variables parameters with significantly higher percentages of values out of range in the male subgroup were total bilirubin B12 RBC and MPV whereas those with significantly lower percentages of values out of range were MCV ESR and MCHC Table a0The correlation between the biochemical and hematological data with age was also explored in both study groups As shown in Table a0 for the control population urea creatinine total cholesterol and AST showed a significant positive correlation with age while eGFR showed a significant negative correlation For the DS population Na urea creatinine TG total cholesterol AST AF MCV ESR MCH and RDW showed a significant positive correlation with age while eGFR ALT B12 hemoglobin hematocrit RBC MCHC and platelet count showed a significant negative correlationDiscussionThe present study evaluated several biochemical and hematological parameters in a large sample of adults with DS Several studies exploring laboratory tests in DS patients exist but their focus is limited to specific areas of metabolism11 DS is among the most complex genetic conditions compatible with life characterized by accelerated aging and affecting gene expression beyond chromosome The sheer number of affected genes and epigenetic changes suggests that numerous pathways of human metabolism are altered and subsequently might be reflected in laboratory test parameters Here we performed a comprehensive approach by analyzing parameters related to different physiological mechanisms We found significant differences with respect to nontrisomic controls in both biochemical and hematological parameters even after adjusting for potential confounding factors Furthermore we found age and sexrelated differences in several of the parameters The fact that women with DS experience menopause earlier than healthy women24 may explain some of these sexrelated differencesClinically and as previously described48925 our DS cohort presented with a higher incidence of congenital heart disease gastrointestinal pathology dermatological pathology bone pathology hypothyroidism otolaryngology pathology ophthalmological pathology epilepsy and AD than the control population Arterial Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT UL ¤ a0years a0years Females Males Females Males Females Males Females Males Females Males Total proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count 109LMPV fLWBC 109LFemales Males Females Males Females Males nn OOR n OOR ControlDown syndromeReference valuesMedian P25P75nMedian P25P75pvalue categoricalpvalue continuouspvalue continuous adjusted Table Biochemical and hematological parameters in the control group and the cohort of patients with Down Syndrome Differences between groups were analyzed using Wilcoxon Ranksum test ANCOVA models adjusted for age and sex or the Fishers exact test OOR out of range NA not applicablehypertension and dyslipidemia were less prevalent whereas no difference was observed regarding the diabetes mellitus incidence as discussed belowWith respect to laboratory studies the hematological profile was largely altered in DS individuals when compared to the control population Of note significant differences were found for almost all the hematological parameters when comparing males and females suggesting the need to consider sex when evaluating the hematological profile in a DS individual It is well known that trisomy impacts hematopoietic cell biology through multiple and complex pathways In adults the metabolic and redox derangements observed in the RBCs from individuals with DS have been previously linked to alterations in cell survival and size in particular macrocytosis26 Different studies have also proposed that the additional copy of chromosome has a profound impact on fetal hematopoiesis which ultimately impacts the function and number of hematopoietic lineages27 Additionally between and of newborn infants with DS develop transient myeloproliferative disorder32 Although the disease usually resolves without treatment in the first few months of life it is estimated that of individuals with transient myeloproliferative disorder will go on to develop subsequent leukemia3536 Finally the fact that folate concentrations are significantly lower in DS individuals matches the observed hematological alterations Taken together these impaired hematological parameters suggest the existence of abnormalities Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cFemalenVariableBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count 109LMPV fLWBC 109L n OOR Median P25P75n OOR Median P25P75pvalue categoricalpvalue continuousMalen Table Differences between sex in the Down syndrome group Differences between groups were analyzed using Wilcoxon Ranksum test or the Fishers exact test OOR out of range NA not applicablein hematopoiesis and provide information on how an extra copy of chromosome may lead to phenotypic consequencesConcerning the biochemical profile our results support the findings from previous independent studies We showed that of our DS individuals presented with values out of range for TSH level Of those out of were treated for hypothyroidism Impaired TSH and FT4 levels have been largely described in DS populations37 Moreover subclinical hypothyroidism in children with DS is an abundantly common occurrence with a prevalence of approximately and has been attributed to the dysregulation of the hypothalamicpituitarythyroid axis37 Regarding urea metabolism of our DS individuals presented with a high urea concentration which may be due to impaired renal function among other causes Indeed and as previously reported39 almost of our DS individuals also presented with impaired creatinine values Serum creatinine is the most reliable parameter for detecting kidney damage due to its high diagnostic specificity From its concentration and based on formulas in which age sex and weight are taken into account it is possible to estimate the glomerular filtration rate eGFR Our DS cohort also presented with a lower eGFR which is in agreement with a previous study exploring renal disease in DS individuals40 Despite the significantly altered parameters related to renal function our DS individuals presented with a very low frequency of arterial hypertensionConcerning the lipid profile we found significantly lower total cholesterol and TG concentrations in DS individuals compared to the control population It would have been interesting to study the fractioned forms of cholesterol together with their apolipoprotein concentrations however because the current study was not designed to answer questions regarding lipid metabolism lowdensity lipoprotein cholesterol LDLc and highdensity lipoprotein cholesterol HDLc were not measured Several works measuring circulating total cholesterol Scientific RepoRtS 101038s41598020707192Vol1234567890wwwnaturecomscientificreports 0cControlnSpearmans rhoDown syndromenSpearmans rhopvaluepvalueBiochemical parametersTSH mUILNa mmolLK mmolLGlucose mmolLUrea mmolLCreatinine µmolLeGFR mlmin173Total bilirrubin µmolLTG mmolLTotal cholesterol mmolLAST ULALT ULAF ULGGT ULTotal Proteins gLB12 pmolLFolate nmolLHematological parametersHemoglobin gLHematocrit LLRBC 1012LMCV fLESR mmhMCHC gLMCH pgRDW Platelet count 109LMPV fLWBC 109L Table Correlations between biochemical and hematological parameters and age NA not applicableLDLc HDLc and TG concentrations in the DS population exist However they report contradictory results and prevent firm conclusions from being drawn Some studies have reported an unfavorable41 or favorable lipid profile46 However most of the studies reported no change in serum TC LDLc or HDLc in individuals with DS compared to a control group or to population norms414547 In our study these lower total cholesterol and TG concentrations may have translated into a significantly lower prevalence of hyperlipidemia in DS individuals It has been described that DS individuals may be protected against atherosclerosis4752 leading to a low incidence of cardiovascular events53 However a work carried out with individuals with DS found that they were at high risk of cerebrovascular events but a lower risk of coronary events in males55 Therefore risk of major cerebrovascular events in people with DS should not be ruled out Concerning diabetes mellitus a similar incidence of type diabetes mellitus50 and a higher incidence of type diabetes mellitus has been described for individuals with DS56 We found no difference in type diabetes mellitus frequency among our DS and control populations as previously described in a different study16 In regard to arterial hypertension prevalence our results are in line with numerous studies that have described a lower incidence of this condition in DS individuals50515758 Despite these observations cholesterol fractioned forms and glycated hemoglobin HbA1c concentrations were not measured making it difficult to draw conclusions regarding dyslipidemia and diabetes mellitus in our cohort Yet an increased degree of hypolipidemia should not be ruled out Overall future studies elucidating the mechanisms behind the low cholesterol and TG concentrations and lower prevalence of arterial hypertension observed in our DS cohort should be performedIt is important to emphasize that our main goal was to help determining if the observed biochemical and hematological alterations have direct clinical implications for DS individuals While the altered biochemical and hematological profiles may be developmental features ie a consequence of the specific genetic characteristics of individuals with DS or the result of accelerated aging it should be recalled that they may also be reflecting comorbidities or the use of medication From a clinical standpoint to elucidate if the observed differences are consequence of concomitant conditions or features of the syndrome itself could be of help in the management of DS individuals Unfortunately due to the design of our study these questions remain unanswered Future Scientific RepoRtS 101038s41598020707192Vol0123456789wwwnaturecomscientificreports 0cstudies focusing on specific areas of metabolism of DS individuals with different comorbidities could shed some light on this matterOur study has several strengths We collected relevant clinical biochemical and hematological data in a large DS cohort and performed a systematic analysis The fact that our controls were chosen from a healthy background broadens the actual differences and strengthens the present results Ultimately according to the wide inclusion criteria and the broad range of represented ages we believe that the results from our study may help clinicians when interpreting laboratory analyses in DS individuals Some limitations should also be taken into account The control and DS populations were not strictly age and sex matched and the control group had a reduced number of males when compared to females Nonetheless both populations were within the same age range and additional analysis including adjustment for age and sex were performed Furthermore despite our large cohort of DS individuals the number was still not sufficient to perform statistical analysis stratification according to the observed clinical conditions Moreover and as stated previously some of the observed biochemical andor hematological alterations may have been a consequence of the use of drugs for the treatment of other comorbidities Finally our defined clinical biochemical and hematological profiles were somehow general and unable to cover all the possible comorbidities present in DS individualsIn conclusion adults with DS show a specific profile of biochemical and hematological parameters A good understanding of the differences in our cohort with those in the general population might aid in the clinical followup of adults with DS especially considering that the life span of DS individuals can now reach a0years of age in developed countriesReceived March Accepted July References Parker S E et al Updated national birth prevalence estimates for selected birth defects in the United States Birth Canfield M A et al National estimates and raceethnicspecific variation of selected birth defects in the United States Defects Res A Clin Mol Teratol Birth Defects Res A Clin Mol Teratol Besser L M Shin M Kucik J E Correa A Prevalence of down syndrome among children and adolescents in metropolitan Atlanta Birth Defects Res A Clin Mol Teratol Yang Q Rasmussen S A Friedman J M Mortality associated with Downs syndrome in the USA from to A populationbased study Lancet Bittles A H Glasson E J Clinical social and ethical implications of changing life expectancy in Down syndrome Dev Med Child Neurol Morris J K Alberman E Trends in Downs syndrome live births and antenatal diagnoses in England and Wales from to Analysis of data from the National Down Syndrome Cytogenetic Register BMJ b3794 Startin C M et al Health comorbidities and cognitive abilities across the lifespan in Down syndrome J Neurodev Disord Hithersay R et al Association of dementia with mortality among adults with Down syndrome older than years JAMA Neurol Bull M J Health supervision for children with Down syndrome Pediatrics Roizen N J Patterson D Downs syndrome Lancet Hawli Y Nasrallah M ElHajj Fuleihan G Endocrine and musculoskeletal abnormalities in patients with Down syndrome Nat Rev Endocrinol Sakadamis A Angelopoulou N Matziari C Papameletiou V Souftas V Bone mass gonadal function and biochemical assessment in young men with trisomy Eur J Obstet Gynecol Reprod Biol Niegawa T et al Evaluation of uric acid levels thyroid function and anthropometric parameters in Japanese children with Down syndrome J Clin Biochem Nutr Costa R et al Bone mineral density distribution curves in Spanish adults with Down syndrome J Clin | Thyroid_Cancer |
Wntcateninmediated signaling is a key pathway regulating tissue growth and development and tumorigenesis and has received increasing attention in recent years In addition to participating in healthy tissue and an development ectopic activation of the pathway can cause a variety of tumors and other pathologies The pathway plays a critical role in many processes such as proliferation differentiation apoptosis migration invasion epithelialmesenchymal transition and cancer cell stemness The importance of the Wnt signal is selfevident This review describes the underlying mechanism of Wnt signaling pathway and highlights the latest findings on the relationship between Wnt signaling pathway and tumorigenesis In addition the potential relationship between miRNAs and Wnt signaling is presented Furthermore we discuss the intrinsic link between Wnt signaling and cancer cell stemness which shed light on the malignant progression of tumor cells Finally cancer treatment strategies based on the canonical Wnt signaling pathway are summarized hoping to help clinical development Keywords Wntcatenin tumor miRNAs cancer stem cell target therapeutic strategyIntroductionWnt signaling pathway plays a crucial role in embryonic development adult tissue homeostasis and cancer1 Due to the role in cell fate and tissue development increasing attention has been paid to Wnt signaling pathway in regenerative medicine2 Moreover the pathway is also involved in many pathological processes such as proliferation differentiation apoptosis migration invasion epithelial mesenchymal transition EMT and cancer cell stemness Notably the aberrant activation of Wntcatenin signaling results in the expression of several modulators that antagonize the antitumor activity of T cells which might lead to the failure of cancer immunotherapy3 Since Wnt signaling plays a pivotal role in tumorigenesis it is a promising target for the development of cancer therapeuticsSince the discovery about years ago many reports on the relationship between Wnt signaling and malignant tumor progression have been published The Wnt family consists of cysteinerich protein species which deliver canonical Wnt signaling through the interaction with Frizzled FZD and coreceptor low density lipoproteinrelated receptors and LRP56 thereby regulating cell fate growth and tissue repair4 After a series of posttranslational modifications such as porcupine palmitoylation lipid modification and glycosylation in the endoplasmic reticulum the Wnt precursor gradually maturates into the functional Wnt ligand Then the transmembrane protein Wntless Wls binds Wnt ligand and reaches to Cancer Management and Research Wen This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphp and incorporate the Creative Commons Attribution Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the work you hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed For permission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWen Dovepressthe plasma membrane via the Golgi apparatus Finally Wnt is detached from the cell membrane as components of exosomes or lipid protein ps15 As shown in Figure Wnt ligandbinding membrane receptor complex induces constitutive activation of Wntcatenin signaling which delivers downstream signaling LRP receptor phosphorylation recruits Axin to degrade Dishevelled DVL protein activation leads to inactivation of destruction complex which leads to the stability and accumulation of catenin6 Subsequently catenin is translocated into nucleus and interacts with the transcription coactivators like Tcell factor TCF and lymphoid enhancer factor LEF proteins to regulate the expression of Wnt target genes such as AXIN2 cycling and cMYC7 When Wnt signal is turned off the scaffold protein Axin adenomatous polyposis coli APC glycogen synthase kinase 3 GSK3 and casein kinase 1α CK1α form a multimeric destruction complex which induces catenin phosphorylation and ubiquitination degradation8Other mechanisms have been identified downstream of the canonical Wnt signaling pathway including the Hippo signaling cascade downstream nuclear effectors YAPTAZ Under Wnt OFF conditions YAPTAZ is an essential component of the destruction complex It recruits E3 ubiquitin ligase transducin repeatcontaining protein TrCP to promote catenin degradation in which YAP TAZ is a negative regulator of Wnt signaling However when Wnt is ON LRP56 induces the dissociation of YAP TAZ from Axin resulting in nuclear accumulation of YAP TAZ and enhancement of the expression of proproliferative genes in which YAPTAZ is a positive regulator of Wnt signaling9 Furthermore members of the Rspondin Figure An overview of Wnt signaling pathway As illustrated on the upper left side of Figure in the absence of Wnt ligand destruction complex induces the phosphorylation of catenin and the phosphorylated catenin is recognized and ubiquitinated by YAPTAZrecruited TrCP and subsequently subjected to proteasomal degradation In addition the E3 ubiquitin ligases Cullin 3KLHL12 recognize DVL and induce its degradation On the membrane ZNRF3RNF43 targets Frizzled receptor for ubiquitination and lysosomal degradation In the nucleus the inhibitory complex Groucho and histone deacetylases HDAC bind to TCFLEF transcription factors resulting in the inhibition of the Wnt signaling gene expression as shown in the lower left side of Figure On the upper right side of Figure the phosphorylated LRP receptor recruits Axin and DVL protein to the plasma membrane Activated DVL inactivates the destruction complex In this process the combination of DVL and ASPM inhibits the recognition of Cullin 3KLHL12 catenin with the assistance of histone modifying coactivators such as Pygopus Pygo and CREB binding protein CBPp300 interacts with the transcription factors TCFLEF to regulate target gene expression Frizzled ubiquitination is inhibited through the interaction of RSPO with LGR46 and ZNRF3 RNF43 Ub stands for ubiquitination and ps stands for phosphorylation Abbreviations LRP56 lowdensity lipoproteinrelated receptors and DVL dishevelled TCF Tcell factor LEF lymphoid enhancer factor APC the adenomatous polyposis coli GSK3 the glycogen synthase kinase 3 CK1α casein kinase 1α TrCP transducin repeatcontaining protein RSPO Rspondin LGR5 leucinerich repeat containing Gproteincoupled receptors ASPM abnormal spindlelike microcephaly associated HDAC histone deacetylases Pygo Pygopus CBP CREB binding proteinsubmit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alleucinerich RSPO ligand family were identified as positive effectors of Wnt signaling10 In the absence of RSPO the two homologues E3 ubiquitin ligases ZNRF3RNF43 ubiquitinated Frizzled inducing Fzd endocytosis When RSPO forms complex with repeatcontaining Gproteincoupled receptors LGR and ZNRF3 RNF43 however the Frizzled ubiquitination process is inhibited thereby inducing the activation of the Wnt signaling cascade Recently the abnormal spindlelike microcephaly associated ASPMDishevelled signal axis was proposed to participate in the generation of canonical Wnt signaling ASPM interacts with Wnt regulator DVL blocking the recognition for DVL by the ubiquitin ligase complex Cullin 3KLHL12 inhibiting the ubiquitination and subsequent degradation of DVL and maintaining a high level of Wnt activity11Besides the canonical pathway the Wnt signal also has an additional noncanonical activation pathway which is independent of catenin The noncanonical Wnt signaling pathway targets different receptors and activates multiple intracellular targets among which the most widely studied are Wntplanar cell polarity PCP and WntCalcium Ca2 pathways As shown in Figure in the WntPCP pathway Wnt ligand binds to receptor proteins to recruit and activate DVL which subsequently activates the small GTPases Rho or Rac triggering downstream Rhoassociated kinase ROCK and cJun Nterminal kinase JNK recruitment allowing cytoskeleton reanization12 In the WntCa2 pathway Fzd receptor is activated which induces the activation of DVL Then DVL recruits phospholipase C PLC which converts phosphatidylinositol 45bisphosphate PIP2 to diacylglycerol DAG and inositol 145triphosphate Figure An overview of noncanonical Wnt signaling pathway Noncanonical Wnt signaling is divided into WntPCP and WntCa2 pathways In WntPCP Wnt ligand binding receptor proteins trigger the activation of the ROCK and JNK downstream kinases allowing cytoskeletal reanization In WntCa2 noncanonical Wnt signaling increases the intracellular levels of DAG and IP3 by recruiting DVL stimulates the intracellular Ca2 release and activates downstream PKC CaN and CaMKII thereby regulating intracellular calcium fluxes and downstream calciumdependent cytoskeletal andor transcriptional responses Abbreviations ROCK Rhoassociated kinase JNK cJun Nterminal kinase PLC phospholipase C PIP2 phosphatidylinositol 45bisphosphate DAG diacylglycerol IP3 inositol 145triphosphate PKC protein kinase C CAN calcineurin CaMKII Ca2calmodulindependent protein kinase II DAAM1 the formin protein dishevelled associated activator of morphogenesis RHOA Ras homolog family member A RAC1 Rasrelated C3 botulinum toxin substrate AP1 activator protein1 CDC42 Cell division cycle protein NFAT nuclear factor of activated T cellsCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cWen DovepressIP3 IP3 stimulates intracellular Ca2 release and DAG and Ca2 together activate downstream protein kinase C PKC calcineurin CNA and Ca2calmodulindependent protein kinase II CaMKII thereby regulating intracellular calcium fluxes and downstream calciumdependent cytoskeletal and or transcriptional responses1314 Although noncanonical Wnt signaling is also an indispensable factor in the development of cancer this review mainly focuses on canonical Wnt signalingThere is growing evidence that Wntcatenin signaling is one of the main driving factors of some malignant tumors such as colorectal cancer liver cancer and breast cancer This review therefore focuses on the latest research progress on the correlation between the Wnt signal and tumors and the potential relationship between the Wnt signal and miRNAs In addition the possible relationship between the Wnt signal and CSC is discussed Finally cancer treatment strategies based on the canonical Wnt signal are summarizedWnt Signaling in Colorectal CancerColorectal cancer CRC is the third most common cancer in the world and the fourth most deadly cancer after lung liver and stomach cancer with the highest incidence in developed countries Constitutive activation of Wnt catenin signaling is a functional marker of colorectal cancer in which APC mutations occur in approximately of CRCs APCbased therapy may be therefore a promising strategy for the treatment of colorectal cancer15 It was reported that APC silencing drives hyperproliferation in the intestine and eventually forms colon adenomas Restoring APC gene led to continuous regression without recurrence whereas reestablished crypt homeostasis was found in tumor tissues carrying Kras and p53 mutations8 Wnt signaling is thus a promising target for the development of cancer therapy It was recently found that metastasisassociated in colon cancer MACC1 served as a transcriptional target of Wntcatenin signaling The DBC1 Deleted in breast cancer1 coactivator promotes selfrenewal capacity and drug resistance in colon cancer by regulating LEF1catenindependent enhancer in the MACC1 intron16 Furthermore Deptor The DEP domain containing mTOR interacting an mTOR inhibitor promotes cancer cell proliferation and survival in CRC and is a potential target for novel cancer therapies Deptor is a direct target gene for WntcatenincMyc signaling17 Silencing Deptor induces differentiation and inhibition of CRC cells by increasing ketone production and decreasing the expression of B lymphoma MoMLV insertion region Bmi1 while increasing mTOR activation A combination of AktmTOR and Wntcatenin inhibitors thus can exert a potent antitumor effect In addition to inhibition of positive regulators for Wnt signaling a negative regulator was also found as a possible target for Wnt signaling18 Vset and transmembrane domain containing 2A VSTM2A is a secreted protein that inhibits Wntcatenin signaling in colon cancer by directly inhibiting LRP6 activity and inducing LRP6 endocytosis and degradationCancer metastasis is the main cause of death in CRC patients Tocopherol alpha transfer proteinlike TTPAL and chromatin anizer special ATrich binding protein SATB1 are possibly involved in the invasion and metastasis of CRC Gou found19 that the upregulation of TTPAL is associated with a poor prognosis of colon cancer in which TTPAL directly interacts with thyroid receptorinteracting protein TRIP6 and inhibits the ubiquitination and degradation of TRIP6 TRIP6 competitively binds to the membraneassociated guanylate kinase with inverted domain structure1 MAGI1 tumor suppressor and dissociates catenin from MAGI1 Free catenin enters the nucleus to activate the oncogenic Wntcatenin signal increasing the ability of cancer cells to migrate and invade In addition overactivation of Wnt signaling promotes the interaction between TCF7L2catenin complex and SATB1 leading to SATB1 expression20 SATB1 regulates the expression of tumor growth and metastasisassociated genes regulating the interconversion of colon cancer in the invasion phenotypeWntcatenin signaling affects the malignant progression of colon cancer by regulating tumor suppressoractivator It is therefore pivotal to further explore Wnt signal related targets with a view to revealing a network of Wnt signaling in cancer and providing more options for cancer treatments The roles of Wnt signaling in the development of colon cancer were summarized in this section hoping to understand Wnt signaling from a new perspectiveWnt Signaling in Liver CancerAccording to the survey by World Health anizations International Cancer Research Center in liver cancer is one of the worlds highmortal cancers and hepatocellular carcinoma HCC is the second leading cause of cancer death worldwide catenin is expressed in the adult liver which mediates Wnt signaling and regulates the transcription and translation of genes around liver cells affecting various aspects of liver biology Here we mainly describe the roles submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alto regulate of Wnt signaling in hepatic metabolic partitioning and liver regeneration RSPOLGR45ZNRF3RNF43 module is the main signaling complex in the Wntcatenin signaling pathway liver metabolism development and regeneration21 Knocking down LGR45 in the mouse liver resulted in a loss of catenin signaling and a defect in hepatic metabolic partitioning Supplementing RSPO1 or knocking down Znrf3Rnf43 extends Wntcatenin signal gradient in the liver in a reversible and LGR45dependent manner resuming the liver defect phenotype In addition liver regeneration is possibly regulated by type I transmembrane protein TMEM9 In the model of liver injury induced by CCl4 TMEM9 enhances the rapid assembly of the vATPase lysosomal proton pump activates cathepsin activity in lysosomes induces APC degradation and activates Wntcatenin pathway leading to the promotion of liver regeneration22 Wntcatenin signaling is therefore essential for liver development whereas the abnormal activation of the catenin signal is also a sign of promoting the development of hepatocellular carcinoma and other liver diseases As shown in Figure approximately of HCC exhibited Wntcatenin signaling activity among them about of hepatocellular carcinomas had Catenin 1 CTNNB1 mutations23 which highlighted the importance of CTNNB1 in the progression of liver cancer Together with the activation levels of other signals in HCC we might hypothesize that the crosstalk between Wnt signaling and multiple signaling pathways promotes the development of liver cancer24 In addition in a mouse hepatocyte model catenin activation per se is not sufficient to induce liver cancer and other mutation events must be involved in the development of liver cancer such as mutations in a telomerase reverse transcriptase gene TERT25 or those in the gene mesenchymal epithelial transition factor MET26 This phenomenon may be related to the weak catenin activity in the mouse hepatocyte model We found that there are weak mutations in the CTNNB1 in HCC and HCA hepatocellular adenoma for example the mutation takes place at K335N387 S45 or T41 of the CTNNB127Wnt signaling plays a crucial role in the growth and development of liver However its aberrant activation may promote the occurrence of hepatocellular carcinoma and other liver lesions It is noteworthy that a liverspecific Wntcatenin signaling pathway exists in liver cells Glypican3 GPC3 is a heparan sulfate proteoglycan specifically expressed on the surface of liver cancer cells Li reported28 that GPC3 regulates the activation of Wnt signaling by dual mechanisms and promotes proliferation of hepatocellular carcinoma When FZD is not abundant GPC3 acts as an alternative Wnt receptor and recruits Wnt at the cell surface through the Nterminal CRD domain leading to the promotion of Wnt signaling When FZD is abundant GPC3 and FZD interact through their heparin sulfate HS chains and the two molecules form a triple complex with Wnt to synergistically stimulate Wnt signaling GPC3 might function as a bridge to stabilize the interaction between Wnt and FZD Further studies promote the elucidation of the roles of Wnt signaling in the development of liver cancer and provide a theoretical basis for GPC3 targeted therapyWnt Signaling in Breast CancerWnt signaling is involved in the malignant progression of breast cancer such as proliferation invasion metastasis and drug resistance In healthy breast tissue inhibition of Figure Status of Wntcatenin signal activation in HCC A Activation ratio of Wntcatenin signaling in HCC B Mutation frequency of common highmutation genes in HCCCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cWen DovepressWntcatenin activity leads to developmental disorders reduced cell proliferation during pregnancy29 and However there is increasing evidence that aberrant activation of the Wnt signal promotes the development of breast cancer It was demonstrated30 that bone marrow tyrosine kinase on chromosome X BMX is upregulated in breast cancer It inhibits the degradation of catenin by promoting GSK3 phosphorylation thereby disrupting normal Wnt signaling and promoting the malignant progression of breast cancer Rational regulation of Wntcatenin signaling is therefore a key to the treatment of breast cancer although it is difficult to specifically control the signaling In theory inhibition of Wntcatenin signaling can prevent tumor progression Surprisingly it is likely that silencing canonical Wnt signaling drives cancer cells into dormancy resulting in the hindrance of tumor cells from cancer immunoediting by the host immune system31 It was reported that Dickkopf1 DKK1 inhibits lung metastasis by antagonizing noncanonical Wnt signal of breast cancer DKK1 however could promote breastto bone metastasis by regulating canonical Wnt signal in osteoblasts Taken together it is not straightforward to manipulate canonical Wnt signaling for the treatment of metastatic cancerWnt Signaling and MicroRNAsIn recent years several studies have revealed the potential connection between noncoding RNA and Wnt signaling Table summarizes the effect of miRNAs on Wntcatenin signaling in cancerIt has been reported that miRNA expression is also controlled by Wnt signaling In HCC CTNNB1 binds to the miR18396182 promoter region and increases the transcription of the miRNAs thereby enhancing tumor cell invasion It is noteworthy that there is a mutual feedback loop between some miRNAs and Wntcatenin signaling In colon cancer miR452 induces Wnt signaling by targeting GSK3 Since T cell factorlymphatic enhancement factor is an effective transcription factor in the miR452 promoter in turn miR452 transcription is promoted by Wnt signaling forming a positive feedback loop46 In addition a negative feedback loop was found between the miRNA and Wnt signaling pathway miR145 a tumor suppressor miRNA can interact with TCF4catenin complex recruit polycomb repressive complex PRC2 histone trimethylase and suppress Wnt signal transduction Recruitment of PRC2 however results in a high level of methylation at the miR145 promoter region which downregulates the expression of miR14547 It was further demonstrated that abnormally downregulated miR145 was inversely correlated with its inhibitory regulators TCF4 and SUZ12 Suppressor of Zeste Protein Homolog indicating that miR145 forms a double negative regulatory loop with the negative regulator of colorectal cancer It is therefore imperative to further analyze the network and regulation of miRNA and Wnt catenin signaling pathway which would provide insights into the development of miRNAbased drugsWnt Signaling in Cancer StemnessIt is hypothesized that malignant tumors grow in a layered manner and cancer stem cells CSCs having selfrenewal characteristics and multidirectional differentiation ability play important roles in the tumor development and progression CSCs continuously maintain the replacement of new CSC tumorigenic subpopulations and differentiate into nonCSC progenies with high proliferative ability48 It is assumed that cancer originates in part from CSCs de Sousa e Melo demonstrated that in colorectal cancer Lgr5GFP tumor cells had higher tumorigenicity than Lgr5GFP tumor cells showing that Lgr5 cells had tumorinitiating ability49 In addition depletion of Lgr5 cells triggers differentiated cancer cells to continuously reverse to cells in a tumorpropagating state and replenish the Lgr5 CSC pool In another study a similar conclusion was provided Mature leukemia cells could reacquire clonogenic and leukemogenic properties through dedifferentiation It is worthy of note that CSC plasticity contributes to the interconversion of leukemia status through interfering with endogenous PU1 an ETS family pioneer transcription factor required for myelopoiesis50 However CSCs that transformed from differentiated cells require additional factors mutations inflammation or changes in the microenvironment for their stemness features51 For example after hepatotoxin induction hepatocytes produce tumor nodules and express progenitor cell markers52 Targeting CSCs and preventing the generation of CSCs from nonCSCs are thus a key for the development of cancer therapyThere is a wealth of evidence that CSCs are closely related to tumor invasion metastasis and drug resistance The differentiation gradient between CSCs and their non CSC progenies results in tumor heterogeneity which indicates that cells of different genotypes can exist in the same tumor including subgroups with transferability Owing to the unique nature of tumors CSCs have become the leading cause of drug resistance in cancer treatment Wnt submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Wen et alTable Effect of miRNAs on WntCatenin Signaling in CancermiRNAmiR100miR125bmiR181a5pmiR3773pmiR504miR1301miR31943pmiR186miR4543pmiR27amiR221222miR1945pmiR31miR5905pTargetsUpTMEM170BEcadherinDownDKK1 ZNRF3ZNRF3 RNF43 DKK3 APC2catenin TCF4XIAP ZEB2FZD7BCL9BCL9catenin MCRS1RPRD1A AXIN2 DKK3 SFRP1cateninWIF1 SFRP2 DKK2 AXIN2SOX17Dkk1 AXIN1 GSK3Wnt1 catenin NcadherinEffectCell TypeActivationActivationInhibitionInhibitionInhibitionInhibitionInhibitionInhibitionActivationInhibitionActivationactivationActivationInhibitionColorectal cancerColorectal cancerColorectal cancerColorectal cancerHCCHCCHCCHCCBreast cancerBreast cancerBreast cancerBreast cancerBreast cancerBreast cancerRef[][][][][][][][][][][][][][]Abbreviations XIAP Xlinked inhibitor of Apoptosis ZEB2 zinc finger Ebox binding homeobox BCL9 Bcell CLLlymphoma MCRS1 microspherule protein RPRD1A regulation of nuclear premRNA domaincontaining 1A SFRP1 secreted frizzledrelated protein TMEM170B transmembrane protein 170B WIF1 Wnt inhibitory factor Ecadherin epithelial cadherin Ncadherin neuralcadherincatenin signaling plays an essential role in the reprogramming and cancer cell stemness and is an indispensable cytokine network for promoting the progression of CSCs Reilein found that the cell types of CSCderivatives depend in part on the magnitude of spatially graded Wnt pathway activity53 The latest report54 proves the remarkable heterogeneity in Wnt activity in HCC cells among which WntactivityhighALDH1EpCAM triplepositive cells are the most stemlike and highly tumorigenic cells in all CSC populations This type of cells is called superpotent CSCs spCSC The ASPMDishevelled signal axis is highly activated in spCSCs and is heterogeneous in HCC tissues Future studies would reveal the underlying mechanism of Wnt and stem cell heterogeneity in liver cancer and provide new insights into CSCbased tumor treatment strategyWnt signaling contributes to reprogramming and maintenance of the CSC status such as the epithelialmesenchymal transition Chang found that in epithelial cells the SRYrelated highmobilitygroup box gene Sox15 tumor suppressor interacted with the cateninEcadherin complex and then interacted with the proximal promoter region of caspase3 CASP3 Caspase3 inactivates the Twist family bHLH transcription factor Twist which inhibits Wnt signaling for CSC phenotype acquisition In interstitial cells Twist1 forms a complex with cateninTCF4 and binds to the proximal promoter region of ATPbinding cassette G2 ABCG2 which induces the expression of stem cellrelated genes and promotes Wnt signaling to induce the CSC phenotype In the presence of Wnt signaling EMT regulates the CSC phenotype by inducing the conversion of two types of complexes55 In addition to activated Wnt signaling inactivated Wnt signaling can also promote malignant progression of CSCs especially tumor recurrence Autocrine inhibition of WNT signaling induces the resting state of CSCs and tumor dormancy After the primary tumor undergoes periodic proliferation and cancer immunoediting a small fraction of the progenies are transformed into latency competent cancer LCC cells and these cells exhibit Soxdependent stem phenotype LCC cells actively silence WNT signaling to enter into a quiescent state by expressing Sox2 target gene DKK1 The cancer cells then gain longterm viability through immune escape When the function of cancer immunoediting is somehow hampered LCC cells randomly enter cell cycle triggering the next metastatic exp | Thyroid_Cancer |
"Pancreatic cancer PC is one of the most aggressive cancers and has an extremely poor prognosisworldwide Long noncoding RNA lncRNA has been reported to be a potential prognostic biomarker in theinitiation and prognosis of PC Nevertheless the biological functions and the detailed molecular mechanism ofLINC00514 in PC remain unclearMethods We measured the expression level of LINC00514 in PC tissues and cell lines by quantitative realtime PCRGain and lossoffunction experiments were performed to explore the bioeffects of LINC00514 on PC developmentboth in vitro and in vivo Subcellular fractionation luciferase reporter assay RNA immunoprecipitation assay pulldown assay and western blotting were performed to investigate the oncogenic molecular mechanisms ofLINC00514Results In this study LINC00514 was shown to be upregulated in PC tissues and cell lines Increased LINC00514expression was significantly associated with the clinical progression and prognosis of PC patients In additionsilencing LINC00514 inhibited PC cell proliferation migration and invasion while LINC00514 overexpressionpromoted these processes Moreover LINC00514 knockdown remarkably inhibited PC development and metastasisin vivo Deeper investigations indicated that LINC00514 acted as a sponge for microRNA285p miR285p in PCand that Rap1b was a downstream target of miR285p Furthermore the positive correlation of LINC00514 andRap1b and the negative correlation between miR285p and LINC00514 or Rap1b were revealed Based on therescue assays Rap1b inhibition partially suppressed the oncogenic effect of LINC00514 overexpression on PC cellproliferation migration and invasionConclusions This study is the first to characterize the oncogenic function of the long noncoding RNA LINC00514in pancreatic cancer progression by acting as a competing endogenous RNA ceRNA of miR285p to upregulateRap1b expression Understanding this molecular mechanism might contribute to further discoveries of betterdiagnostic and therapeutic options for pancreatic cancerKeywords LINC00514 Pancreatic cancer Proliferation Invasion miR285p Rap1b Correspondence songzhiwangtongjieducnDepartment of Oncology the First Affiliated Hospital of Nanchang University Yongwaizheng Street Nanchang Jiangxi Peoples Republic ofChina The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cHan Journal of Experimental Clinical Cancer Research Page of Table Primers involved in the studyGeneLINC00514Rap1bMiR285pGAPDHForward primerGAGGCAGGAGAATCGCTTGAACCACAGCAATGAGGGATTTATACTGGTGTCGTGGGTCGACGCTCTCTGCTCCTCCTGTTCU6Abbreviation LINC00514 long intergenic nonprotein coding RNA GAPDH glyceraldehyde 3phosphate dehydrogenaseCTCGCTTCGGCAGCACAReverse primerGAGGCAGGAGAATCGCTTGAACCTGACCTTGTTCCTTCCCTACCTCGCTTCGGCAGCACAATCCGTTGACTCCGACCTTCACAACGCTTVACGAATTTGCGTFig LINC00514 was upregulated in PC and predicted a poor prognosis a LINC00514 expression was detected in PC tissue and adjacentnormal tissue by qRTPCR bd Associations between LINC00514 expression and tumor size Lymph node metastasis or clinical stage weredetected by qRTPCR e qRTPCR was applied to confirm the expression level of LINC00514 in PC cell lines and normal pancreatic epithelial cellline f KaplanMeier analysis was used to assess the relation between LINC00514 expression level and overall survival in PC patients p05p01 p001 All experiments were repeated at least for three times and mean ± SD was used to represent the final result PC pancreaticcancer qRTPCR quantitative realtime polymerase chain reaction SD standard deviation 0cHan Journal of Experimental Clinical Cancer Research Page of BackgroundAs an extremely aggressive cancer worldwide pancreatic cancer PC has shown an increasing incidencerate in recent years [] Due to its high level of malignancy PC has become the fourth leading cause ofdeath from malignanttumors with poor prognosisand the 5year survival rate is less than [ ]Early diagnosis of PC has been a considerable challenge due to its complicated pathological process andintricate molecular mechanism While some advancesin imaging and clinical treatment have improved diagnosis and therapy [] the outcome of PC patients remains unsatisfactory Hence a better understandingofthe underlying molecular mechanism is essentialfor seeking a novel therapeutic target for PChavestudiesfunctions HoweverLong noncoding RNA lncRNA is a ribonucleotidechain with a coding length of more than nucleotides[] In the past it was thought that since lncRNAs didnot have the ability to encode proteins they lacked biologicalin recent years scientistshave found that lncRNAs execute their biological effectsin epigenetics [] at the histone modification [] tranlevels [] Accumuscriptional and posttranscriptionallatedelucidatedtheextraordinarysignificance of lncRNAs in the progression of a widerange of diseases such as cardiovascular diseases []diabetes [] neurodegenerative diseases [] and human cancers For instancelncRNA SNHG1 whichcan be positively regulated by miR21 activates theAKT pathway to promote sorafenib resistance in hepatocellular carcinoma cells [] lncRNA EPB41L4AAS1 suppresses the Warburg effect and plays a significant role in metabolic reprogramming of cancer[]intestinalstem cells and promote tumorigenesis of colorectalcancer []lncGata6 could maintain stemness ofLINC00514 is a newly identified lncRNA and very fewreports about it are found in the literature Research byLi [] proved that LINC00514 could be an inhibitor of malignant behaviors of papillary thyroid cancer Inaddition another study has also shown a relationship between LINC00514 and neuroendocrine prostate cancer[] In our study we explored the function and mechanism of LINC00514 in PC We discovered thatLINC00514 expression was increased in PC tissue andPC celllines and that the upregulated expression ofLINC00514 was associated with PC cell proliferationmigration and invasion in vitro and tumor growth andmetastasis in vivo Mechanistically LINC00514 accelerated pancreatic cancer progression via the miR285pRap1b axis AllthatLINC00514 might act as a potential prognostic biomarker of PC occurrence and provide a novel target forPC therapythe evidence above suggestsMethodsClinical samplesPC tissue and adjacent normal tissue were collectedfrom the First Affiliated Hospital of Nanchang University with the informed content of the enrolled patientsin this research Patients received neither chemotherapynor radiotherapy before surgery Our study was approved by the Human Research Ethics Committee ofNanchang UniversityQuantitative realtime PCRRNA was extracted by TRIzol reagent Invitrogen fromtissue samples and cells Extracted RNA was later reverse transcribed into complementary DNA cDNA byPrimeScript RT Reagent Takara Japan A SYBR GreenKit Takara Japan was utilized to perform RTPCRGAPDH and actin were used as internal controlsGene expression levels were calculated by the ÎÎCtmethod The primer sequences are shown in Table Cell lines and cell cultureThe normal pancreatic epithelial cell line HPDE andPC celllines BxPC3 SW1990 PANC1 AsPC1Capan2 and MIAPaCa2 were purchased from ATCCCells were cultured in Dulbeccos modified EaglesTable Correlation between LINC00514 expression level andclinical featuresCharacteristicsN2P valueAllAge years ¥ GenderMaleFemaleTumor size cm ¥ DifferentiationPoorModerateWellLymph node metastasisAbsentPresentClinical stage AJCCIIIIIIIVLINC00514 expressionHighLowAbbreviation LINC00514 long intergenic nonprotein coding RNA p was considered statistically significant 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted PC cell proliferation migration and invasion ab Transfection efficiency of LINC00514 overexpression plasmids andshRNA in BxPC3 and SW1990 cells were evaluated by qRTPCR cd CCK8 assay was performed to detect cell BxPC3 SW1990 proliferationability with LINC00514 overexpression and LINC00514 silencing e Colony formation assay was carried out to further detect cell proliferationcapacity fg Transwell migration and invasion assay were carried out to detect cell migration and invasion under LINC00514 overexpression andknockdown h Western blot was conducted to evaluate the impact of LINC00514 on EMT progression p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8 EMT epithelialmesenchymal transitionmedium DMEM containing fetal bovine serumFBS at °C with CO2 in humidified airCell transfectionLINC00514 overexpression plasmid and shRNAs againstLINC00514 and Rap1b were purchased from GenePharma Shanghai China with scramble plasmid andshRNA used as negative controls MiR285p mimics andmiR285p inhibitors were acquired from Gene Pharma aswell All of the above reagents were transfected into cellsvia TF3000 Transfection Reagent Invitrogen accordingto the manufacturers recommendationsColony formation assayCells à cells per well were seeded in 6well platesand then incubated for days After being washed withPBS three times colonies were stained with hematoxylinand countedViability assayTo evaluate cell viability a CCK8 assay was carried outCells à cells per well were plated in 96well platesfor h h h and h The cell growth rate was analyzed by Cell Counting Kit8 Solarbio China reagentaccording to the manufacturers instructions The opticaldensity value was measured by a microplate reader at nmMigration and invasion assaysA transwell chamber Corning Tewksbury MA wasused to detect cell migration and invasion capacitiesCells à were seeded on the upper chamber covered with Matrigel Corning Tewksbury MA whileDMEM with FBS was placed on the lower chamberAfter h of transfection cells that passed from theupper chamber onto the lower chamber were fixed withmethanol stained with crystal violet and imaged under alight microscopeIn vivo analysisFiveweekold female nude mice were purchased fromthe National Laboratory Animal Center Beijing Chinaand maintained under specific pathogenfree conditionsSubsequently the mice were randomly separated intotwoLINC00514groups Cells à oftheoverexpression group and NC group were subcutaneously injected into the right axillary of nude miceTumor volume was measured every days and weightwas measured at the end of the experimentTo further evaluate the effects of LINC00514 we carried out pulmonary metastasis analysis PC cells à were injected into nude mice via the caudal vein After days the mice were euthanized The lungs of micewere removed to observe tumor metastasis All experiments were approved by the Animal Research EthicsCommittee of Nanchang UniversitySubcellular fractionation assayThe PARIS Kit Life Technologies was used to isolatenuclear and cytoplasmic RNAs according to the manufacturers protocol Reverse transcription of extractedRNAs and RTPCR were conducted as described beforeLuciferase reporter assayThe online software StarBase30 httpstarbasesysueducnwas used to predict the binding sites of LINC00514to miRNA285p Wildtype LINC00514 and mutantLINC00514 of the putative binding sites were clonedinto a luciferase vector Promega and cotransfected withmiR285p mimics into PC cells via LF3000 transfectionreagent After h cells were harvested for luciferase activity analysisPulldown assayWtmiR285p and NCmiRNA were labeled with biotinand transfected into BxPC3 and SW1990 cells The celllysates were incubated with streptavidin magnetic beads at °C for h After that the beads were rinsed with precooled lysis buffer and salt buffer The pulldown RNAswere extracted to detect LINC00514 levelsRNA immunoprecipitation assayThe Magna RIP RNABinding Protein ImmunoprecipitationKit Millipore MA was used to conduct the RIP assay according to the manufacturers protocol The cells were lysedand incubated with Ago2 and IgG Then cell lysates weremixed with antiAgo2 and antiIgG in RIP buffer MilliporePrecipitated RNAs were collected for RTPCR analysis 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 promoted tumor growth and pulmonary metastasis in vivo ab The images of subcutaneous tumors were obtained on Day cf The tumor volumes and weights of shLINC00514 group compared with NC group and LINC00514 overexpression group compared withempty group were quantified Tumor volumes were analyzed by ANOVA gh qRTPCR was used to assess the transfection efficiency ij Theimage of pulmonary metastasis was photographed at the endpoint kl Pulmonary metastasis of LINC00514 silencing and LINC00514overexpression compared with their control groups were evaluated p05 p01 p001 The mean±SD was used to represent the finalresults of experiments repeated at least three times PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction SDstandard deviation NC negative control ANOVA analysis of variancePVDF membranesWestern blotProtein was extracted from cells and transferred topolyvinylidene difluorideafter sodium dodecyl sulfate polyacrylamide gel electrophoresis After that membranes were blocked with nonfat milk and incubated overnight at °C withprimary antibodies After rinsing the membranes threetimes with PBS a secondary antibody labeled withhorseradish peroxidase was used to incubate membranesroom temperature Antibodiesagainst ECadherin NCadherin and Vimentin wereall purchased from CST company and actin andRap1b antibodies were purchased from Abcam Thedilution ratio was determined accordingto theinstructionsfor h atStatistics analysisAll data are presented as the mean ± standard deviationAll experiments were repeated at least three times Students t test ANOVA Spearmans rank correlation testand Ï2 test were used for statistical analysis A value ofp was considered statistically significantResultsLINC00514 was upregulated in PC and predicted a poorprognosisFirst we analyzed the LINC00514 profile in PC Wefound that LINC00514 was remarkably increased in PCtissues compared with the corresponding normal tissuesFig 1a The upregulated expression of LINC00514 wassignificantly associated with the LINC00514 level andtumor sizelymph node metastasis and clinical stageFig 1bd while no significant correlation was foundbetween LINC00514 expression and age gender ortumor differentiation Table Additionally LINC00514expression was increased in PC cell lines compared withthe normal pancreatic epithelial cell line Fig 1e Furthermore KaplanMeier survival curves revealed thathigh LINC00514 expression was related to a lower overallthe lowLINC00514 level group Fig 1f Overall LINC00514was increased in PC and might be associated with clinical progression and a poor prognosis of PC patientsrate compared with that ofsurvivalcellandpromotedLINC00514 promoted cell proliferation migration andinvasionTo investigate whether LINC00514 is involved in cellproliferation migration and invasion we carried outgain and lossoffunction assays The LINC00514 overexpression plasmid and LINC00514 shRNA were stablytransfected into BxPC3 and SW1990 cells with ascramble plasmid and shRNA used as negative controlsFig 2ab According to the results of CCK8 and colony formation assays LINC00514 overexpression significantlySW1990proliferation capacity while suppression of LINC00514remarkably inhibited these processes Fig 2ce Moreovertranswell assays were utilized to prove thatLINC00514 increased cell migration and invasion capabilities Fig 2fg For further confirmation westernblotting was performed to measure the expression ofEMT markers in both BxPC3 and SW1990 cells As expected Ecadherin was observed to be strikingly downregulated by LINC00514 overexpression whereas Ncadherin and Vimentin were obviously upregulated andthe shLINC00514 group showed the opposite resultsFig 2h In summary LINC00514 promoted PC cellproliferative migratory and invasive capacitiesBxPC3LINC00514 knockdown inhibited tumor growth andpulmonary metastasis in vivoTo further identify the bioeffects of LINC00514 ontumor growth we constructed a subcutaneous xenografttumor model BxPC3 cells transfected with LINC00514shRNA compared with NC shRNA or transfected withthe LINC00514 overexpression plasmid and comparedwith the empty plasmid were subcutaneously injectedinto nude mice The tumors were measured every daysafter injection After euthanizing the mice we obtainedimages of the tumors Fig 3ab Compared with thoseof the NC group the volume and weight of tumors inthe LINC00514 shRNA group were significantly reducedwhileresults were observed in theLINC00514 overexpression group Fig 3cf QRTPCRwas used to assess the transfection efficiency Fig 3ghThen we further investigated the role of LINC00514 inPC metastasis in vivo Nude mice were injected withBxPC3 cells transfected with LINC00514 shRNA compared with NCLINC00514the oppositeshRNA orthe 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig LINC00514 was a sponge for miR285p ab Subcellular fractionation assay was used to determine the subcellular localization ofLINC00514 a BxPC3 cells b SW1990 cells C Sequence of WTLINC00514 MutLINC00514 and miR285p were conducted dg Luciferasereporter assay and RIP assay was performed to demonstrate that miR285p was a downstream target of LINC00514 h Pulldown assay wasconducted to detect the reaction between miR285p and WTLINC00514 or MutLINC00514 ij Relative miR185p expression level in BxPC3and SW1990 were determined by qRTPCR K The expression of miR285p in PC tissue and normal tissue were detected by qRTPCR LSpearmans rank correlation test was utilized to analyze the correlation between the levels of LINC00514 and miR285p MN The miR285pexpression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo op CCK8 assay was performed to detectproliferation of cells transfected with LINC00514 shRNA and cells cotransfected with LINC00514 and miR285p inhibitor qr Transwell migrationand invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experiments were repeated atleast for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chainreaction RIP RNA immunoprecipitation SD standard deviation WTlINC00514 wild type LINC00514 MutLINC00514 mutant LINC00514 CCK8cell counting kitoverexpression plasmid compared with the empty plasmid into the tail vein Images of pulmonary metastasiswere acquired at the endpoint Fig 3ij There was anobviously lower incidence of pulmonary metastasis and asmaller number of metastatic tumors per lung in the shLINC00514 group compared with the NC groupwhereas the LINC00514 overexpression group showedthe opposite results Fig 3klin the celldetected the expression level of miR285p in the tumorswe collected before and the results showed that miR285p expression was higherlines withLINC00514 knockdown while miR285p was lower inthe cells with LINC00514 overexpression Fig 4mnLINC00514 promoted cell proliferation migration andinvasion at least partially by sponging miR285p Fig4or In summary LINC00514 accelerates PC progression by sponging miR285pLINC00514 acted as a sponge for miR285pTo explore the underlying molecular mechanism of theoncogenic effects of LINC00514 on PC we determinedthe subcellular localization of LINC00514 The resultsshowed that LINC00514 was mostly distributed in thecytoplasm Fig 4ab which suggested that LINC00514might exert its biological function by sponging miRNAStarBase30 was utilized to identify a candidate microRNA miR285p and predict the potential downstreamtargets of LINC00514 Fig 4c The luciferase reporterassay results confirmed that the luciferase activity ofWTLINC00514 was clearly decreased by miR285pmimics while the luciferase activity of MutLINC00514did not change significantly Fig 4de In addition theRIP assay further revealed that LINC00514 and miR285p were enriched in beads conjugated to Ago2 comparedwith the IgG group Fig 4fg Furthermore overexpression of WTLINC00514 but not MutLINC00514 decreased miR285p expression in BxPC3 and SW1990cells Fig 4h Additionally overexpressing LINC00514dramatically decreased miR285p levels in both BxPC3and SW1990 cells while silencing LINC00514 increasedmiR285p levels with NC shRNA used as an internalreference Fig 4ij Then we further detected miR285p expression in tumor tissue The results revealed alower level of miR285p in PC tissue than in normal tissue and a negative correlation between LINC00514 expression and miR285p levels Fig 4kl To obtainmore evidence in vivo experiments were performed WeRap1b was a downstream target of miR285p in PCThe posttranscriptional function of miRNAs is usually toinhibit protein synthesis by base pairing with the ²untranslated region [] Next to ascertain the detailed regulatory mechanism of LINC00514 in PC we searchedStarBase30 and observed that Rap1b was predicted to bea downstream target of miR285p Fig 5a MutRap1bor WTRap1b and miR285p or NCmiRNA were transfected into BxPC3 and SW1990 cells A luciferase reporter assay and RIP assay were used to confirm thehypothesis that Rap1b is a direct target of miR285p Fig5be Then we found that Rap1b expression was downregulated by LINC00514 silencing while cotransfectingmiR285p and shLINC00514 inhibited the effect ofLINC00514 knockdown on Rap1b at both the transcriptional and translational levels Fig 5fgThen we detected Rap1b levels in tumor tissue Rap1bwas obviously increased in PC tissue compared with adjacent normal tissue and there was a positive relationshipbetween Rap1b expression and LINC00514 levels while anegative correlation was observed between Rap1b expression and miR285p levels Fig 5hj In addition we alsodetected the expression of Rap1b in vivo and the transfection efficiency was examined previously As expected theexpression of Rap1b was clearly decreased in BxPC3 cellsby LINC00514 knockdown while increased expressionwas observed in LINC00514overexpressing cells Fig 5kl Thus far we have proven that Rap1b is a direct target 0cHan Journal of Experimental Clinical Cancer Research Page of Fig See legend on next page 0cHan Journal of Experimental Clinical Cancer Research Page of See figure on previous pageFig Rap1b was a downstream target of miR285p in PC a The sequence of WTRap1b MutRab1b and miR285p were conducted efLuciferase reporter assay and RIP assay were performed to determine the association between miR285p and Rap1b fg QRTPCR and westernblot were used to detect Rap1b expression in cells of LINC00514 silencing and cells of cotransferring miR285p and LINC00514 shRNA attranscription and translation level h Relative Rap1b expression in tumor tissue and normal tissue were detected by qRTPCR ig Thecorrelation between Rap1b and LINC00514 as well as the correlation between Rap1b and miR285p were analyzed by Spearmans rankcorrelation test kl The Rab1b expression levels under LINC00514 silencing and LINC00514 overexpression were evaluated in vivo mn CCK8assay was performed to detect proliferation of cells transfected with miR285p inhibitor and cells cotransfected with miR285p inhibitor andRap1b shRNA OP Transwell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01p001 All experiments were repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtime polymerase chain reaction RIP RNA immunoprecipitation SD standard deviationof miR285p Thereafter functional experiments werecarried out to investigate the bioeffects of Rap1b The resultsdemonstrated that Rap1b silencing remarkably suppressedthe promoting effects of the miR285p inhibitor on cell proliferation migration and invasion capacities Fig 5mppatients which indicated that LINC00514 might be involved in PC progression In addition it was determinedthat LINC00514 facilitated PC cell proliferation migration and invasion in vitro and tumor growth and metastasis in vivo The underlying molecules however havenot yet been revealedRap1b inhibition inhibited the tumorigenesis effects ofLINC00514Finally we explored the role of LINC00514 mediatedby Rap1b in promoting tumor growth We knockeddown Rap1b in BxPC3 and SW1990 cells to determine whether Rap1b inhibition can reverse the oncogenic effects of LINC00514 Based on the rescueassays Rap1b inhibition partially inhibited the effectof LINC00514 overexpression on cell proliferationmigration and invasion Fig 6adIn conclusionthesethatLINC00514 acted as a key tumor promotor of PC bycompetitively binding to miR285p and then upregulating the expression of Rap1bdemonstratedcollectivelyresultsthrough the miR1883pBRD4 axisDiscussionIn recent years PC has received increasing attention dueto its high incidence and extremely poor prognosis []Accumulated studies have shown that lncRNAs play animportant role in the initiation and development of cancers including PC [] For instance LINC00346 accelerated PC progression and gemcitabineresistancepartially[]lncRNA GLSAS mediated the feedback loop of Mycand GLS and provided a potential therapeutic strategyfor metabolic reprogramming in PC [] AFAP1AS1was shown to exert inhibitory effects on the stemness ofPC cells and ultimately PC tumorigenicity in vivo via themiR384ACVR1 axis [] LINC00514 has been previously reported in papillary thyroid cancer [] and neuroendocrine prostate cancer [] butthere are noreports in PC Our study revealed that LINC00514 expression was markedly elevated in PC tissues and PC celllines and that increased expression of LINC00514 wasassociated with the progression and prognosis of PCIn recent years increasing evidence has proven the hypothesis that lncRNAs exert their biological impact by acting as competitive endogenous RNAs ceRNAs to affectthe development of cancers [] There has been considerable progress in the study of ceRNAs in PC For examplelncRNAPVT1 promotes PC cell proliferation and migration by sponging miR448 [] cucurbitacin B inhibits PCcell proliferation both in vitro and in vivo throughlncRNAAFAP1AS1 binding with miR146b5p [] andPXNAS1 acts as a ceRNA of miR3064 which upregulates PIP4K2B expression and suppresses the progressionof pancreatic cancer [] In our research subcellular fractionation assays indicated that LINC00514 was mostly located in the cytoplasm which provided a basis forLINC00514 to act as a ceRNA in the initiation and progression of PC Then the online software StarBase30 wasutilized to predict the possible downstream target miR285p for LINC00514 Luciferase reporter assay RIPassay and pulldown assay were used to confirm the interaction between LINC00514 and miR285p Overexpression of LINC00514suppressed miR285p whileLINC00514 silencing upregulated miR285p expressionFurther investigation was carried out to demonstrate themigration and invasion promoting effect of miR285p inthe initiation and development of PC which suggested atumorpromotingeffectthat wasdependent on miR285pLINC00514ofAccording to the ceRNA hypothesis mRNA expression is upregulated due to lncRNA competitively bindingto miRNA Rap1b was first reported in the study of Chajut [] and was found to be related to various cancers such as thyroid cancer [] breast cancer []gastric cancer [] and colorectal cancer [] Howeverthere is only a limited number of reports about Rap1b inPC [] In our current study Rap1b was predicted to be 0cHan Journal of Experimental Clinical Cancer Research Page of Fig Rap1b inhibition restrained the tumorigenesis effects of LINC00514 ab CCK8 assay was performed to detect proliferation of cellstransfected with LINC00514 overexpression plasmids and cells cotransfected with LINC00514 overexpression plasmids and Rap1b shRNA cdTranswell migration and invasion assay were carried out to detect cell migration and invasion abilities p05 p01 p001 All experimentswere repeated at least for three times and mean±SD was used to represent the final result PC pancreatic cancer qRTPCR quantitative realtimepolymerase chain reaction SD standard deviation CCK8 cell counting kit8a direct target of miR285p by StarBase30 Luciferase reporter assay and RIP assay confirmed thedirect binding of Rap1b with miR285p Rap1bacting as a cancerpromoting gene had a positivecorrelation with LINC00514 while there was anegative relationship between Rap1b and miR285p Moreover silencing Rap1b partially abolishedthe tumorigenic effects of LINC00514 based on therescue assayIn conclusion ourstudy provides evidence thatLINC00514 promotes PC development by spongingmiR285p and increasing Rap1b expression Thishighlights the LINC00514miR285pRap1b axis as anovel diagnostic and therapeutic strategy for PCpatientsresults highlighted the significantConclusionsOurtheLINC00514miR285pRap1b axis in PC progressionsuggesting that LINC00514 may serve as a potential biomarker and therapeutic target in PCrole ofAbbreviationsPC Pancreatic cancer LncRNA Long noncoding RNA LINC00514 Longnoncoding RNA00514 MiR285p MicroRNA285p CeRNA Competingendogenous RNA EMT Epithelialmesenchymal transition QRTPCR Quantitative realtime PCR RIP RNA immunoprecipitation 0cHan Journal of Experimental Clinical Cancer Research Page of AcknowledgmentsNot applicableAuthors contributionsQH and ZWS designed the study QH JHL and JPX collated the data carriedout data analyses and produced the initial draft of the manuscript QH andZWS contributed to drafting the manuscript All authors have read andapproved the final submitted manuscriptFundingThis study was funded by Jiangxi Provincial Education Fund Project youth Science Foundation of Jiangxi Province20202BAB216027Availability of data and materialsAll the data and materials supporting the conclusions were included in themain paperEthics approval and consent to participateThe study was conducted in accordance with the Declaration of Helsinkiprinciples It was approved by the Ethics Committee of the First AffiliatedHospital of Nanchang UniversityConsent for publicationNot applicableCompeting interestsThe authors declare no competing interestsReceived May Accepted July ReferencesPei X Song F Wang Z Emerging incidence trends and application ofcurative treatments of pancreatic cancer in the USA Medicine e17175Ansari D Tingstedt B Andersson B Holmquist F Sturesson C Williamsson CSasor A B D Bauden M Andersson R Pancreatic cancer yesterdaytoday and tomorrow Future Oncol Shin SJ Park H Sung YN Yoo C Hwang DW Park JH Kim KP Lee SS RyooBY Seo DW Prognosis of pancreatic Cancer patients with synchronousor Metachronous malignancies from other ans is better than those withpancreatic Cancer only Cancer Res Treat Halbrook CJ Lyssiotis CA Employing metabolism to improve the diagnosisand treatment of pancreatic Cancer Cancer Cell Beermann J Piccoli MT Viereck J Thum T Noncoding RNAs indevelopment and disease background mechanisms and therapeuticapproaches Physiol Rev Wei | Thyroid_Cancer |
role of miR1179 in the development of cancer has been proved by different studies However the expression profile and role of miR1179 is yet to be explored in human oral cancer Consistently this study was undertaken to explore the molecular role of miR1179 in regulation of the human oral cancer development and progression The results showed miR1179 to be significantly p overexpressed in all the oral cancer cell lines relative to normal cells The repression of miR1179 transcript levels not only suppressed the proliferation of oral cancer cells but also increased their sensitivity to vincristine The decline in proliferative rates was attributed to induction of autophagy in oral cancer cells as confirmed by transmission electron microscopic analysis Western blot analysis showed that the expression of LC3BII increased and that of beclin decreased while LC3BI expression remained constant upon miR1179 inhibition Inhibition of miR1179 caused significant decrease in the migration and invasion of the oral cancer cells The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition At molecular level the miR1179 was shown to exert its anticancer effects via deactivation of MEKERK and PI3KAKT signalling cascades In the findings point towards the potential of miR1179 in the treatment of oral cancerKeywords Oral cancer Micro RNA Proliferation Autophagy MetastasisIntroductionDespite advancement in science and technology the current strategies employed for the treatment of human oral cancer are still far from descent As such presently the oral cancer is still ranked 6th most prevalent type of cancer globally Peers et a0al The survival rates of oral cancer are comparatively lower than breast and prostate cancers The overall 5year survival rate of oral Correspondence MonicaHendrixaxoyahoocom Yanmei Gao and Hanmei Xu contributed equally to this workDepartment of Stomatology Affiliated Hospital of Jilin Medical University NO of Road Huashan Fengman Area Jilin Chinacancer is only as against and for breast and oral cancers respectively Gupta et a0al Additionally the recurrence of human oral cancer further adds to the problem Borsetto et a0al Hence it is need of the hour to look for the alternative approaches for the management of oral cancer As revealed by the recent research reports the molecular regulators of human cancers have emerged as vital targets to be studied for their usage against these deadly ailments Cao et a0 al Among these molecular agents the small RNA entities called microRNAs miRs which do not code for proteins but have molecular regulatory roles have been shown to be involved in the development and tumorigenesis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 0cGao a0et a0al AMB Expr Page of of number of human cancers including the human oral cancer Gaezon et a0 al Wu et a0 al The miRs have not only been shown to regulate the proliferation of human cancers but have also been shown to have influence the metastasis to neighbouring tissues Peng et a0al MicroRNA1179 miR1179 has been implicated in the regulation of proliferation and metastasis of different human cancers Jiang et a0al Krutovskikh et a0al Song et a0 al Lin et a0 al The miR1179 has been shown to inhibit the growth of the glioblastoma cells by inducing cell cycle arrest Xu et a0al The inhibition of metastasis of hepatocellular carcinoma has been reported to be due to interaction of miR365 with ZEB2 Gao et a0al In yet another study miR targets HMGB1 to suppress the proliferation of gastric cancer cells Li and Qin Nonetheless the role and therapeutic implications of miR1179 has not be reported This study was therefore designed to investigate the role of miR1179 in human oral cancer cells via modulation of the MEKERK and PI3KAKT signalling pathwaysMaterials and a0methodsCell lines and a0culture conditionsThe oral cancer cell lines SCC4 SCC9 SCC15 and SCC25 along with normal EBTr cell line were procured from ATCC USA The culturing of cell lines was performed using Dulbeccoʼs modified Eagleʼs medium DMEM Thermo Scientific The cell lines were maintained in a CO2 incubator at a0°C with CO2 concentration and relative humidity of TransfectionTo stably transfect the oral cancer cells with miRNC and miRinhibitor Lipofectamine Thermo Scientific was used and the procedure was carried as per the manufacturer guidelinesExpression analysisThe RNeasy Mini Kit Qiagen and miScript Reverse Transcription Kit Qiagen were respectively used to isolate the RNA from cancer and normal cell lines and for cDNA synthesis The SYBR Green mix Thermo Scientific was used for performing the expression analysis of miR1179 through quantitative realtime polymerase chain reaction qRTPCR on QuantStudio real time PCR Thermo Scientific The cycling conditions were as follows °C for a0 s followed by cycles of °C for a0s and °C for a0min The expression values were quantified using ddCt method The real primer sequences were miRF ² GCG GAA GCA TTC TTT CAT ² and miRR ² CAA GGG CTC GAC TCC TGT ²Cell viability assayTo assess the proliferation rates of the oral cancer cells transfected with miRNC and miRinhibitor for a0 h and administered withwithout a0 µM vincristine the cells were cultured in 96well plate for a0h a0h or a0h at °C Following this the 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT reagent Thermo Scientific was added at final concentration of After a0 h a0 µl dimethyl sulfoxide DMSO was added for dissolving the crystals of formazan Absorbance at a0 nm was taken using spectrophotometer to determine the proliferation rates of oral cancer cellsElectron microscopyFor the assessment of induction of cell autophagy the cancer cells transfected with miRNC or miRinhibitor for a0 h were examined using Transmission electron microscope TEM Trypsin treatment was used to make the collection of cancer cells which were then washed and then fixed with glutaraldehyde in phosphate buffer a0m The Osmium tetroxide was then used for postfixing of cells The cells were then treated with ethanol and embedded in resin The ultramicrotome was used for section cutting which was followed by electron microscopyTranswell chamber assayThe migration and invasion of oral cancer cells transfected with miRNC or miRinhibitor were determined by using Transwell chamber without or with matrigel coating Here the cell suspension containing approximately cells was poured into the upper portion of transwell chamber and lower portion was supplemented with a0µl DMEM medium with fetal bovine serum FBS Following a0 h incubation at a0 °C and CO2 concentration the cancer cells from the surface of membranes upper side were swabbed away with cotton swabs while the cells sticking to lower surface of membrane were fixed with ethanol and the stained with crystal violet The cells were then examined and photographs were taken using light microscopeWestern blottingFor the estimation of protein concentrations the western blotting technique was used Precisely after transfection with miRNC or miRinhibitor the oral cancer cells were cultured for a0h at a0°C Centrifugation was used to collect the cells which were then washed using PBS buffer This was followed by treatment with RIPA buffer containing a0 mM TrisHCl pH a0 mM NaCl Triton X100 SDS a0mM EDTA a0mM Na2HPO4 a0mM NaF a0mM NaVO4 a0mM phenyl 0cGao a0et a0al AMB Expr Page of methylsulfonyl fluoride and protease cocktail inhibitor The total protein concentrations were estimated using Bradford assay Equal protein concentrations were loaded on the Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDSPAGE from each sample The gel was blotted to PVDF membrane followed by treatment with primary antibodies Santa Cruz Biotechnology Inc Dallas TX USA overnight at °C The blots were washed in tris buffered saline TBS incubated with horseradish peroxidaseconjugated secondary antibody Santa Cruz Biotechnology Inc for a0h at °C washed again three times with TBS and chemiluminescence was captured on hyperfilm following incubating the blots in enhanced chemiluminescence reagentStatistical analysisThe experiments were performed in triplicate and expressed as mean ± standard deviation SD The Students ttest was performed through GraphPad Prism software The pvalues were taken as statistically significant differenceResultsmiR is a0upregulated in a0oral cancer cellsThe qRTPCR analysis showed miR1179 to be significantly upregulated in all the oral cancer cell lines studied SCC4 SCC9 SCC15 and SCC25 relative to normal oral cell line EBTr The maximum transcript upregulation of miR1179 was observed in SCC9 oral cancer cells 65fold and SCC25 cells 45fold relative to normal EBTr cells Fig a01a As such SCC9 and SCC25 cell lines were taken for further experimentationmiR inhibition suppresses the a0proliferation and a0enhances chemosensitivity of a0oral cancer cellsTo reveal the molecular functionality of miR1179 in oral cancer the suppression of miR1179 was achieved by transfecting the SCC9 and SCC25 cancer cells with miR1179 inhibitor Using miRNC transfected as control the repression of miR1179 transcript levels was confirmed by qRTPCR method which showed significant p decrease of miR1179 expression in SCC9 and SCC25 cells Fig a01b Additionally the results showed that suppression of miR1179 resulted in remarkable decline of proliferation rates of SCC9 and SCC25 cancer cells Fig a01c Interestingly the antiproliferative effects of vincristine were shown to be greatly enhanced when SCC9 and SCC25 cancer cells were transfected with miRinhibitor construct Fig a0 Together the results clearly indicate that miR1179 inhibition suppresses proliferation and enhances the chemosensitivity of the human oral cancer cellsFig miR1179 regulates the proliferation of human oral cancer cells a Expression of miR1179 in human oral cancer and normal cells b Expression of miR1179 in miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells c Cell viability of miRNC and miR1179 inhibitor transfected SCC9 and ACC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 enhances the Vincristine sensitivity of the human SCC9 and SCC25 oral cancer cells Individual experiments were performed in triplicate and expressed as mean ± SD P by the western blotting of LC3BI LC3BII and beclin autophagy related proteins The LC3BI protein levels remined constant beclin protein levels declined and the LC3BII protein levels increased in miR1179 inhibitor transfected SCC9 and SCC25 oral cancer cells Fig a03b Hence it is evident that the transcript repression of miR1179 in human oral cancer cells declines the cell proliferation rates via induction of cell autophagymiR suppression reduces oral cancer cell metastasisThe assessment of migration and invasion capabilities of SCC9 and SCC25 oral cancer cells transfected with miR inhibitor or miRNC constructs by transwell chamber assay showed that the miR1179 inhibitor mediated suppression of miR1179 expression resulted in significant decline in the migratory and invasive potential of SCC9 and SCC25 cancer cells Fig a0 The migration and invasion found to be and for SCC9 and and for SCC25 cells upon miR1179 inhibition Taken together the results indicate that miR1179 has a regulatory role on cancer cell metastasismiR modulates MEKERK and a0PI3KAKT pathways in a0oral cancerIn order to analyse the molecular mechanics of miR1179 in oral cancer the repression of miR1179 was made in SCC9 and SCC25 oral cancer cells The assessment of MEKERK signalling pathway revealed that the decline in miR1179 expression reduced the phosphorylatedMEK and ERK pMEK and pERK protein levels Fig a0 5a However the protein levels of nonphosphorylated versions of MEK and ERK remained almost unchanged Similarly the reduction in phosphorylated protein versions of PI3K and Akt proteins pPI3K and pAkt was Fig Inhibition of miR1179 induces autophagy in oral cancer cells a Ultrastructural analysis of the miRNC and miR1179 transfected SCC9 and SC25 cells b Western blot analysis of miRNC and miR1179 transfected SCC9 and SCC25 cells showing the expression of LC3B I II and Beclin Individual experiments were performed in triplicateInhibition of a0miR induces autophagy in a0the a0oral cancer cellsThe electron microscopic examination of SCC9 and SCC25 human oral cancer cells transfected with miR inhibitor or miRNC clearly revealed the presence of autophagy vesicular structures in miRinhibitor transfected cancer cells Fig a0 3a However such structures were totally lacking from the cancer cells transfected with miRNC The results were further supported 0cGao a0et a0al AMB Expr Page of Fig Inhibition of miR1179 inhibits the migration and invasion of miRNC and miR1179 transfected SCC9 and SC25 cells Individual experiments were performed in triplicate and expressed as mean ± SD P observed under miR1179 repression Fig a05b The protein level of PI3K and Akt remained unchanged Taken together the results reveal that the suppression of miR expression in oral cancer results in the blocking of phosphorylation of MEK ERK PI3K and Akt proteinsDiscussionOral cancer is one of the lethal human disorders and this malignancy is responsible for a considerable level of human mortality and morbidity Warnakulasuriya Peterson Overall the oral cancer ranks 6th in terms of its incidence rates globally One more worrying fact about human oral cancer is its recurrence and development of drug resistance Silva et a0 al So an urgency is felt in the scientific community to devise more robust measures for the oral cancer management Recently miRs have emerged as potent regulators of various human cancers as they have been seen to have profound role in human physiology and disease development Gong et a0al The deregulation of miR1179 has been implicated to be associated with a number of human cancers Peng et a0al In the present study miR1179 was found to be upregulated in oral cancer cells which is in accordance with several previous studies Peng et a0 al Krutovskikh et a0 al Further miR1179 downregulation was previously been shown to decline the viability of ovarian cancer cells Zhihong et a0al Similar observations were made from the results of the current study The miR1179 repression was found to inhibit proliferation and enhance the drug sensitivity of oral cancer cells to vincristine Such implications have been drawn also previously Zhihong et a0 al The characterization of miR1179 in oral cancer in this study revealed that autophagy is induced in oral cancer cells when miR1179 transcript levels are repressed The autophagy was confirmed by the presence of autophagy vesicles which was further implicated by the enhancement of LC3II conjugated protein expression level and declining of LC3I and Beclin expression The miR repression in oral cancer cells further declined the migration and invasion rates of cancer cells which is in conformity with the previous studies on miR1179 Jiang et a0al Lastly among the most important molecular events of cancer progression the activation of MEKERK 0cGao a0et a0al AMB Expr Page of upregulated in human oral cancer cells Inhibition of miR1179 overexpression resulted in decline of proliferation and metastasis and enhancement of chemosensitivity of human oral cancer cells Additionally miR1179 also resulted in the blockage of the MEKERK and PI3KAKT signalling pathway pointing towards the therapeutic implications of miR1179 in oral cancer treatmentAcknowledgementsWe acknowledge the Affiliated Hospital of Jilin Medical University Jilin China Shandong China for providing necessary laboratory faciality and supportAuthors contributionsYG and TP designed the protocol of the study YG and HX performed the experimental work and collect the data for presented study YG and HX involve in the statistical analysis TP supervised the work and drafted the manuscript although all author contributes for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived June Accepted August ReferencesBorsetto D Higginson JA Aslam A AlQamachi L Dhanda J Marioni G Franchella S Frigo A Praveen P Martin T Parmar S Factors affecting prognosis in locoregional recurrence of oral squamous cell carcinoma J Oral Pathol Med Cao M Tang Y Tang Y Liang XH Noncoding RNAs as regulators of lymphangiogenesis in lymphatic development inflammation and cancer metastasis Front Oncol Gao HB Gao FZ Chen XF MiRNA1179 suppresses the metastasis of hepatocellular carcinoma by interacting with ZEB2 Eur Rev Med Pharmacol Sci Garzon R Calin GA Croce CM MicroRNAs in cancer Ann Rev Med Gong H Liu CM Liu DP Liang CC The role of small RNAs in human diseases potential troublemaker and therapeutic tools Med Res Rev Gupta N Gupta R Acharya AK Patthi B Goud V Reddy S Garg A Singla A Changing Trends in oral cancera global scenario Nepal J Epidemiol Huang M Huang B Li G Zeng S Apatinib affect VEGFmediated cell proliferation migration invasion via blocking VEGFR2RAFMEKERK and PI3KAKT pathways in cholangiocarcinoma cell BMC Gastroenterol Jiang L Wang Y Rong Y Xu L Chu Y Zhang Y Yao Y miR1179 promotes cell invasion through SLIT2ROBO1 axis in esophageal squamous cell carcinoma Int J Clin Exp Pathol Krutovskikh VA Herceg Z Oncogenic microRNAs OncomiRs as a new class of cancer biomarkers Bioessays Fig Inhibition of miR1179 blocks MEKERK and PI3K and AKT signalling pathways a Western blots showing the effects of miR1179 inhibition on expression of MEK pMEK ERK and pERK proteins in SCC9 and SCC25 cells b Western blots showing the effects of miR1179 inhibition on expression of PI3K pPI3K AKT and pAKT proteins in SCC9 and SCC25 The experiments were performed in triplicateand PI3KAKT pathways hold a prime essence as these pathways enable the transcription of various downstream regulators of cancer growth and proliferation Huang et a0al These pathways have been shown to be aberrantly activated in different cancer types For example PI3KAKT pathway has been shown to be activated in pancreatic cancer Lan et a0 al In thyroid cancer cells PI3KAKT and MAPKERK pathway has been shown to significantly overexpressed Su et a0 al Similarly MEKERK pathway has shown to be activated in ovarian cancer Zhang et a0al As such the blockage of these signalling pathways is a vital asset to keep the cancer progression at check Zhihong et a0 al The miR1179 suppression renders the MEKERK and PI3KAkt pathways to proceed at fairly diminished rates by preventing the phosphorylation of crucial signalling components like MEK ERK PI3K and AKT Summing up the current study deduced the molecular functionality of miR1179 and implicated its molecular targeting in the management of human oral cancer To conclude the present study showed miR1179 to be significantly 0cGao a0et a0al AMB Expr Page of Lan CY Chen SY Kuo CW Lu CC Yen GC Quercetin facilitates cell death and chemosensitivity through RAGEPI3KAKTmTOR axis in human pancreatic cancer cells J Food Drug Anal Lin C Hu Z Yuan G Su H Zeng Y Guo Z Zhong F Jiang K He S MicroRNA1179 inhibits the proliferation migration and invasion of human pancreatic cancer cells by targeting E2F5 Chem Biol Interact Li Y Qin C MiR1179 inhibits the proliferation of gastric cancer cells by targeting HMGB1 Hum Cell Peng X Guo W Liu T Wang X Tu XA Xiong D Chen S Lai Y Du H Chen G Liu G Identification of miRs143 and145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT PLoS ONE 275e20341Peres MA Macpherson LM Weyant RJ Daly B Venturelli R Mathur MR Listl S Celeste RK GuarnizoHerre±o CC Kearns C Benzian H Oral diseases a global public health challenge Lancet Petersen PE Oral cancer prevention and controlthe approach of the World Health anization Oral Oncol Silva SD Hier M Mlynarek A Kowalski LP AlaouiJamali MA Recurrent oral cancer current and emerging therapeutic approaches Front Pharmacol Song L Dai Z Zhang S Zhang H Liu C Ma X Liu D Zan Y Yin X MicroRNA1179 suppresses cell growth and invasion by targeting spermassociated antigen 5mediated Akt signaling in human nonsmall cell lung cancer Biochem Biophy Res Co Su X Shen Z Yang Q Sui F Pu J Ma J Ma S Yao D Ji M Hou P Vitamin C kills thyroid cancer cells through ROSdependent inhibition of MAPKERK and PI3KAKT pathways via distinct mechanisms Theranostics Warnakulasuriya S Global epidemiology of oral and oropharyngeal cancer Oral Oncol Wu BH Xiong XP Jia J Zhang WF MicroRNAs new actors in the oral cancer scene Oral Oncol Xu X Cai N Zhi T Bao Z Wang D Liu Y Jiang K Fan L Ji J Liu N MicroRNA1179 inhibits glioblastoma cell proliferation and cell cycle progression via directly targeting E2F transcription factor Am J Cancer Res Zhang R Shi H Ren F Feng W Cao Y Li G Liu Z Ji P Zhang M MicroRNA3383p suppresses ovarian cancer cells growth and metastasis implication of Wntcatenin beta and MEKERK signaling pathways J Exp Clin Cancer Res Zhihong Z Rubin C Liping L Anpeng M Hui G Yanting W Zhenxiu S MicroRNA1179 regulates proliferation and chemosensitivity of human ovarian cancer cells by targeting the PTENmediated PI3KAKT signaling pathway Arch Med Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | Thyroid_Cancer |
lack of COVID19 diagnostic tests for the whole Spanish population thecurrent strategy is to identify the disease early to limit contagion in the communityAimTo determine clinical factors of a poor prognosis in patients with COVID19 infectionDesign and settingDescriptive observational retrospective study in three primary healthcare centres with anassigned population of MethodExamination of the medical records of patients with COVID19 infections confirmed by polymerase chain reaction Logistic multivariate regression models adjusted for age and sexwere constructed to analyse independent predictive factors associated with death ICUadmission and hospitalizationResultsWe included patients mean age years female aged � years were health workers doctors nurses auxiliaries Predictors of ICUadmission or death were greater age OR 95CI to male sex OR 95CI to autoimmune disease OR 95CI to bilateral pulmonary infiltrates OR 95CI to elevated lactatedehydrogenase OR 95CI to elevated Ddimer OR 95CI to and elevated Creactive protein OR 95CI to Myalgia or arthralgia OR 95CI to was protective factor against ICU admission andPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsdeath Predictors of hospitalization were chills OR 95CI to feverOR 95CI to dyspnoea OR 95CI to depressionOR 95CI to lymph ia OR 95CI to andelevated Creactive protein OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting forage and sexConclusionDetermining the clinical biological and radiological characteristics of patients with suspected COVID19 infection will be key to early treatment and isolation and the tracing ofcontactsIntroductionOn December the health authorities of Wuhan city Hubei Province China reporteda cluster of cases of pneumonia of unknown aetiology with onset of symptoms on December including severe cases with a common exposure identified in a city market [] whichwas closed on January On January the Chinese authorities identified a newCoronaviridae family virus initially named coronavirus 2019nCoV and later coronavirusSARSCoV2 as the causal agent [] The genetic sequence was shared by the Chinese authorities on January On January the first case was detected in the USA in Washingtonstate [] On January the World Health anization declared the SARSCoV2 outbreak in China a public health emergency of international concern [] Subsequently the outbreak has spread outside China with Europe especially affected []The first positive case diagnosed in Spain was confirmed on January on the islandof La Gomera while the first death occurred on February in Valencia city the date was confirmed twenty days later The first confirmed case in Barcelona was on February and fromthen until June there have been confirmed cases in Spain []The most common signs of infection are respiratory symptoms fever cough and shortnessof breath In more severe cases the infection may cause pneumonia severe acute respiratorysyndrome renal failure and death [] Transmission appears to be mainly persontopersonvia the airway through respiratory droplets measuring microns when the patient has respiratory symptoms cough and sneezing and contact with fomites [] Most estimates of theincubation period of COVID19 range from to days with most around five days Evidenceon the transmission of the virus before symptom onset is unclear There is currently no specifictreatment for COVID19 infections To date the most important scientific efforts have focusedon three areas strategies to contain the spread of the disease the initiation of clinical trialswith antivirals and multiple therapies and the design of a new vaccine which is still unclearThese strategies include some of a community nature where primary healthcare plays a centralrole in disease prevention and control [] Few studies have described the clinical characteristics of the disease fewer the predictive factors and virtually none have described the Mediterranean population compared with the rest of the world Therefore this study aimed todescribe the clinical biological and radiological manifestations the evolution treatments andmortality rate of patients with COVID19 infection in the population of Barcelona city anddetermine the most important predictors of a poor prognosisPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsMaterials and methodsA multicentre observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of with one reference hospital The studyincluded all consecutive adult patients with COVID19 confirmed by polymerase chain reaction PCR from nasal and pharyngeal samples during the study period of February to April Diagnostic confirmation was made in the hospital laboratories as PCR is not available in primary healthcare centres Signs and symptoms the main available haematologicaland biochemical data and the results of imaging tests were recorded as were comorbiditiesthe evolution the hospitalization rate intensive care unit ICU admission and the treatmentsreceived The study population was divided into four age groups years years years and � years Other variables recorded were the type of followup the need fortemporary work disability and the source of possible contacts The time to first medical visitwas defined as the difference in days between symptom onset and medical visit by a familyphysician The factors that determined a poor prognosis hospitalization ICU admissiondeath were collected The data were obtained from the electronic medical record Missingdata were collected by telephone interviews with patients when possible Patients from nursinghomes were excluded as the rate of infections and mortality has been shown to be muchhigher than in the noninstitutionalized population The study was approved by the EthicsCommittee of the Hospital Clinic of Barcelona registration number HCB20200525 Thestudy was conducted according to the Helsinki Declaration and Spanish legislation on biomedical studies data protection and respect for human rightsStatistical analysisCategorical variables are presented as absolute frequencies and percentages and continuous variables as means and standard deviations SD Predictors of death ICU admission andhospitalization were determined using the students t test for continuous variables and the chisquare test for categorical variables Logistic multivariate regression models adjusted for ageand sex were constructed to analyse independent predictive factors associated with death ICUadmission and hospitalization Odds ratios OR and their confidence intervals 95CIobtained in the adjusted regression analysis were calculated Forest plots were used to represent OR and 95CI Values of p005 were considered statistically significant The statisticalanalysis was performed using the R version for WindowsResultsClinical characteristics and comorbiditiesWe included patients mean age years female aged � yearsThe mean time from symptom onset to the medical visit was SD days Clinical characteristics are shown in Table Notably were health workers doctors nurses auxiliaries The most frequentclinical symptoms were cough fever general malaise fatigue myalgia or arthralgia dyspnoea diarrhoea headache anosmia and dysgeusia Physical examination in patients showed had auscultatory alterations tachypnoea and an oxygen saturation of � ICU admission and death were associated with a greater mean age years vs yearsp male sex vs p dyspnoea vs p fever vs p auscultatory alterations vs p and low oxygen saturation vs p Table Myalgia or arthralgia vs PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Clinical and exploratory factors predicting hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationAgeyearsDistributionno years years years� yearsMaleno Occupationno n No n YesPAdjusted ORNoYesPAdjusted OR ± ± n ± [][ CI]n ± n ± [][ CI] [] []Other type of exposure Health professionalOther health workersSmoking exsmokersmokernototal no Temperature at admission ËC Patients with fever �ËCnototal no Time from symptom onset tomedical visitdays¡Symptomsno ¶ ± ± ± ± ± ± NANANA [] [] [] [] ± ± ± [] [] ± [] NANANA []CoughGeneral malaiseFatigue [] [] [] [] [] []Myalgia or arthralgia [] []DyspnoeaDiarrhoeaHeadacheAnosmiaDysgeusiaSore throatBlocked noseNausea or vomitingSputum productionChillsAstheniaChest painAlterations in physical examinationnototal no §Auscultatory alterationsTachypnoeaTachycardiaPharyngitis [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] NA []Continued PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable ContinuedVariablesTotalDeath or ICU admissionHospitalizationPrognostic factors in Spanish COVID19 patientsOxygen saturation �nototalno n n No n YesPAdjusted ORNoYes[ CI]n n [] PAdjusted OR[ CI] []In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex Temperature distribution was ËC ËC ËC and ËC ¡ In patients data on period between symptom onset and medical visit were lacking¶ Symptoms with a frequency of patients were disorientation n conjunctivitis n haemoptysis n and cutaneous lesions n § patients had a physical examination The alterations with a frequency of patients were cutaneous lesions n and tonsillopharyngitis n Ref reference NA not applicable101371journalpone0237960t001p headache vs p dysgeusia vs p andanosmia vs p were less frequent in patients admitted to the ICU or whodied than the remaining patients Age OR 95CI to and male sexOR 95CI to were independent predictors of ICU admission and deathMyalgia or arthralgia OR 95CI to was the only significant protective factor against ICU admission and death after adjusting for age and sex Fig The best clinicalpredictors of hospitalization were chills OR 95CI to fever OR 95CI to and dyspnoea OR 95CI to Anosmia OR 95CI to was the only significant protective factor for hospitalization after adjusting for age and sex Table and Fig Comorbidities were presented by patients the most common were hypertension in diabetes mellitus in and obesity in Table Heartdisease vs p autoimmune disease vs p diabetes vs p hypertension vs p and chronic kidney disease vs p were the comorbidities significantly associated with ICUadmission and death Table Autoimmune disease was the only significant predictivecomorbidity for ICU admission and death after adjusting for age and sex OR CI to Fig Depression was the best predictor of hospitalization among allcomorbidities OR 95CI to Fig Having � comorbidity was associated with ICU admission and death OR 95CI to and hospitalizationOR 95CI to independently of age and sexImaging and laboratory testsChest Xray was necessary in patients and showed lobar pulmonary infiltrates in bilateral pulmonary infiltrates in and an interstitial pattern in Table Chest CT was required in patients and pulmonary ultrasound in Biologically of patients had lymph ia mm3 Likewise had a lactate dehydrogenase LDH Uml and liver test alterations were commonelevated ASTGOT in and ALTGPT in In of cases Ddimer waselevated 500mgL The most important factors for ICU admission and death were bilateralpulmonary infiltrates OR 95CI to elevated lactatedehydrogenaseOR 95CI to elevated Ddimer OR 95CI to andelevated Creactive protein OR 95CI to Fig Significant predictivePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for death and ICU admission �The upper limits of the confidence intervals were restricted to in order not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g001factors associated with hospitalization after adjusting for age and sex were lymph iaOR 95CI to and elevated Creactive protein OR 95CI to Fig Treatment complications and evolutionTreatment included hydroxychloroquine in patients azithromycin in lopinavirritonavir in glucocorticoids in and tocilizumab in among others Table and of patients required hospitalization Phone follow up was registered in patients patients were monitored at homePLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsFig Prognostic factors for hospitalization �The upper limits of the confidence intervals were restricted to inorder not to mask the significant effects of other variables with smaller ranges101371journalpone0237960g002 of the patients of working age sought work disability due to COVID19 TheICU admission rate was The evolution included pneumonia in patientsadult respiratory distress syndrome in severe renal failure in pulmonarythromboembolism in and sepsis in patients Occupational contact with persons with confirmed or suspected COVID19 infection was reported by patientswhile reported that contact occurred in the family setting Occupational contactwas a protective factor against hospitalization OR 95CI to ICU admission and death OR 95CI to after adjusting for age and sex The mortalityrate to date was PLOS ONE 101371journalpone0237960 August PLOS ONE 0cTable Comorbidities associated with hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admissionHospitalizationn NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Prognostic factors in Spanish COVID19 patientsComorbiditiesno Any comorbidityHypertensionDiabetesObesityDyslipidaemiaCancer [] [] [] [] [] [] [] [] Chronic kidney disease Heart disease Autoimmune disease Chronic obstructive pulmonary diseaseDepressionCardiac arrhythmiaThyroid alterationsAsthmaLiver diseaseCerebrovascular diseaseAlzheimer disease [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] [] []In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex Comorbidities with a frequency of patients were bronchiectasis n fibromyalgia n anaemia n arthritis n HIV n syphilis n andtuberculosis n 101371journalpone0237960t002DiscussionThis study summarizes the clinical biological and radiological characteristics evolution andprognostic factors of patients with COVID19 disease in primary and community healthcareTo date we are aware of three published Spanish studies [] The first reported data from patients on ICU admissions in a region where the pandemic was reported early [] Thestudy by Borobia [] describes the first adult patients with COVID19 consecutivelyadmitted to a University Hospital in Madrid The third focuses on the differences by agedependent categories in the clinical profile presentation management and shortterm outcomes [] Although there have been two systematic reviews and metaanalysis that analysethe clinical characteristics of COVID19 they are limited to Chinese cohorts or case series [] and a large USA cohort [] that did not analyse clinical predictors of a poor prognosisClinically the same main symptoms of cough and fever are reported in all series Howeverin Barcelona city we have observed diarrhoea anosmia and dysgeusia which is hardlyreported in the Chinese series [] which unlike ours comes principally from hospitals diarrhoea occurred in of cases very similar to the in New York [] and clearly higherthan the reported in China Nearly of patients had anosmia and dysgeusia similar tothe results obtained in French patients [] In contrast expectoration was found in only compared with in the Chinese seriesPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Analytical and radiological predictors of hospitalization and ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted ORNoYesn n [ CI]n n PAdjusted OR[ CI]Alterations in chest Xraynototalno Bilateral pulmonary infiltratesInterstitialground glass patternLobar pulmonary infiltrateAlterations in chest CAT scannototal no ¡ [] [] [] [] [] []Bilateral pulmonary infiltrates Interstitialground glass pattern Laboratory parametersnototalno [] [] [] []Leukocytes mm3 [] []Lymphocytes mm3Platelets mm3 [] [] [] []Haemoglobin gdl [] []Creactive protein mglitreProcalcitonin ngmlLactate dehydrogenase UlitreAminotransferase aspartate UlitreAlanine aminotransferase UlitreTotal bilirubin mgdL [] [] [] [] [] [] [] [] [] [] [] []Creatine kinase Ulitre [] []Creatinine 15mgdL Ddimer mglitreSodium mEqlitrePotassium mEqlitre [] [] [] [] [] [] [] []In bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex patients had a chest Xray The alterations with a frequency patients were pneumothorax n and pleural effusion n Chest Xray resultswere not available in patients¡ patients had a chest CAT scan Alterations with a frequency of patients were pulmonary thromboembolism n emphysema n lobarpulmonary infiltrates n pneumonia n atelectasis n and pleural effusion n CAT scan results were not available in five patients101371journalpone0237960t003PLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsTable Predictors of the evolution complications and treatment in patients hospitalized or with ICU admissiondeathVariablesTotalDeath or ICU admission n Hospitalization n n NoYesPAdjusted OR [NoYesPAdjusted OR [n n CI]n n CI]Complicationsno Any complicationPneumonia NA [] [] []Adult respiratory distress [ []syndromeRenal failurePulmonary thromboembolismTreatmentsno ¡HydroxychloroquineAzithromycinLopinavirRitonavir [] [] []NA] [] [] [] [] [] []Oxygen therapy [] []Intravenous antibiotics [] []GlucocorticoidsTocilizumabCephalosporinsLow molecular weight heparin Remdesivir Covid19 infectionno [] [] [] [] [] [] [] [] [] []Any cohabitant [] []Any work colleague [] []Any contact person in other [] []settingsIn bold statistically significant independent predictive factors associated with hospitalization death or ICU admission logistic multivariate regression adjusted for ageand sex Complications in patients were sepsis n multian failure n electrolyte alterations n hematologic alterations n and lung cancer n ¡ Treatments with a frequency of patients except remdesivir were amoxicillin n interferon n rituximab n darunavir n and entecavirn NA not applicable101371journalpone0237960t004Chinese patients had a mean age of years ten years lower than our series and ofour patients were aged � years compared with and in China Germany andthe USA respectively but in Italy [ ] Older age and male sex predisposed to ahigher mortality rate in our and all large series [ ] In our patients comorbidities werethree times higher than in the Chinese cohort [] and were similar to the findings of the NewYork study [] Any comorbidity was a risk factor for hospitalization ICU admission anddeath Depression was an independent risk factor for hospitalization which has not beenobserved in other cohorts studied Depression was often accompanied by a vulnerable socialsituation which may have justified hospitalization Likewise autoimmune diseases were independent risk factor for ICU admission and death Various hypotheses have been postulated onpossible autoimmune alterations in the pathogenic evolution of the disease With respect toPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientstreatment no drug has proved effective against Covid19 until now Moreover many treatments were unavailable in the outpatient setting Currently we are only certain that treatmentwith tocilizumab showed better survival rates in retrospective cohorts [] although its efficacy has not been tested in randomized clinical trials Therefore the results on the outcomesassociated with treatment should be interpreted with cautionThe same comorbidities were identified with hypertension and diabetes being the twomost common while in the USA and Italy obesity seems to be higher Our results show thatobesity was close to being an independent risk factor for hospitalization OR CI to Strikingly of our patients were healthcare workers compared with in Wuhanand in Germany [ ] Although these studies recognized an important degree ofunderreporting of cases in health workers the difference remains important There are at leasttwo possible explanations first the lack of personal protective equipment in the initial phaseof the epidemic a constant revindication of health professionals who felt undersupplied Secondly many cases were health professionals from primary healthcare or the reference hospitalwho reside in the same area where they workIn all reported series bilateral pneumonia was the most common radiological finding waspresent in more than half the cases [] and was a factor of a poor prognosis and mortality Incontrast an interstitial radiological pattern did not confer an increased risk of mortality TheWuhan study reported a CAT scan use of compared with in Barcelona In contrast chest Xrays were carried out in and respectively the availability of diagnostic means was higher in China A recent international consensus states that radiologicalassessment is not necessary in asymptomatic patients or those with mild disease but is requiredin patients with moderate or severe disease regardless of whether a definite diagnosis ofCOVID19 has been made [] In addition simple chest Xrays are preferable in a resourceconstrained environment with difficulties in accessing CAT scans [] The possible use of pulmonary ultrasound for the pointofcare diagnosis of COVID19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability []In fact the regional Catalan government has recently acquired ultrasound machines toenable family physicians to make doctors can make pointofcare home or nursing homediagnoses of pneumonia [] Biologically lymph ia and increased CRP LDH and Ddimer were usually constant and similar in all series and were associated with an increased riskof mortality A differential variable in our series is a greater number of alterations in liver testswhich was present in of patients data similar to the USA and Italian cohorts but different from the Chinese cohort where it was [] We also found hypokalaemia in of patients a factor not reported in other studiesWe found a hospitalization rate of compared with in the USA and inChina and an ICU admission rate of which was similar to the Chinese USA and German results While the protocols of action and admission are similarand depend on the level of clinical involvement the therapeutic protocols differ between hospitals cities and countries There remain many unknowns in the treatment of COVID19The only truth is that we do not have a vaccine an etiological treatment or a treatment withsufficient scientific evidence to generalize its use Currently the systematic review of antiretroviral treatments has not offered conclusive results [] and despite in vitro results for hydroxychloroquine COVID19 infections are currently intractable [ ]The mortality rate in our study was compared with in New York in hospitalized patients in China in Germany and in Italy Different informationand recording systems the availability of diagnostic tests and above all the anization ofnational health systems may have contributed to the differences observedPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsThe study had some limitations due to the observational retrospective design However itis sufficiently representative of the population with confirmed COVID19 to permit betteridentification of the factors of a poor prognosis of the disease from a clinical perspective Wecannot rule out some heterogeneity in data codification due to observers interpretations of themedical records However this bias is minimal as most clinical factors included are clearlydefined in the electronic medical record Another limitation of this study is the percentage ofpatients without laboratory parameters more than Even though in real clinical practicethese percentages may be expected the results corresponding to laboratory parameters shouldbe interpreted with cautionFour months after the declaration of the pandemic there is not a sufficiently reliable available and generalizable diagnostic test that can analyse the seroprevalence of COVID19 evenin the most industrialized countries Given this lack determining the clinical biological andradiological characteristics of probable cases of COVID19 infection will be key to the initiation of early treatment and isolation and for contact tracing especially in primary healthcareSupporting informationS1 DatasetXLSXAcknowledgmentsThe authors thank David Buss for editorial assistanceAuthor ContributionsConceptualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuData curation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuFormal analysis Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuInvestigation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuMethodology Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuProject administration Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuResources Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuSupervision Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuValidation Antoni Siso´Almirall Belchin Kostov Minerva MasHeredia Sergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana August AnguitaGuimetJaume BenaventÃreuPLOS ONE 101371journalpone0237960 August PLOS ONE 0cPrognostic factors in Spanish COVID19 patientsVisualization Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting original draft Antoni Siso´Almirall Belchin Kostov Jaume BenaventÃreuWriting review editing Antoni Siso´Almirall Belchin Kostov Minerva MasHerediaSergi VilanovaRotllan Ethel SequeiraAymar Mireia SansCorrales Elisenda SantArderiu Laia CayuelasRedondo Angela Martı´nezPe´rez Noemı´ Garcı´aPlana AugustAnguitaGuimet Jaume BenaventÃreuReferences World Health anization Pneumonia of unknown causeChina wwwwhointcsrdon05january2020pneumoniaofunkowncausechinaen accessed April Centers for Disease Control and Prevention Novel Coronavirus 2019nCoV Situation Summarystackscdcgovviewcdc84806cdc_84806_DS1pdf accessed April Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H First Case of Novel Coronavirus in the United States N Engl J Med 101056NEJMoa2001191 PMID World Health anization Novel Coronavirus2019nCoV wwwwhointdocsdefaultsourcecoronavirusesituationreports20200130sitrep10ncovpdfsfvrsnd0b2e480_2 accessed AprilJohns Hopkins University CSSE COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University JHU gisanddatamapsarcgiscomappsopsdashboardindexhtmlbda7594740fd40299423467b48e9ecf6 accessed April Ministerio de Sanidad Gobierno de España Situacio´n del COVID19 en España covid19isciiies accessed April Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical Characteristics of Coronavirus Disease in China N Engl J Med 101056NEJMoa2002032 PMID Yeo C Kaushal S Yeo D Enteric involvement of coronaviruses is faecaloral transmission of SARSCoV2 possible Lancet Gastroenterol Hepatol 101016S2468 PMID Chang BB Chiu TY Ready for a long fight against the COVID19 outbreak an innovative model oftiered primary health care in Taiwan BJGP 103399bjgp 20X101068PMID Barrasa H Rello J Tejada S Martı´n A Balziskueta G Vinuesa C SARSCov2 in Spanish Intensive Care Early Experience with 15day Survival In Vitoria Anaesth Crit Care Pain Med 101016jaccpm202004001 PMID Borobia AM Carcas AJ Arnalich F A´ lvarezSala R MonserratVillatoro J Quintana M A Cohortof Patients with COVID19 in a Major Teaching Hospital in Europe J Clin Med Martı´nSa´nchez FJ Del Toro E Cardassay E Valls Carbo´ A Cuesta F Vigara M Clinical presentation and outcome across age categories among patients with COVID19 admitted to a Spanish Emergency Department Eur Geriatr Med 101007s41999020003592 PMIDFu L Wang B Yuan T Chen X Ao Y Fitzpatrick T Clinical characteristics of coronavirus disease COVID19 in China A systematic review and metaanalysis J Infect 101016jjinf202003041 PMID RodriguezMorales AJ CardonaOspina JA Gutie´rrezOcampo E VillamizarPeña R HolguinRiveraY EscaleraAntezana JP Clinical laboratory and imaging features of COVID19 A systematicreview and metaanalysis Travel Med Infect Dis 101016jtmaid2020101623PMID Richardson S Hirsch JS Narasimhan M Crawford JM McGinn T Davidson KW PresentingCharacteristics Comorbidities and Outcomes Among Patients Hospitalized With COVID19 inthe New York City Area JAMA 101001jama20206775 PMID Goyal P Choi JJ Pinh | Thyroid_Cancer |
"Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAURKA a cell cycleregulated kinase is associated with malignant transformation and progression in many cancer types Weanalyzed the expression change of AURKA in pancancer and its eï¬ect on the prognosis of cancer patients using the TCGAdataset We revealed that AURKA was extensively elevated and predicted a poor prognosis in most of the detected cancer typeswith an exception in colon cancer AURKA was elevated in colon cancer but the upregulation of AURKA indicated betteroutcomes of colon cancer patients Then we revealed that undermethylation of the AURKA gene and several transcriptionfactors contributed to the upregulation of AURKA in colon cancer Moreover we demonstrated that AURKA overexpressionpromoted the death of colon cancer cells induced by Oxaliplatin whereas knockdown of AURKA significantly weakened thechemosensitivity of colon cancer cells to Oxaliplatin Mechanistically AURKA inhibited DNA damage response by suppressingthe expression of various DNA damage repair genes in a TP53dependent manner which can partly explain that ARUKA isassociated with a beneï¬cial outcome of colon cancer This study provided a possibility to use AURKA as a biomarker to predictthe chemosensitivity of colon cancer to platinum in the clinic IntroductionAurora Kinase A AURKA is a cell cycleregulated kinaseinvolved in centrosome maturation mitotic entry bipolarspindle assembly and chromosome separation [] The elevated expression of AURKA is frequently reported in manycancer types [] AURKA alone or combined with otherfactors can trigger cell malignant transformation [ ] andpromote the malignant phenotype of cancer cells [ ]AURKA shows oncogenic activity by regulating multipleoncogenic and tumorsuppressive proteins [] Of theseproteinstumor suppressor TP53 has been intensivelystudied Phosphorylation of TP53 at Ser215 and Ser315 byAURKA results in TP53 degradation by MDM2mediatedubiquitination and abrogation of TP53 DNA bindingtransactivation activity respectively [ ] In turn TP53downregulation increases the expression of ARUKA atboth transcriptional and posttranslationallevels [ ]Negative feedback regulation between AURKA and TP53greatly promotes carcinogenesis and progressionMaintaining genome stability by transactivating theDNA damage response DDR genes is the critical mediatorof TP53dependent tumor suppression [ ] thus TP53deï¬ciency causes the loss of various DDR mechanisms and 0cBioMed Research Internationalthereby facilitates genome instability and cancer development [] Meanwhile platinuminduced DNA damage cantrigger the DDR which is a major contributor to chemoresistance to platinum [] In view of the association betweenAURKA TP53 and DDR the upregulated AURKA in cancer might promote the cancer progression but meanwhileenhance the chemosensitivity of DNA damageinducingdrugs in the clinicIn this study we analyzed the expression state and regulation mechanism of AURKA in colon cancer We also testedthe eï¬ect of dysregulated AURKA on chemosensitivity to theplatinum drug and explored the underlying molecular mechanism in colon cancer These results provided a novel insightinto the function of AURKA in cancer Material and Methods Dataset and Processing The data of AURKA mRNAexpression in types of cancers and matched normal tissueswere downloaded from The Cancer Genome Atlas TCGAdatabase including Bladder Urothelial Carcinoma BLCABreast Invasive Carcinoma BRCA Cervical Squamous CellCarcinoma and Endocervical Adenocarcinoma CESCColon Adenocarcinoma COAD Head and Neck SquamousCell Carcinoma HNSC Kidney Renal Clear Cell Carcinoma KIRC Kidney Renal Papillary Cell CarcinomaKIRP Liver Hepatocellular Carcinoma LIHC Lung Adenocarcinoma LUAD Lung Squamous Cell CarcinomaLUSC Pancreatic Adenocarcinoma PAAD Prostate Adenocarcinoma PRAD Rectum Adenocarcinoma READSarcoma SARC Skin Cutaneous Melanoma SKCM Stomach Adenocarcinoma STAD Thyroid Carcinoma THCAand Uterine Corpus Endometrial Carcinoma UCEC Thecorrelation between the AURKA level and the overall survivalOS of cancer patients was also analyzed through the GEPIAhttpgepiacancerpkucnindexhtml The mRNA expression data are shown in Supplementary We compared theexpression level of AURKA mRNA by calculating the meanvalue and standard deviation The eï¬ect of AURKA copynumber variant CNV on AURKA expression level was alsoanalyzed based on the Colon Adenocarcinoma COAD datafrom the TCGA database The eï¬ect of methylation on theexpression of AURKA was assessed using the MEXPRESS datamexpressbe The transcription factor TF targeting ARUKA was screened based on the ChipSeq data in theUCSC databank httpgenomeucsceduENCODE Thetargeted regulatory capacity of TP53 on DDR genes wasassessed using the Cistrome Data Browser httpcistromedb Meanwhile the correlation between AURKAand TF genes was analyzed in colon cancer through theGEPIA httpgepiacancerpkucnindexhtml Cell Culture Two colon cancer cell lines SW1116 andHCT116 and 293TN cell line were used in this study Missense mutation presents in TP53 in SW1116 whereasHCT116 has a wildtype TP53 according to the Cancer CellLine Encyclopedia CCLEportalsbroadinstituteccleabout Additionally all the DDR genes involvedin our study are the wildtype but for BRCA2 which has aframeshift in HCT116 They were cultured using DulbeccosModiï¬ed Eagle Medium DMEM HyClone Logan UTUSA with fetal bovine serum FBSInvitrogenCarlsbad CA USA μgml streptomycin and IUml°penicillin at C in a humidiï¬ed atmosphere containing CO2 Cells used to detect phosphorylated TP53 were treatedwith a speciï¬c proteasome inhibitor MG132 Sigma StLouis MO USA μM for six hours The Construction of Stable Cell Lines Overexpression orknockdown of AURKA was achieved by using lentivirus ps to infect colon cancer cells described as before [] Inbrief the ORFs of AURKA cloned by PCR and synthesizedshRNA against AURKA were inserted into PlvxPuro andSHC201 vectors respectively The scramble sequences wereinserted into these vectors to be used as control Thesevectors were transfected into 293TN cells with the packingvectors System Bioscience Mountain View CA USA toget pseudo lentiviral ps After being ï¬ltered andconcentrated by PEG precipitation System Biosciencelentiviral ps were added to the culture medium toinfect colon cancer cells for h After routine culture for h the stable cells were selected and puriï¬ed by puromycin μgml MTT Assay Colon cancer cells were seeded in 96wellplates at a density of cells per well and incubated overnight The culture medium was replaced with fresh culturemedium containing a diï¬erent concentration of Oxaliplatin and μgml with replicates each After h of incubation μl MTT gl was added to each wellfor h in the incubator The supernatant was removed and μl DMSO was added to each well After being vibratedfor min the plate was read on a microplate reader at nm to calculate the cell viability rate All assays werereplicated three times The result was analyzed using the cellviability percentage the total number of viable cells at eachdrug concentration relative to the number of viable cellstreated with the solvent control Western Blot Total proteins were extracted from coloncancer cells using the RIPA buï¬er Beyotime Institute ofBiotechnology Shanghai China μg protein was separated in SDSPAGE gel by electrophoresis and transferredonto PVDF membrane The blots were blocked by BSA°at C overnight The membrane was incubated with primaryantibodies AURKA rabbit polyclonal antibody ProteinTech Wuhan China No 102971AP diluted at TP53 rabbit polyclonal antibody Proteintech WuhanChina No104421AP diluted at phosphoTP53Ser315 mouse monoclonal antibody Santa Cruz Biotechnology Nosc135772 MDM2 rabbit polyclonal antibodyProteintech Wuhan China No 190581AP and GAPDHmouse monoclonal antibody ProteinTech Wuhan ChinaNo 600041Ig diluted at After washingthemembranes were incubated with peroxidaseconjugatedsecondary antibody Santa Cruz Biotechnology for h at°C The ECL system Thermo Scientiï¬c Rockford IL 0cBioMed Research InternationalUSA was used to visualize the blots All assays were replicated three times RealTime PCR Total RNA was extracted from coloncancer cells using TRIzol Invitrogen Carlsbad CA USAEasyScript® Reverse Transcriptase TransGen Biotech CoBeijing China was used to reverse RNA into cDNA Thelevel of DDR gene ATR XLF XRCC1 RPA1 BRCA2 andRAD51 was quantiï¬ed using the SYBR Green PCR mixBioresearcher Beijing China through CFX96TM RealTime System BioRad The reaction mixture underwent° cycles consisting of denaturation for s at C and°annealing and prolongation for s each at C GAPDHwas used as the endogenous controls All assays were replicated three times The primers used for PCR are shown inSupplementary Statistics Analysis The expression of AURKA in a diï¬erent type of tumors and the diï¬erential expression of genesbetween two groups were analyzed by a twosided Studentsttest Survival analyses were conducted with the KaplanMeier method using the logrank test and the median valueseparation model based on the AURKA expression is presented The hazard ratio was calculated based on the CoxPH model The correlation between methylation statusand AURKA expression was analyzed using the Pearsoncorrelation and Wilcoxon ranksum test Pearsons correlation and Z test were used to analyze the correlationbetween AURKA and TFs The eï¬ect of CNV on AURKAexpression was assessed by the KruskalWallis test MTTresults were analyzed using variance analysis p p p Results AURKA Was Upregulated and Predicted a Beneï¬cialOutcome in Colon Cancer To explore the eï¬ect of AURKAon cancer progression and prognosis we ï¬rstly employedthe TCGA dataset to analyze the mRNA expression ofAURKA in types of tumors Compared with the matchednormal tissues AURKA was significantly upregulated incancer tissues in out of cancer types Figure 1a Nextwe assessed the correlation between the AURKA level andoverall survival OS in cancer types using the GEPIAWe showed that the AURKA level was adversely correlatedwith OS in of cancers including LUAD KIRP PAADSKCM and LIHC However a high level of AURKA wasassociated with a longer OS in COAD Figure 1b Theseresults suggested that AURKA overexpression might playan important role during the carcinogenesis and progressionof cancer however the elevated expression of AURKA predicted a beneï¬cial outcome only in colorectal cancer DNA Undermethylation and Several TranscriptionFactors Might Contribute to the Elevated Expression ofAURKA in Colon Cancer To explore the mechanism bywhich AURKA was upregulated in colon cancer we ï¬rstlyanalyzed the eï¬ect of methylation status on AURKA expression By using the MEXPRESS there were methylationsites in the AURKA gene identiï¬ed Of them methylationsites were significantly adverse correlated with the level ofAURKA Figure 2a Meanwhile we screened the potentialTFs activating AURKA expression based on the ChipSeqdata using the UCSC database and found that a total of TFs potentially regulate AURKA transcription Of themthe expression of TFs was positively correlated with thelevel of AURKA in colon cancer tissues according to theGEPIA correlation analysis Moreover of them have beenidentiï¬ed to be overexpressed in colon cancer tissues compared with the matched normal tissues through the GEPIAexpression analysis Figure 2b The top four TFs highlycorrelated with AURKA r p were E2F1MYBL2 MYC and BRCA1 The expression and correlationwith AURKA of these four TFs are shown in Figures 2cand 2d We also analyzed the eï¬ect of AURKA CNV onthe expression level of AURKA The result indicated thatthe expression level of AURKA in the AURKA CNV gaingroup was much higher than that in the AURKA CNV neutral group in COAD whereas there was no diï¬erencebetween the AURKA CNV loss and CNV neutral groupFigure 2e But the incidence of CNV gain was lower incolon cancer patients These results indicated that undermethylation the elevated TFs and gene ampliï¬cation mightcontribute to the elevated expression of AURKA in coloncancer AURKA Increased the Chemosensitivity of Colon CancerCells to Oxaliplatin We found that upregulated AURKAwas associated with the improved prognosis of colon cancerpatients thus we speculated that if AURKA increases chemosensitivity of platinum by increasing the genomic instability in colon cancer We ï¬rstly constructed the stable cell lineswith AURKA overexpression or knockdown Figure 3aand then assessed the eï¬ect of AURKA on the chemosensitivity of colon cancer cells The resultindicated thatAURKA overexpression promoted the death of HCT116and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened theresponse of colon cancer cells to Oxaliplatin Figures 3band 3c These results showed that AURKA may improvethe prognosis of colon cancer patients by increasing the chemosensitivity of colon cancer cells to the DNAdamagingdrug AURKA Downregulated the Expression of DDR Genes byInhibiting TP53 Previous research showed that AURKAinhibits the expression of TP53 which mediates the expression of DDR genes at the transcriptional level We detectedthe eï¬ect of AURKA on TP53 expression by immunoblotin colon cancer cells The result indicated that TP53 wasdownregulated when AURKA was overexpressed whereasupregulated when AURKA was knocked down in coloncancer cells Figures 4a and 4b Next we screened a setof DDR genes that play an important role in DNA damageinduced by chemotherapeutics Meanwhile most of themfunction after the activation of TP53 [] Using theCistrome Data Browser we assessed the transcriptionalregulatory potential of TP53 on these genes and found someof them had higher scores in two sets of data with high 0c AKRUA fo level evitaler ecid2BioMed Research InternationalCESCCOADHNSCKIRCKIRPLIHCLUADLUSCPAADPRADREADSARCSKCMSTADTHCAUCECTNBLCABRCATumorNormalCOAD Overall survivalLogrank p0034HRhigh06pHR0036nhigh135nlow135lavivrus tnecrePaLUAD Overall survivalLogrank p0047HRhigh13pHR0049nhigh238nlow239lavivrus tnecrePKIRP Overall survivalLogrank p0007HRhigh23pHR00088nhigh141nlow141MonthsMonthsMonthsLow AURKA TPMHigh AURKA TPMPAAD Overall survivalLogrank p00059HRhigh18pHR00068nhigh89nlow89Low AURKA TPMHigh AURKA TPMSKCM Overall survivalLogrank p0014HRhigh14pHR0014nhigh229nlow229lavivrus tnecrePlavivrus tnecrePLow AURKA TPMHigh AURKA TPMLIHC Overall survivalLogrank p000022HRhigh19pHR000028nhigh181nlow181lavivrus tnecrePlavivrus tnecrePMonthsLow AURKA TPMHigh AURKA TPMMonthsMonths Low AURKA TPMHigh AURKA TPMbLow AURKA TPMHigh AURKA TPMFigure AURKA was upregulated in colon cancer and predicted a beneï¬t outcome a Compared with the matched normal tissuesAURKA was significantly upregulated in cancer tissues in out of cancer types b AURKA expression level was adversely correlatedwith OS in of cancers but positively correlated with OS in COADquality control Supplementary We then applied realtime PCR to verify the expression of six representative genesATR XLF XRCC1 RPA1 BRCA2 and RAD51 The resultsindicated that the six DDR genes were downregulated incolon cancer cells with AURKA overexpression but upregulated when knocking down AURKA in colon cancer cellsFigures 4c and 4d which implied that AURKAincreased the chemosensitivity of colon cancer cells to DNAdamageinducing drugs by inducing the degradation ofTP53 and then decreasing the expression of DDR genes 0cBioMed Research InternationalGpG islandAURKA chr2054967393chr2054944445 bpchr2054966401 chr2054967165 chr2054967495 chr2054967671 chr2054967718 r r r r r p p p p p TFE2F1MYBL2MYCBRCA1CBX3rapTFZNF217ELK1EZH2ZBTB33SMARCC1MYBLbrMYCpTFARID3ATCF3YY1FOXM1TEAD4rpBRCA1E2F1COADnumT275 numN349COADnumT275 numN349COADnumT275 numN349COADnumT275 numN349cFigure Continued 0cBioMed Research International MPTFEgol MPTCYMgolp value r p value r MPTLBYMgollog2AURKA TPMlog2AURKA TPMp value r log2AURKA TPMp value r MPTACRBgollog2AURKA TPMdnoisserpxe AKRUACNV loss CNV neutral CNV gainCNV_TypeCNV_TypeCNV LossCNV NeutralCNV gaineFigure Undermethylation upregulation of TFs and gene ampliï¬cation potentially contributed to the elevated expression of AURKAa Five methylation sites in AURKA DNA were significantly adversely correlated with the level of AURKA b Based on the public data analysisa total of TFs potentially regulated AURKA transcription The expression of TFs was positively correlated with the level of AURKAMoreover of them have been identiï¬ed to be overexpressed in colon cancer tissues compared with the matched normal tissues c d Theexpression of the top four TFs highly correlated with AURKA was higher in colon cancer tissues compared with normal tissues e Theexpression level of AURKA in the AURKA CNV gain group was significantly higher than that in the AURKA CNV neutral group in COAD 0cBioMed Research InternationalCtrol AURKACtrolCtrol AURKACtrolshAURKAshAURKAAURKAGAPDH ytilibaiv lleC ytilibaiv lleCAURKAGAPDHSW1116aHCT116p00009Drug concentration ðgmlControlAURKAIC50 ðgml ðgml ytilibaiv lleCSW1116p00427Drug concentration ðgmlControlAURKAIC50 ðgml ðgmlb ytilibaiv lleCcHCT116HCT116p00029Drug concentration ðgmlControlshAURKA1037shAURKA1184IC50 ðgml ðgml ðgmlSW1116p00043Drug concentration ðgmlControlshAURKA1037shAURKA1124IC50 ðgml ðgml ðgmlFigure AURKA increased the chemosensitivity of colon cancer cells to Oxaliplatin a AURKA was upregulated or knocked down in twocancer cell lines b c AURKA overexpression promoted the death of HCT116 and SW1116 colon cancer cells induced by Oxaliplatinwhereas knockdown of AURKA significantly weakened the response of colon cancer cells to Oxaliplatin DiscussionIn this study we evaluated the expression of AURKA in types of tumor tissues and matched normal tissues Theresult indicated that AURKA was upregulated in most testedcancer types compared with their normal tissues OS analysisshowed that higher AURKA was correlated with a worse outcome of most of the cancer types whereas it only indicated afavorable outcome in colon cancer The prognostic role ofAURKA has ever been assessed in colorectal cancer patientsby a research team in [] Despite the lack of statisticalsigniï¬cance they still put forward that AURKA may have a 0cBioMed Research InternationalCtrolAURKACtrolCtrolAURKA CtrolshAURKAshAURKAAURKAMDM2TP53GAPDHpTP53GAPDHAURKAMDM2TP53GAPDHpTP53GAPDHGMGMANRm fo level evitaler ecid2ANRm fo level evitaler ecid2SW1116aATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1SW1116 CtrolSW1116 AURKAATRBRCA2 RPA1 XRCC1 RAD51 NHEJ1HCT116 CtrolHCT116 AURKAANRm fo level evitaler ecid2cANRm fo level evitaler ecid2dGMGMHCT116bATRRPA1BRCA2SW1116 CtrolSW1116 shAURKA1024SW1116 shAURKA1037XRCC1RAD51NHEJ1 ATRRPA1BRCA2HCT116 CtrolHCT116 shAURKA1024HCT116 shAURKA1037XRCC1RAD51NHEJ1Figure AURKA downregulated the expression of DDR genes by inhibiting TP53 a b Overexpression of AURKA promoted thephosphorylation of TP53 and decreased the level of total TP53 whereas knockdown of AURKA reduced the phosphorylation of TP53 andincreased the level of total TP53 in colon cancer cells by immunoblot AURKA had no eï¬ect on the expression of MDM2 c d Sixrepresentative DDR genes were downregulated in colon cancer cells with AURKA overexpression but upregulated when knocking downAURKA in colon cancer cells by realtime PCRpositive eï¬ect on survival and emphasized the necessity tostudy the eï¬ect of AURKA on response to treatment []Further study showed that undermethylation and upregulation of TFs potentially contribute to the elevated expressionof AURKA in colon cancer at least partly Some studiesindicated that gene ampliï¬cation is another contributor tothe elevated AURKA [ ] We also identiï¬ed that geneampliï¬cation in colon cancer patients can result in AURKA 0cBioMed Research Internationalupregulation however its incidence rate was very low incolon cancer patients Finally we demonstrated that AURKAmight improve the prognosis of colon cancer patients byincreasing the chemosensitivity of colon cancer to Oxaliplatin via inhibiting the DDR Our results uncovered thedoubleedged sword eï¬ects of AURKA by inhibiting TP53in colon cancerThe genomic instability has been recognized as a hallmark of cancer and it is associated with carcinogenesis andprogression of cancer [ ] AURKA functions as an oncogene during the development of multiple malignant tumorsby inducing centrosome ampliï¬cation and genomic instability [ ] In colon cancer the overexpressed AURKA is thecontributor to chromosomal instability [ ] MoreoverAURKA has been revealed to impair the function of DNAdamage repair through inhibiting the expression of DDRgenes such as RAD51 and BRCA12 [] In additionto the DDR genes involved in Homologous RecombinationRepair HRR TP53 showed the transcriptional regulatorypotential on Mismatch Repair MMR genes according tothe binding scores from the ChipSeq data Supplementary The inhibitory eï¬ect of AURKA on TP53 which has beendemonstrated to transcriptionally activate many DDR genesenlarges the potential of AURKA facilitating DNA damage[] Some studies also indicate that TP53 is essential for chemoresistance rendered by AURKA [ ] In order to verifythe function of TP53 during this process we respectivelyassessed the correlation between AURKA level and OS inpatients with wildtype or mutant TP53 The results indicated that the patients with the higher AURKA had a longerOS time in TP53 wildtype groups although only a marginalsigniï¬cance was achieved due to the reduced number ofsamples But no diï¬erence was found in TP53 mutant groupsSupplementary The current research supports that AURKA is involved incolon carcinogenesis through promoting genomic instabilitybut the increased AURKA provides a good chance forenhancing the sensitivity of chemotherapy based on DNAdamageinducing drugs The eï¬ect of AURKA on chemosensitivity has been studied in diï¬erent cancer types Up to nowthey all concluded that AURKA impaired the chemosensitivity which is the exact opposite of our ï¬nding Forexample it was reported that inhibiting AURKA enhancesthe chemosensitivity of cancer cells to the taxane and paclitaxel [ ] cisplatin [] doxorubicin [ ] and5ï¬uorouracil 5Fu [] In particular platinum chemosensitivity is inhibited by AURKA in various cancers includingovarian cancer [] hepatocellularcarcinoma [] medulloblastoma [] acute myeloid leukemia []as well as headand neck cancer [] Our ï¬nding that AURKA increasedthe platinum chemosensitivity in colon cancer was diï¬erentfrom the previous studies in other cancer types which coincided with our ï¬nding that higher AURKA indicated betterprognosis only in colon cancer but not in other cancersThough cancer stem cell is a small subpopulation of cancercells AURKA silencing sensitized the response of colorectalcancer stem cell CRCSC to Oxaliplatin by upregulatingantiapoptotic factors [] which is diï¬erent from our ï¬ndings in colon cancer cells The diï¬erence might be associatedwith heterogeneity induced by tumor microenvironment andgenomic instability [] AURKAmediated TP53 inhibitionmight result in diï¬erent consequence in diï¬erent geneticcontexts However this hypothesis might be determined byfurther experiments ConclusionAURKA was upregulated in various cancer types but onlypositively correlated with the prognosis of colon cancerpatients The mechanism might be that AURKA improvesthe chemosensitivity of colon cancer cells to Oxaliplatin byinhibiting the expression of TP53regulated DDR genes andthen facilitating DNA damage This study provides a possibility to use AURKA as a biomarker to predict the chemosensitivity of colon cancer to platinum in the clinicData AvailabilityAll the data used to support the ï¬ndings of this study areincluded within the Conflicts of InterestThe authors declare that there is no conï¬ict of interestregarding the publication of this paperAuthors ContributionsBaocong Shan Ran Zhao Jian Zhou and Minghui Zhangcontributed equally to this workAcknowledgmentsThis work was partly supported by the National NaturalScience Foundation of China to Xiaobo Li Natural Science Foundation of Heilongjiang Province H2018009to Tianzhen Wang H2018010 to Yiqi Wu the FundamentalResearch Funds for the Provincial Universities to Ran Zhao to Yuanyuan Zhu and KYYWF0289 to Weiwei Yang and Heilongjiang Postdoctoral Fund LBHZ17136 to Weiwei YangSupplementary MaterialsSupplementary Figure The correlation of AURKA leveland OS in patients with wildtype TP53 or mutant TP53 AThe patients with the higher AURKA had a longer OS timein TP53 wildtype groups although only a marginal signiï¬cance was achieved due to the reduced number of samplesB No diï¬erence was found in TP53 mutant groups C Mostof the mutations in TP53 were missense variant followed bystop gained and frameshift variant Supplementary Table We compared the expression level of AURKA mRNA by calculating the mean value and standard deviation The eï¬ect ofAURKA copy number variant CNV on AURKA expressionlevel was also analyzed based on the Colon adenocarcinomaCOAD data from the TCGA database SupplementaryTable The primers for PCRSupplementary Table Theassessment of transcriptional regulatory potential of p53 on 0cBioMed Research InternationalDDR genes based on ChipSeq data from cell lines Supplementary Table The assessment of transcriptional regulatory potential of p53 on Mismatch Repair MMR genesbased on ChipSeq data from celllines SupplementarymaterialsReferences[] B Goldenson and J D Crispino The aurora kinases in cellcycle and leukemia Oncogene vol no pp [] A S Nikonova I Astsaturov I G Serebriiskii R L DunbrackJr and E A Golemis Aurora A kinase AURKA in normaland pathological cell division Cellular and Molecular Life Sciences vol no pp [] H Zhou J Kuang L Zhong Tumour ampliï¬ed kinase_STK15_ _BTAK_ induces centrosome ampliï¬cation aneuploidy and transformation Nature Genetics vol no pp [] A H SillarsHardebol B Carvalho M Tijssen TPX2and AURKA 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"Laryngology COVID19 Coronavirus Head and Neck Cancer Otolaryngology Key Points Journal Preproof 0c The landscape of ever evolving information about COVID19 during the pandemic has hindered the transition to normal clinical volume and efficiency COVID19 should not be a reason for delay in diagnosis or treatment with patients who have upper aerodigestive tract pathology or malignancy The approach to resection reconstruction and surveillance for patients with head and neck cancer may need to be altered to consider severity of disease patient comorbidity and prevalence of regional COVID19 infections amongst other factors In light of the significant number of prolonged intubations there may be an increase in the number of patients who develop early and late sequelae of treatment for COVID19 Tracheostomy should be performed in a safe and efficient manner when specific indications are met Synopsis This review explores the changes to practice associated with COVID19 for providers treating patients with head and neck cancer and laryngeal pathology The aim of the review is to highlight some of the challenges and considerations associated with treating this patient population during the pandemic Additionally it seeks to discuss some of the areas of concern related to ramping up clinical volume IntroductionHistoryDefinitionsBackground The downstream effects of COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 have now pervaded most aspects of society and have made an indelible mark on the way that medicine and specifically otolaryngology is being practiced Of note the Journal Preproof 0cdisease represents a threat to an aging population throughout the world but also has dangerous implications for providers Among the most atrisk group of medical providers may be those within the fields of otolaryngology and ophthalmology An otolaryngologist was among one of the first providers to succumb to the illness in its early days as it spread through Wuhan China In light of the risk to both patients healthcare workers and society atlarge a push has been made to mitigate the risk of transmission within the field of Otolaryngology Head and Neck Surgery As of June 22nd there are a total of COVID19 cases reported worldwide with a total of deaths The United States has the highest number of cases at with the total number dead at Given the high mortality associated with the novel virus much of the world has enacted significant social distancing restrictions and facial covering mandates to curb the spread of the disease The origin of the virus is not well understood but it is thought that a bat or pangolin vector might have served as the primary reservoir The disease tends to be marked by fever of patients and cough of patients however a litany of other symptoms have also been described including gastrointestinal upset diarrhea shortness of breath headache loss of smelltaste among others Severe disease is characterized by an acute respiratory distress syndrome ARDS with a mortality for patients that require mechanical ventilation The disease has a slight male predominance at Severity of disease seems to correlate to age as patients who are aged have a mortality while those over present with a mortality approaching in early studies Journal Preproof 0cThe nasal cavity and nasopharynx seem to harbor the highest viral load concentration and thus the nasopharynx is the preferred location for acquisition of samples for diagnostic testing Nasal swabs oropharyngeal swabs bronchial alveolar lavage saliva and tracheal aspirates have also been suggested as possible testing sites The current preferred diagnostic assay is RTPCR which has a variable sensitivity of depending on the institution and type of test During the months of May and June many cities states and countries have focused on a return to normal activity and a ramp up of commercial activities During this time many otolaryngology practices have aimed to ramp up activity as well while employing telehealth social distancing and utilization of personal protective equipment PPE The American Academy of Otolaryngology recently published return to practice guidelines which are detailed below As the world continues to move forward during the COVID19 era considerations such as testing including preoperativepreprocedure COVID testing surgical triage clinic workflow and practice management continue to evolve as more information becomes available This review is intended to highlight some of the current recommendations for patient care within the Laryngology and Head and Neck Surgical Oncology scope of practice Discussion Laryngology Journal Preproof 0cAs cases continue to rise increased emphasis has been placed on protection for the provider in the clinical setting Over the last decade officebased management of many common laryngeal disorders has significantly expanded This includes but is not limited to officebased laser ablation of papilloma or dysplasia transoral or transcervical injection laryngoplasty for vocal fold paralysis and EMGguided injection of Botox for spasmodic dysphonia Given the high number of clinicbased aerosolgenerating procedures practiced by todays laryngologists many providers have seen a marked reduction in their ability to treat patients and their clinical productivity Within the category of aerosolgenerating procedures is flexible fiberoptic laryngoscopy one of the most widely used diagnostic tools for all otolaryngologists and speech pathologists A consensus statement reported by Rameau et al from a virtual webinar attended by approximately participants in the American laryngology community recommended flexible laryngoscopy should be reserved for critical cases in which the findings may have an immediate impact on diagnosis or treatment Indications include hemoptysis odynophagia limiting hydration and nutrition or airway compromisenotably secondary to infectious and malignant conditions Some have advocated for preclinic COVID testing prior to any aerosol generating procedure however given the high false negative rate of many available tests the use of universal personal protective precautions is recommended According to Givi and colleagues examinations should take place in negative pressure rooms if possible with avoidance of topical lidocaine spray although other groups recommend administration of a topical anesthetic to theoretically decrease the coughsneeze reflex unpublished online chats A substitute to standard aerosolized anesthesia may be pledgets soaked in lidocaine and Journal Preproof 0c oxymetazoline The group also suggests using videolaryngoscopy whenever possible to keep the practitioner and the patient farther apart Disposable laryngoscopes should be used whenever possible Most studies universally recommend the following personal protective equipment PPE N95 mask or powered airpurifying respirators PAPRs gloves gown eye shield or goggles and cap It has also been suggested that the patients wear a mask covering the mouth during flexible laryngoscopy to reduce aerosolization from phonatory maneuvers and in case of coughing or sneezing At this time transoral rigid and mirror laryngoscopy are discouraged unless flexible laryngoscopy cannot be performed due to the increased risk of gagging and coughing as well as the need for the patient to phonate with the mouth uncovered to allow visualization of the larynx Additionally universal masking is encouraged in all clinical spaces in accordance with many state policies Patients in the waiting rooms are encouraged to physically distance or wait in their car for a phone call prior to presenting for their appointment Crosby also suggests offering personal protective equipment for the friends and family accompanying the patient during laryngoscopy and other sites prevent friends and family from accompanying patients inside for the visit Some alternatives to flexible laryngoscopy have been raised including transcervical laryngeal ultrasound which has a reported concordance of in identifying vocal fold motion abnormalities Another key consideration for the laryngologist in the COVID19 era is the approach to sanitization and room turnover after aerosol generating procedures AGPs Laryngoscope turnover should include highlevel disinfection including the use of such chemical disinfectants as glutaraldehyde chlorine dioxide or orthophthaladehyde OPA Some authors recommend immediate placement of the scope after use into a covered receptacle for transport from the Journal Preproof 0cexamination room to the sterile processing areas After completion of laryngoscopy room sanitization with an EPAregistered hospitalgrade disinfectant is recommended with a hydrogen peroxide solution gL chlorine disinfectant or alcohol According to the CDC website it is unknown how long the air inside a particular examination room remains infectious and likely relates to the room size rapidity of air exchange patient factors like viral shedding amount of coughingsneezing and length of time patient was in the room The CDC suggests that rooms with air changeshour ACH take about and minutes to purify the air with and efficiency respectively As the number of air changeshour decreases the time between patients should be increased to allow for appropriate dissipation of theoretical infectious agents As such many hospitals have recommended a turnover time of 4x the time it takes to purify the air with efficiency which may be either minutes or minutes depending on the level of air turnover or could be no additional time if any additional HEPA filtration system and negative pressure has been added Limited data exists to support this approach for SARSCoV2 Laryngology patients are quite diverse with respect to their level of acuity Some patients require more urgent intervention while others may have their care deferred1221 Most guidelines advocate for a tiered approach to ramping up both clinicbased and surgical activity The American Academy of Otolaryngology published guidelines for ramping up clinical activity on May The AAO recommends limiting patient care to individuals with timesensitiveurgent and emergent medical conditions This approach is also echoed in the care of head and neck cancer patients see below According to the guidelines emergent conditions include impending airway obstruction due to infection neoplasm stenosis foreign body which may Journal Preproof 0cwarrant the following intervention flexible and rigid laryngoscopy with intervention direct laryngoscopysuspension laryngoscopy bronchoscopy and tracheostomy Urgent conditions include moderate or impending airway obstruction progressive dysphonia progressive dysphagia glottic incompetence causing aspiration or impaired pulmonary toilet which warrant the previously described procedures in addition to stroboscopy functional endoscopic evaluation of swallow esophagoscopy with or without intervention open airway procedures for cancer Time sensitive conditions include T1 glottic carcinoma or carcinoma in situ stablemild dysphonia stable dysphagia which adds transcervical Botox injection to the above list of procedures Routine conditions which may be deferred for days or more include mildmoderate dysplasia nonobstructive benignphonotraumatic lesions of the vocal folds glottic incompetence glottic incompetence with mild to moderate dysphonia gender affirmation globuscough without alarm signs Comparing acuity of patients also raises an important point about the subset of patients who are typically seen for benign phonotraumatic voice disorders Many live vocal performance venues have shut down concerts have been cancelled or postponed and some studies point to live singing as being a potential source of massive spread For this reason one might assume that the percentage of patients being seen for acute phonotraumatic voice disorders diminishes somewhat Conversely as patients continue to recover from hospitalizations related to COVID19 it is anticipated that there may be a number of patients with sequelae of prolonged intubation including posterior glottic stenosis vocal fold granulomas and trachealsubglottic stenosis Laryngeal surgery in the era of COVID has had to undergo some significant changes in the approach to patient triage surgical technique and management of the airway Preoperative Journal Preproof 0cevaluation of patients must weigh the risk of delaying surgery with the risk of complications related to COVID19 infection Lei et al studied a group of operative patients in whom all were COVID19 positive within the incubation period Mortality was for this group and required ICU admission25 Of note all patients in this study underwent surgery about days prior to demonstrating signs or symptoms of COVID19 pneumonia This suggests there is significant risk associated with elective surgery in seemingly asymptomatic patients who are infected with COVID19 For this reason many authors have suggested preoperative COVID testing although it is a subject of some debate Some advocate for a negative test within hours followed by selfquarantine until the time of surgery while others favor a negative test 48hrs from the time of surgery and a point of care negative test on the day of surgery17 This is not always possible given the limitations of the institution where the patient is undergoing surgery Just as discussed earlier with regard to personal protective equipment in clinic universal precautions should be taken including full PPE and all patients should be presumed positive Airway management in the COVID19 era has become a point of focus for quality improvement and safety groups Endotracheal intubation is cited as one of the procedures which seems to have the highest aerosol generating burden It is recommended that intubation be performed by the most experienced practitioner Additionally some recommend early intubation for patients that are high risk for decompensation while others have advocated delaying intubation in favor of noninvasive means of ventilation This may include high flow nasal cannula which actually has minimal dispersion of exhaled air if appropriately fitted according to Cheung It is recommended that flexible fiberoptic intubation be avoided whenever possible Journal Preproof 0cAdditionally excessive bagmask ventilation should be avoided due to the risk of dispersion of exhaled air Furthermore jet ventilation is considered particularly high risk and should be avoided if possible Management of the surgical airway and the topic of tracheostomy has been well represented in the recent literature During the SARS outbreak in open tracheostomy was the most common surgical procedure performed on infected patients Most studies seem to favor open tracheostomy over percutaneous tracheostomy however consideration may be given for percutaneous dilatation tracheostomy in some patients if the anatomy is favorable and the practitioner has sufficient expertise with the procedure Tay and colleagues advocate for use of PAPR during tracheostomy based on the experience of countries during the SARS crisis Other authors have suggested the use of an N95 mask appropriate eye protection gown double gloves and cap To decrease the risk of autocontamination some have recommended an infection control nurse be available to monitor donning and doffing procedures during tracheostomy Additional proposals include trach teams which may consist of a surgeon anesthetist and scrub nurse to increase efficiency and create an environment of consistent verbal and nonverbal communication especially important given the burdens of communicating through a mask or PAPR Portugal et al discuss a surgical safety checklist for performing tracheostomy in patients with COVID1932 This includes performing tracheostomy in the ICU whenever possible decreasing the number of personnel in the room and having a specific tracheostomy bundle in the ICU room to decrease the number of times providers and nurses need to break scrub to leave the room They also recommend donning inner gloves prior to gown and outer gloves after donning gown to maintain clean inner gloves for the removal and Journal Preproof 0cdisposal of the rest of the PPE Two universally agreed upon maneuvers include stopping ventilation prior to entrance into the airway and holding ventilation until after the tracheostomy tube cuff has been inflated Givi and colleagues suggest that a smaller tracheotomy cuffed may be preferred to decrease the spread of aerosolized particles Miles discusses the New York experience suggesting that for intubated patients the cuff pressure should be checked every hours with a goal of cm H2O greater pressure predisposes tracheal pressure necrosis33 The group also suggests delaying the timing of tracheostomy until days after onset of symptoms when feasible Finally some have advocated for the use of specific air containment setups including plastic draping smoke evacuator tubing or specifically designed negative pressure box The field of laryngology has had to undergo significant change in the setting of the COVID pandemic As the numbers of COVID19 patients have continued to increase during the month of June it is clear that practice of laryngology in the postCOVID era will need to be carefully ramped up to protect patients and providers alike Additionally one would expect a continued increase in the number of recovered patients being seen for sequelae of prolonged intubation Decisions to relax restrictions on flexible laryngoscopy and other AGPs will depend on the local incidence of COVID19 infection availability and accuracy of preprocedure testing sustainable supply of PPE the ability to properly sanitize rooms and ultimately development of an effective vaccine Head and Neck Surgical Oncology Journal Preproof 0cSimilar to laryngology the approach to head and neck surgical oncology continues to evolve as more information becomes available during the COVID era During the early weeks of the pandemic the aspect of cancer care most concerning to patients and providers involved potential delays in therapy Finley suggests that delaying cancer surgery should be done with extreme caution despite COVID1937 Additionally delays beyond weeks could significantly affect longterm outcomes and morbidity of treatment Among patients diagnosed with severe COVID19 requiring ICU admission patients with cancer deteriorated faster than noncancer patients Desai and colleagues discovered a higher risk of severe events in patients recently treated with chemotherapy or surgery in the past days compared to noncancer COVID16 patients38 Additionally patients with advanced stage cancer tended to have a higher rate of severe events when compared to early stage cancer Cancer patients undergoing active treatment are predisposed to COVID19 related complications and critically ill patients with cancer have a higher predisposition to death Head and neck cancer patients especially are considered a highrisk population for complications associated with COVID19 infection8 making safe coordination of care difficult but imperative Head and neck cancer patients are an atrisk group for a number of reasons Silverman points out that head and neck cancer patients tend to present with advanced age history of tobacco and alcohol abuse and cardiac and pulmonary comorbidities which are similarly found in COVID19 Risk of respiratory sequelae in patients who have received chemotherapy andor radiation therapy are high with increased rates of dysphagia aspiration and pneumonia Additionally head and neck cancer patients have an increased risk of respiratory infections and aspiration pneumonitis These factors may expedite deterioration to Journal Preproof 0csevere adverse events in patients with COVID19 Additionally head and neck patients who are actively receiving chemotherapy or immunotherapy may have depressed immune function malnutrition and older age For this reason the patients need to be carefully selected and comorbidities strongly considered when constructing a treatment plan for patients with head and neck cancer Within the United States mortality for patients of color African American and Latinx with COVID19 is significantly higher than for Caucasian patients40 Unfortunately this is a consequence of inequality within society and the healthcare system rather than a biological or pathological difference41 Correspondingly these communities also tend to present with more advanced disease and have significantly worse mortality compared to their Caucasian fellow citizens This pandemic has laid to bear some of the gross inequities within the American health care system and highlighted the need for equitable decision making for all patients with a diagnosis of head and neck cancer during the COVID19 era Another consideration for the head and neck cancer patient during the COVID19 era may include the financial burden and cost of survivorship associated with undergoing cancer treatment and financial hardship related to COVID19s effect on the world economy and increasing levels of unemployment Given the significant job losses across the United States there is preliminary data to suggest that there will be at least million newly unemployed people who will also lose their insurance coverage in the wake of the pandemic Increased financial strain has been associated with decreased quality of life scores and subsequently mortality in head and neck cancer patients Journal Preproof 0c Recommendations for head and neck clinic are similar to what was previously discussed for laryngology Providers are expected to take universal precautions regardless of the patients COVID status Flexible fiberoptic laryngoscopy is considered a highrisk aerosol generating procedure Due to this laryngoscopy should be reserved for instances in which it is likely to change management One of the beneficial consequences of the COVID19 era is the increased access of care through the widespread adoption of telehealth clinics among most hospitals49 Providers may use telemedicine as an initial preoperative assessment or prescreen for patients that will later be seen in clinic or prior to surgery While telehealth is wonderful for obtaining a detailed history reviewing dataimaginglabs and discussing surgical optionsrisksbenefits a big drawback is the inability to perform a comprehensive head and neck physical exam Physical examination with or without fiberoptic laryngoscopy is important to define the extent of tumor and formulating an ablative and reconstructive plan Fortunately some workarounds include anatomic and physiologic imaging for ablative planning and CT angiography and virtual planning sessions for microvascular reconstruction Telemedicine also serves a vital role in triage of posttreatment head and neck cancer patients who may not be able to be seen as frequently due to the pandemic Telemedicine also serves a vital role in the coordination of care between multiple oncologic disciplines Dharmarajan and colleagues highlighted the University of Pittsburgh approach to a virtual multidisciplinary tumor board clinic MDC This strategy has been adopted by multiple institutions and works quite well to coordinate care between specialties In their study they found that of virtual tumor board participants preferred virtual multidisciplinary clinic to Journal Preproof 0cthe inperson format Additionally about of participants indicated that they would prefer to continue the virtual multidisciplinary format once in person meeting restrictions had been lifted Through multiple virtual meeting applications practitioners can share imaging and laryngoscopy which may assist with decision making for patients Similar to the guidelines published for laryngeal surgery the American Academy of Otolaryngology has published recommendations for ramping up clinical volume as it relates to triage for head and neck surgical oncology Setzen et al note that most head and neck cases fall within the urgent category The guidelines list emergent procedures as being tumor obstructing airway significant bleeding acute or impending neurological change and salivary gland or deep neck abscesses Urgent procedures within days include all head and neck squamous cell carcinomas of the upper aerodigestive tract benign tumors with impending complications or morbidity anaplastic thyroid cancer medullary thyroid cancer bulky differentiated thyroid cancer with regionaldistant metastasis locally aggressive or large nodules 4cm Bethesda high grade salivary malignancies skin cancers and parathyroid carcinomas with significant systemic effects Time sensitive procedures include lowrisk differentiated thyroid cancer lowgrade salivary neoplasms and slower growing basal cell carcinomas in favorable locations Routine procedures include benign thyroid pathology parathyroid disease without significant systemic effects benign salivary lesions and low risk skin cancers and posttreatment disorders Ranasinghe and colleagues recommend a tiered approach to surgical triage with more aggressive pathology being prioritized in a similar fashion to the AAO guidelines Similarly the review recommends considering alternatives to longduration microvascular reconstructive cases It is recommended that the focus shift to simplifying reconstruction and Journal Preproof 0creducing surgical duration when its feasible and appropriate However it is also acknowledged that this approach may lead to an increase in downstream complications Endocrine surgery is similarly tiered in a memo by Ashok Shaha which outlines a strategic approach to thyroid surgery during the pandemic Similar to other strategies anaplastic thyroid cancer medullary thyroid cancer and locally aggressive differentiated thyroid cancer specifically with impending concern for airway obstruction take precedent However some alternatives are also discussed for instance in patients with BRAF V600E mutations who may have surgery delayed while being treated with appropriate targeted therapies Additionally deescalation of surgical care is advocated for benign conditions like thyroid goiters that are nonobstructive and even papillary thyroid microcarcinoma which may be followed with serial ultrasonography until resource allocation has returned to preCOVID levels As institutions attempt to weigh the pros and cons of elective and essential surgery in the midst of the pandemic some authors have advocated for creating rating systems to allow for appropriate surgical triage during periods of limited clinical output and resource reallocation The medically necessary timesensitive MeNTS procedures scoring system aims to ethically and efficiently manage resource reallocation and risk to healthcare providers during the COVID19 pandemic51 The scoring system which uses procedural disease and patient factors within a 5point Likert scale to determine the potential risk of proceeding with surgery The cumulative MeNTS score may range between and with score above being considered within the high risk or resource heavy procedures either due to patient factors AgeComorbidities or procedure factors head and neck surgical site high anticipated blood loss ICU admission requirement Using scoring systems like MeNTS should help hospitals more Journal Preproof 0cappropriately and objectively triage elective and essential surgeries in the setting of a resurgence of caseslimiting of resources Given the significant lack of available knowledge regarding SARSCoV2 and its associated complications it is difficult to characterize risk for patients undergoing ablative and reconstructive head and neck surgery As mentioned earlier in asymptomatic COVID19 positive patients undergoing elective surgery mortality approached COVID19 has demonstrated myriad manifestations which might interfere with the success and management of patients undergoing head and neck surgery Tang demonstrated that coagulopathy was more common in patients with severe disease and nonsurviving COVID patients52 In this study Ddimer fibrin | Thyroid_Cancer |
Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptorKevin M Tveter1 Jose A Villa Rodriguez Alrick J Cabales1 Li Zhang2 Fiona G Bawagan1 Rocio M Duran1 Diana E Roopchand To cite Tveter a0KM Villa Rodriguez a0JA Cabales a0AJ et a0al Polyphenol induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptor BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 º Additional material is published online only To view please visit the journal online http dx bmjdrc KMT and JAV R contributed equallyReceived March Revised June Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Food Science Rutgers The State University of New Jersey New Brunswick New Jersey USA2Key Laboratory of Genomic and Precision Medicine Beijing Institute of Genomics Chinese Academy of Sciences Beijing Branch Beijing ChinaCorrespondence toDr Diana E Roopchand roopchand sebs rutgers eduIntroduction Bile acid BA biotransformation by gut bacteria impacts BA profile and signaling to nuclear receptors such as the farnesoid X receptor FXR regulating glucose metabolism Altered BA FXR signaling was therefore investigated as a potential mechanism linking polyphenol induced gut bacterial changes and improved glucose metabolismResearch design and methods Diabetic dbdb were fed low fat diet LFD or LFD supplemented with a proanthocyanidin rich extract of grape polyphenols LFD GP for weeks Metabolic phenotypes serum BAs gut microbiota composition and gene expression markers relevant to gut barrier and glucose metabolism were assessed Gut anoids were used to investigate effects of individual BAs on ileal FXR activityResults Compared with LFD fed controls GP supplemented dbdb mice showed improved glucose metabolism decreased relative abundance of gut bacteria associated with production of secondary BAs SBAs and depleted serum levels of SBAs taurohyodeoxycholic acid THDCA Ïmuricholic acid ÏMCA and tauroÏmuricholic acid TÏMCA Serum levels of primary BAs PBAs increased consistent with higher gene expression of PBA synthesis enzyme Cyp7a1 GP induced BA changes associated with FXR inhibition as evidenced by reduced expression of FXR responsive genes Shp Fgf15 and Fabp6 in ileum tissue as well as hepatic Shp which negatively regulates PBA synthesis GP treatment did not affect expression of hepatic Fxr or expression of Abcb11 Slc51b and Obp2a genes controlling BA transport Ceramide biosynthesis genes Smpd3 Sptlc2 and Cers4 were decreased in liver and intestine suggesting lower tissue ceramides levels may contribute to improved glucose metabolism THDCA ÏMCA and TÏMCA behaved as FXR agonists in ileal anoid experiments therefore their depletion in serum of GP supplemented dbdb and wild type WT mice was consistent with FXR inhibitionConclusion These data suggest that by altering the gut microbiota GPs modify BA FXR signaling pathways to promote glucoregulationINTRODUCTIONDietary polyphenols in plant based foods contribute to improved glycemic control in Significance of this studyWhat is already known about this subject º Dietary polyphenols such as proanthocyanidins alter the gut microbial community and are associated with improved metabolic resilience in humans and mice º Bile acids BAs signal to farnesoid X receptor FXR a nuclear transcription factor that regulates hepatic BA synthesis and glucose metabolismWhat are the new findings º Grape polyphenol GP supplementation decreased abundances of gut bacterial taxa associated with secondary BA SBA production concomitant with depletion of SBAs and increased primary BAs PBAs in murine serum º GP supplementation suppressed expression of FXR regulated genes Fgf15 Fabp6 and Shp an inhibitor of PBA synthesis which was consistent with increased serum PBAs º GP induced FXR inhibition was associated with decreased expression of genes required for biosynthesis of ceramides which impair glucose homeostasis º The SBAs depleted in GP treated mice were revealed as FXR agonists in ileal gut anoidsHow might these results change the focus of research or clinical practice º This study highlights BA FXR signaling pathways as an important mechanism for further investigation in human intervention studies of dietary polyphenols and metabolic healthmice and humans1 Improved glucose metabolism in mice supplemented with berryfruit extracts was related to a proanthocyanidin PAC induced bloom of Akkermansia muciniphila1 a microbe shown to attenuate symptoms of metabolic syndrome MetS and type2 diabetes T2D in obese mice and humans5 We hypothesized that metabolic improvements also result from changes in BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismhost derived bacterial metabolites regulating host energy metabolismBile acids BAs are signaling molecules linking the gut microbiota to host energy metabolism Primary BAs PBAs synthesized in the liver are conjugated with taurine mice or glycine humans7 Bacterial derived bile salt hydrolases deconjugate taurine or glycine from the sterol core of PBAs followed by bacterial transformations such as αdehydroxylation dehydrogenation and epimerization which generate secondary BAs SBAs7 PBAs and SBAs signal to key regulators of energy metabolism such as nuclear transcription factor farnesoid X receptor FXR and Takeda G protein coupled receptor TGR57 Fxr mice were protected from high fat diet HFD induced obesity and had altered BA and gut microbiota higher Bacteroidetes less Firmicutes profiles compared with wild type WT mice8 Transfer of cecal microbiota from HFD fed Fxr mice to germ free WT mice resulted in less adiposity and improved glucose metabolism compared with mice that received microbiota from HFD fed WT mice suggesting the altered gut microbial and BA profiles in FXR deficient mice contributed to metabolic improvements8 Studies using tissue specific FXR knockout mice showed that intestinal FXR activity was required to mediate HFD induced metabolic dysfunctions9 Pharmacological inhibition of intestinal FXR using tempol antibiotics metformin or glycinemuricholic acid Gly MCA led to gut microbial remodeling and improved glucose and lipid homeostasis9In addition to the A muciniphila bloom mice fed HFD supplemented with PAC rich grape polyphenols GPs showed other profound bacterial community changes raising the possibility that altered BA profile and signaling could contribute to observed improvement in glucose homeostasis1 The present study provides compelling evidence in support of this hypothesisRESEARCH DESIGN AND METHODSDietsA complex of grape polyphenols and soy protein isolate GP SPI was prepared as previously described1 Nutritional profiles for SPI and GP SPI are provided in online supplementary table Isocaloric ingredient matched diets Research Diets New Brunswick New Jersey USA used in this study were previously described4 Mice were fed low fat diet LFD containing SPI LFD formulated with GP SPI delivering GP LFD GP HFD containing SPI or HFD formulated with GP SPI delivering GP HFD GP Online supplementary table provides diet formulation detailsAnimalsFour week old dbdb mice B6BKSD LeprdbJ stock no Jackson Laboratory Bar Harbor Maine USA were single housed on a hour lightdark cycle to hours light with ad libitum access to LFD and water in a controlled temperature room °C±°C for week for acclimation Mice were randomized to receive LFD n7 or LFD GP n7 for days Metabolic phenotyping included food intake body weight body composition EchoMRI in1 system Echo Medical Systems Houston Texas USA and oral glucose tolerance tests as previously described1 On day mice were euthanized by CO2 inhalation followed by cardiac puncture and collection of tissues as previously described1 Fecal and cecal samples were collected for microbial community profiling Wild type C57BL6J mice n10 aged weeks were acclimated on LFD for week and randomly divided into two groups and fed HFD or HFD GP n5 per group for weeks after which mice were euthanized by CO2 inhalationTissue gene expression analysisRNA was extracted from ileum jejunum colon and liver mg with RNeasy Plus Universal Mini Kit QIAGEN followed by RNA cleanup Machery Nagel RNA purification kit RNA µg was reverse transcribed to cDNA and qPCR was performed using TaqMan primers online supplementary table as previously described4 Data were analyzed using 2ÎCT method using hydroxymethylbilane synthase HMBS as housekeeping geneqPCR of A muciniphila in fecal and cecal samples was performed as previously described1 Briefly gDNA extracted from fecalcecal samples was diluted to ngµL for quantification of A muciniphila abundance relative to total bacteria and archaea by qPCR using A muciniphila AM1 AM2 and universal primer U341F U515R sets1 16S rRNA gene sequencingGenomic DNA was extracted from fecal and cecal samples collected from dbdb mice Illumina protocols were used to prepare V4 amplicons of the 16S rRNA gene for sequencing on a MiSeq system resulting in à reads Denoising and clustering were conducted using DADA2 algorithm to differentiate sequences into amplicon sequence variants ASVs for downstream analysis using QIIME Details are available in online supplementary materialsSerum biochemistrySerum for BA analysis was collected by cardiac puncture BAs were analyzed on a Waters Alliance e2695 HPLC system Waters Milford Massachusetts USA coupled to a Waters Acquity QDA mass spectrometer equipped with an electrospray interphase ESI Waters Milford Massachusetts USA and quantified by external calibration curves using pure standards Details are provided in online supplementary materials Serum leptin polypeptide YY PYY interleukin6 IL6 and insulin were determined using a MILLIPLEX MAP Mouse Metabolic Hormone Magnetic Bead kit Millipore with a MagPix instrument Luminex as previously described4anoid experimentsIntestinal crypts were isolated from WT C57BL6J mouse ileum according to established methods16 Crypts were counted and added to Matrigel five crypts per µL BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cCorning growth factor reduced and µL was added per well in well plates and allowed to polymerize °C for min followed by addition of mL of complete growth medium CGM see online supplementary materials CGM without Y27632 dihydrochloride monohydrate was replaced every days anoids were passaged every days ratio Mature anoids days postpassage were treated in triplicate n3 wells with methanol vehicle chenodeoxycholic acid CDCA µM alone or CDCA plus another BA µM in CGM for hours RNA was extracted for qPCR analysis using TaqMan primers as described above4Statistical analysesAnalyses were conducted using Prism GraphPad Software La Jolla California USA Significant differences two groups were assessed with a two tailed unpaired Students t test with Welch correction for unequal variance when needed or by one way or two way analysis of variance groups followed by Sidaks or Tukeys multiple comparison test Statistical analysis of alpha and beta diversity metrics was calculated using QIIME details in online supplementary materials ADONIS and permutation analysis were conducted using R Studio V342 R Studio Software Boston Massachusetts USA and Python RESULTSGPs improve glucose metabolism in dbdb mice independent of obesityLeptin receptor deficient dbdb mice develop obesity gut barrier dysfunction and hyperglycemia independent of HFD feeding17 Compared with LFD fed controls dbdb mice fed LFD containing PAC rich GPs LFD GP for weeks showed significantly improved oral glucose tolerance figure 1A4 Area under the curve remained stable over time for the LFD GP group but increased in the LFD group figure 1A Mice fed LFD GP initially exhibited a transient decrease in food intake presumably due to taste but at later time points LFD and LFD GP groups consumed similar amounts of food ± and ± gdaymouse respectively p005 online supplementary figure 1A The LFD GP group consumed ± mg of GPs per day Both groups had similar body weight gain body composition and liver weights online supplementary figure 1BDGPs promote a bloom in A muciniphila without improving markers of metabolic endotoxemiaCompared with controls GP supplemented dbdb mice had decreased αdiversity as evidenced by richness Shannon index and Faiths phylogenetic diversity index figure 2A Principal coordinate analysis showed that GPs significantly altered fecal community structure within days online supplementary figure As previously observed1 GPs promoted increased cecal mass online supplementary figure Metabolism1E a phenotype common to antibiotic treated and germ free mice and consistent with reported antibacterial properties of PACs18 GP supplemented mice had an increased relative abundance of phylum Verrucomicrobia at the expense of Firmicutes in fecal days and ceca samples day figure 2BC online supplementary figure 3AB Ceca of GP supplemented mice had higher relative abundance of Bacteroidetes 25LFD although except for day fecal Bacteroidetes remained similar between groups figure 2B online supplementary figure 3C GP supplementation did not consistently alter levels of Proteobacteria or Actinobacteria figure 2B online supplementary figure 3DE Consistent with increased Verrucomicrobia quantitative qPCR analyses confirmed that GPs promoted a bloom in Akkermansia muciniphila figure 2C at the expense of other taxa figure 2DReduced abundance of A muciniphila and metabolic endotoxemia characterized by gut dysbiosis compromised gut barrier integrity lipopolysaccharide LPS leakage and intestinal inflammation was associated with impaired glucose metabolism in obese mice and humans5 Oral administration of A muciniphila in obese diabetic mice and humans resulted in improved glucose homoeostasis and attenuated metabolic endotoxemia through improved gut barrier integrity5 GP induced improvement in glucose metabolism in HFD fed mice has therefore been considered a consequence of the A muciniphila bloom leading to reduced inflammation and increased gut barrier integrity1 Reduced metabolic endotoxemia could not however explain the improved glucose tolerance in GP supplemented dbdb mice despite increased A muciniphila in feces day and cecum figure 2C Relative to control GP supplementation did not change intestinal gene expression of markers of inflammation Tnf Il6 iNOS gut barrier integrity Tjp1 Ocln Muc2 peripheral lipid deposition Fiaf or glucose transport Glut2 online supplementary figure 4AC GP supplemented dbdb mice had less Muc3 expression in jejunum and ileum online supplementary figure 4AB suggesting lower mucus secretion There were no differences in serum insulin IL6 or glucoregulatory hormones PYY and leptin online supplementary table These data suggested that other mechanisms were driving improved glucose metabolismGPs reduced gut bacterial taxa associated with production of SBAsAlthough total bacterial and archaeal abundance was not significantly different between LFD and LFD GP groups figure 2E GPs induced profound gut microbial changes online supplementary figure that would be expected to alter BA diversity abundance and signaling Targeted liquid chromatography mass spectrometry LC MS analysis revealed that GP supplemented dbdb mice had higher serum concentrations of BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs improve glycemic control in obese dbdb mice A Oral glucose tolerance tests were performed after mice consumed LFD squares n7 or LFD GP closed squares n7 for and weeks Blood glucose concentrations mgdL expressed as mean±SD were measured at the indicated time points min following administration of glucose gkg Between group difference was determined by unpaired two tailed t test with Welchs correction p01 p001 B Scatter plot of blood glucose AUC determined for individual mice at weeks and where mean AUC±SD are shown as horizontal and vertical bars Difference was determined using two way ANOVA followed by Tukeys intragroup posthoc test different letters indicate significant difference p005 or by Sidaks intergroup posthoc test p001 ANOVA analysis of variance AUC area under the curve GP grape polyphenol LFD low fat dietPBAs driven by increased cholic acid CA and taurocholic acid TCA figure 3A and C online supplementary figure 6A Concentrations of muricholic acid MCA tauroMCA TMCA tauroαMCA TαMCA taurochenodeoxycholic acid TCDCA were similar between groups αMCA was not detected in LFD GP group figure 3A online supplementary figure 6A Increased PBA pool correlated with an overall reduction in SBAs figure 3C where ÏMCA TÏMCA and taurohyodeoxycholic acid THDCA were undetectable in dbdb mice fed LFD GP although deoxycholic acid DCA was increased figure 3A Total serum BAs were similar between groups figure 3BThe GP induced depletion of SBAs was unrelated to the leptin receptor mutation in dbdb mice Compared with HFD fed controls wild type C57BL6J mice fed HFD supplemented with GP HFD GP for weeks also showed serum depletion of SBAs THDCA ÏMCA and TÏMCA reduced overall levels of serum SBAs and no difference in concentration of total serum BAs online supplementary figures 6B and Unlike dbdb mice GP supplemented WT mice had decreased tauro deoxycholic acid TDCA and no significant difference in CA or overall PBA pool although TCA concentration was increased online supplementary figures 6B and In dbdb mice multiple correlation analyses were performed to associate GP induced changes in serum BAs figure to changes in fecalcecal gut bacteria online supplementary figure The GP induced increase of CA TCA PBAs and DCA SBA was positively and significantly associated with increased abundance of Akkermansia Blautia Clostridium ASV59 and S247 figure 3EF Blautia and Clostridium possess BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Supplementation of GPs remodeled gut microbial composition and diversity A Microbial αdiversity metrics of fecal samples collected from mice after consuming LFD squares n7 or LFD GP closed squares n7 for indicated number of days and of cecal Cec samples collected at endpoint day Difference was determined by two way ANOVA followed by Sidaks test B Per cent relative abundance of main bacterial phyla based on Naïve Bayes taxonomic classifier non rarified data Phyla present at relative abundance were classified as Other Percentage of C A muciniphila DNA and D nonA muciniphila DNA relative to total bacterial and archaeal DNA in fecal and cecal samples where relative abundance was determined by qPCR using primers specific for A muciniphila AM1AM2 and universal V4 primers 515F806R E Total 16S bacterial and archaeal gene countsng of gDNA extracted from fecal or cecal samples Group mean±SD at each time point are illustrated by horizontal and vertical lines Difference between diet groups over time panels CE was determined using two way ANOVA followed by Sidaks posthoc test intergroup comparison or Tukeys posthoc test intragroup comparison Different letters a b and c indicate significant difference within diet groups p005 while the same letter indicates no difference Between group differences panels A C D E p005 p001 p0001 p00001 ANOVA analysis of variance ASV amplicon sequence variants AUC area under the curve GP grape polyphenol LFD low fat diet PD phylogenetic diversity7αdehydroxylating activity therefore taxa within these genera may be responsible for DCA production via dehydroxylation of CA21 GP treated mice had lower levels of taxa within the Clostridiales order ASV50 and Ruminococcaceae and Lachnospiraceae families ie ASV56 and and Clostridium genus ASV74 online supplementary figure which are reported to possess 7αdehydroxylating activity required for conversion of PBAs to SBAs21 In agreement with evidence from mice and humans the GP induced decrease in these bacterial taxa encoding 7αdehydroxylation activity was highly correlated to the GP associated depletion of SBAs ÏMCA and TÏMCA figure 3EF21 Finally positive and significant associations were found between reduced αMCA TÏMCA andor ÏMCA and reductions in taxa belonging to RF39 Anaeroplasma Ruminococcus Butyricicoccus Dorea Dehalobacterium Christensenellaceae Lactococcus Streptococcus and Oscillospira figure 3EFGPassociated BA changes promote inhibition of FXR signaling and upregulation of classical BA synthesis pathwayTo investigate the consequences of GP induced serum BA changes gene expression of FXR TGR5 and their downstream targets were analyzed in tissues GP supplementation did not change ileal Fxr gene expression however FXR transcriptional activity was decreased as expression of its target genes fibroblast growth factor Fgf15 small heterodimer partner Shp and ileal BA binding protein I BABP gene Fabp6 were suppressed in ileum figure 4A Intestinal FXR signaling negatively regulates hepatic PBA synthesis through interaction of Fgf15 with hepatic fibroblast growth factor receptor 4Klotho receptor complex or by regulating BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure GPs increased PBAs and reduced SBAs in serum in association with depletion of bacterial ASVs related to SBA production A LC MS and pure standards were used to determine the mean concentration of individual PBAs and SBAs mean±SD in serum samples n7 samples collected from individual mice fed LFD white bars or LFD GP purple bars B Total serum BA concentration mean±SD was determined based on sum of individual PBA and SBA concentrations shown in panel A quantified for each mouse fed LFD n7 or LFD GP n7 C Serum PBA and SBA concentrations mean±SD in LFD versus LFD GP diet groups were calculated by summing the individual PBAs or SBAs shown in panel A D Using data from panels B and C pie charts illustrate pooled PBAs green and pooled SBA gray as a percentage of total serum BA concentration quantified for LFD and LFD GP groups For panels AC significant difference was determined using unpaired two tailed t test followed by Welchs correction p005 p001 Heatmap representation of the Spearmans r correlation coefï¬cient between serum BA profile and significantly changed bacterial ASVs at genera or family level of taxonomy in GP treated mice relative to control diet group from E day27 fecal samples or F day29 cecal samples Shades of red indicate serum BA and bacterial taxa are positively correlated to while shades of blue indicate a negative correlation to Significant positive or negative correlations are shown p005 p001 p0001 ASV amplicon sequence variant CA cholic acid DCA deoxycholic acid GP grape polyphenol LC MS liquid chromatography mass spectrometry LFD low fat diet MCA muricholic acid PBA primary bile acid SBA secondary bile acid TCA taurocholic acid TCDCA taurochenodeoxycholic acid TDCA tauro deoxycholic acid THCDA taurohyodeoxycholic acid TαMCA tauroαMCA TMCA tauroMCA TÏMCA tauroÏMCA TUDCA tauro ursodeoxycholic acidBMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Expression of genes involved in FXR signaling ceramide synthesis and glucose metabolism in response to GP supplementation Scatter plot of relative mRNA levels of indicated genes expressed in A ileum and B liver tissues collected from individual mice fed LFD squares or LFD GP closed squares Group mean±SD n7 samples group is illustrated by horizontal and vertical lines Data represent qPCR of technical duplicates analyzed by ÎCT method Between group difference was determined by unpaired two tailed t test with or without Welchs correction for unequal variance p005 p001 p0001 FXR farnesoid X receptor GP grape polyphenol LFD low fat dietthe hepatic expression and gene repressive function of Shp via liver receptor homologue1 and hepatocyte nuclear factor4α26In agreement with reduced ileal Fgf15 hepatic Shp expression was significantly decreased figure 4B Fgf15 and Shp negatively regulate BA synthesis therefore their reduced expression was consistent with hepatic upregulation of cytochrome P450 family subfamily A member Cyp7a1 the rate limiting enzyme in the classical BA synthesis pathway7 and a trending increase in downstream enzyme Cyp8b1 p006 figure 4B Gene expressions of Cyp27a1 and Cyp7b1 online BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismsupplementary figure 4E involved in the alternative BA synthesis pathway were unaffected by GP treatment To determine if reduced hepatic Shp expression altered hepatic FXR activity we examined FXR target genes Abcb11 encoding bile salt export pump Bsep Slc51b encoding anic solute transporter Ost30 and Obp2a encoding lipocalin Lpn13 an acute phase protein32 Compared with control GP treatment did not affect hepatic expression of Fxr Abcb11 Slc51b or Obp2a figure 4B These data suggest that GP mediated effects on FXR activity are restricted to intestine resulting in suppression of ileal Fgf15 and Shp transcription and increased Cyp7a1 activity and PBA synthesis figure FXR activity regulates Tgr5 expression therefore we measured Tgr5 mRNA levels and downstream targets33 On activation by BAs TGR5 signals the release of incretin glucagon like peptide1 Glp1 to promote euglycemia33 Glp1 is produced when proglucagon protein Gcg is cleaved by prohormone convertase PC13 encoded by Pcsk134 GP supplemented mice showed reduced ileal expression of Tgr5 and no changes in Gcg or Pcsk1 figure 4A GPs did not change expression of Fxr Fgf15 or Tgr5 in colon tissue GPs induced an increase in colonic Gcg however Pcsk1 was unchanged indicating Glp1 levels were unaffected online supplementary figure 4D These data further support the idea that GPs inhibit ileal FXRGPmediated FXR inhibition is associated with downregulation of ceramide synthesis genes and improved markers of hepatic energy metabolismTissue accumulation of ceramides is linked to insulin resistance and diabetes which can be ameliorated by pharmacological or genetic inhibition of ceramide biosynthesis35 FXR activity positively upregulates genes required for ceramide synthesis in ileum which leads to impaired glucose metabolism and hepatic steatosis in mouse models of MetST2D10 Synthesis and accumulation of ceramides in liver contributes to hepatic insulin resistance steatohepatitis and metabolic disease35 We found that GP induced FXR inhibition was associated with lower expression of de novo ceramide synthesis genes specifically sphingomyelin phosphodiesterase Smpd3 in ileum figure 4A and serine palmitoyltransferase long chain base subunit Sptlc2 and ceramide synthase Cers4 in liver figure 4B Consistent with lower expression of ceramide biosynthesis genes GP supplemented mice showed improvements in markers of hepatic energy metabolism evidenced by lower hepatic expression of carbohydrate response element binding protein Chrebp a transcription factor that activates key enzymes of de novo lipogenesis38 glucose phosphatase G6Pase which catalyzes the final step in hepatic glucose production and Idh3a a subunit of the IDH3 isocitrate dehydrogenase heterotetramer complex that regulates fatty acid metabolism and whose inhibition is associated with hepatic glycogen synthesis figure 4B39 GP supplemented mice had reduced expression of Lbp encoding LPS binding protein suggesting decreased liver inflammation and insulin resistance40 Hepatic gene expression of gluconeogenesis enzyme phosphoenolpyruvate carboxykinase Pck1 and CEBP homologous protein Chop which are normally upregulated during hepatic endoplasmic reticulum stress41 were similar between groups online supplementary figure 4ESBAs depleted in GPsupplemented mice are FXR agonists that promote expression of ceramide synthesis genesThe majority of secreted BAs are reabsorbed in ileum and returned to the liver via the portal vein7 Cecum colon and feces have similar BA profiles while the serum BA profile is most closely related to that of ileum and portal vein as a minor fraction of reabsorbed BAs enter systemic circulation25 We sought to investigate how individual BAs altered by GP supplementation might affect ileal FXR signaling Gut anoids were cultured from ileal crypts isolated from WT mice figure 5A and treated with individual PBAs or SBAs that were differentially detected in serum of GP supplemented mice figure 5B To validate the system anoids were treated with CDCA a potent FXR agonist or CDCA in combination with the FXR antagonist TMCA As expected CDCA increased the expression of Fxr Fgf15 and Shp compared with vehicle treated anoids and this effect was attenuated by addition of TMCA figure 5B When anoids were treated with individual BA alone ie µM in the absence of CDCA activator FXR activity was unaffected as Fxr Fgf15 and Shp mRNA expression remained similar to untreated anoids online supplementary figure We therefore investigated individual BAs in the presence of FXR activator CDCA as previously reported11 to reveal agonistic or antagonistic effects on FXR signaling anoid cultures treated with CDCA in combination with ÏMCA TÏMCA THDCA or DCA increased CDCA induced expression of Fxr Fgf15 and Shp indicating that these SBAs are FXR agonists figure 5B In agreement with FXR agonistic activity anoids treated with CDCA in combination with TÏMCA resulted in increased expression of Smpd3 Cers4 and Sptlc2 ceramide synthesis genes Cotreatment with CDCA and ÏMCA THDCA or DCA only upregulated Sptlc2 figure 5B CA a PBA reported to be a weak FXR agonist and detected at higher concentration in GP supplemented dbdb but not WT mice increased CDCA induced activation of Fxr Fgf15 and Shp and increased expression of Cers4 and Sptlc243 TCA a PBA detected at higher concentration in GP supplemented dbdb and WT mice attenuated CDCA induced gene expression of Fxr and Fgf15 but not Shp and reduced CDCA induced gene expression of Cers4 figure 5B These anoid data suggest that GPs led to the depletion of FXR activators ÏMCA TÏMCA and THDCA and increase of an FXR antagonist TCA Consistent with in vivo data the net effect BMJ Diab Res Care 20208e001386 101136bmjdrc2020001386 0cMetabolismFigure Ileal anoids treated with BAs revealed agonistic or antagonistic effects on FXR and ceramide pathway genes A Ileal crypts were isolated and cultured in Matrigel medium detailed in methods Spheroids representative day photo matured into anoids representative day photo B Scatter plot of relative mRNA levels of indicated genes expressed in anoids after hours of treatment with vehicle methanol black diamonds µM CDCA closed red circles a combination of µM CDCA and known FXR inhibitor TMCA µM a combination of µM CDCA and indicated PBA µM red circles a combination of µM CDCA and indicated SBA µM blue squares Data shown were combined from two independent experiments and for each experiment three wells containing mature anoids were treated with indicated BAs Data represent qPCR of technical duplicates analyzed by ÎCT method Group mean±SD n6 wells total per treatment group is illustrated by horizont | Thyroid_Cancer |
"Cardiac arrhythmias Atrial ï¬brillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular ï¬brillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are aï¬icted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial ï¬brillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular ï¬brillation VPCs ventricular premature complexes VT ventricular tachycardia Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial ï¬brillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with conï¬rmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular ï¬brillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial ï¬brillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular ï¬brillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insuï¬ciencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular ï¬bril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial ï¬brillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial ï¬brillation According to a recent survey of electrophysiology professionals atrial ï¬brillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using speciï¬c AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the ï¬rst months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insuï¬ciency and the systemic am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients aï¬icted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with conï¬rmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the ï¬rst documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is signiï¬cantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the ï¬nal weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained signiï¬cantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as deï¬nite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and ï¬uoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary ï¬ndings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpausedependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeï¬ciency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus ï¬ngolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides ï¬uoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to signiï¬cant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs niï¬cant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ¥ ms Changes in ¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ¥ ms or QTc increase of No patients manifested TdP ¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signiï¬ cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no signiï¬cant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identiï¬ed only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of signiï¬cant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this speciï¬c population HCQAZM could not be initiated or had to be interrupted in ¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the ï¬rst manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or forme fruste of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19speciï¬c risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use of various formulas among which the Bazetts correction formula is most commonly used QTc QT RRsec QTc is deï¬ned as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTcQRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F | Thyroid_Cancer |
RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomens Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the seed and soil theory was ï¬rst proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells seeds leave their primary site of origin and spreador metastasize the microenvironment soil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve assoil for tumor development and might also serve as a seed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on theï¬eld of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identiï¬ed receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieï¬yintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and eï¬cienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentiï¬ed as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong TDC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deï¬ciency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKLRANK interactionsand blocking bone resorption The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identiï¬edas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a speciï¬c fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The eï¬cacy of Denosumab has been conï¬rmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspeciï¬c Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeï¬cient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyers plaque structures Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeï¬cient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inï¬ammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL speciï¬cally in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinï¬ammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a signiï¬cant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespeciï¬c deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspeciï¬c rankldeï¬cient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deï¬ciency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a speciï¬c antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to speciï¬cally eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the ï¬rst to reportthat during pregnancy RANKL deï¬ciency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκBcyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and ampliï¬edproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also signiï¬cantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identiï¬ed SNPs which were signiï¬cantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in signiï¬cantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Ampliï¬cation of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also signiï¬cantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which speciï¬cally inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will beneï¬t notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was signiï¬cantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no signiï¬cant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinï¬ltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand signiï¬cantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports ï¬ndings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thismicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas ï¬broblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a vicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs ï¬broblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer prostate cancer and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANKs cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinï¬ltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are signiï¬cantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a signiï¬cantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassigniï¬cantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs Moreover the beneï¬t of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the vicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a signiï¬cantimprovement in overall survival In these patients nostatistically signiï¬cant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab signiï¬cantly improved progressionfree survivalPFS Whether this survival beneï¬t is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the ï¬rst clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a signiï¬cant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ··] p A median followup of months showeda signiï¬cant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI Cox p These data suggest that adjuvant Denosumabcan signiï¬cantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratiï¬cations eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial conï¬rmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso signiï¬cantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment signiï¬cantly reduced the time to bonemetastasis at the site of ï¬rst occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe ï¬eld of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the bodys own immunesystem to seek out speciï¬cally target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as | Thyroid_Cancer |
"Postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in COVID19 patientsMethods We evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans Complete macroscopic and microscopicautopsies were performed on different ans in COVID19 nonsurvivors Presence of SARSCoV2 was evaluatedwith immunohistochemistry IHC in lung samples and with realtime reversetranscription polymerase chainreaction RTPCR test in the lung and other ansResults Pulmonary findings revealed earlystage diffuse alveolar damage DAD in out of patients andmicrothrombi in small lung arteries in patients Latestage DAD atypical pneumocytes andor acute pneumoniawere also observed Four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedThere was no evidence of myocarditis hepatitis or encephalitis Kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients Spongiosis and vascular congestion were the mostfrequently encountered brain lesions No specific SARSCoV2 lesions were observed in any an IHC revealedpositive cells with a heterogeneous distribution in the lungs of of the patients RTPCR yielded a widedistribution of SARSCoV2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brainConclusions In autopsies revealed a great heterogeneity of COVID19associated an injury and theremarkable absence of any specific viral lesions even when RTPCR identified the presence of the virus in many ansKeywords COVID19 SARSCoV2 Autopsy RTPCR Immunohistochemistry Correspondence IsabelleSalmonerasmeulbacbe1Department of Pathology Erasme Hospital Universit Libre de BruxellesULB Route de Lennik Brussels Belgium2Centre Universitaire inter Rgional dexpertise en Anatomie PathologiqueHospitali¨re CurePath CHIREC CHU Tivoli ULB Rue de Borfilet 12A Jumet BelgiumFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cRemmelink Critical Care Page of severeacuteincludingBackgroundCoronavirusesrespiratorysyndrome coronavirus SARSCoV and Middle Eastrespiratory syndrome coronavirus MERSCoV causesevere acute respiratory failure which is associated withhigh mortality rates [] The novel SARSCoV2 strainexhibits phylogenetic similaritiesto SARSCoV andcauses coronavirus disease COVID19 which hasresulted in more than deaths worldwide so farAs the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that SARSCoV2 can directly alter cellfunction by a link to the angiotensin converting enzyme ACE2 receptor which is almost ubiquitous in thehuman body []Nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with COVID19remain poorly understood Controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease []In this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of COVID19 Most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] The aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from COVID19MethodsStudy designIn this postmortem study we included the first adultpatients years who died in our hospital either in aCOVID19 unit or an intensive care unit from March with confirmed SARSCoV2 infection ie positiveRTPCR assay on nasopharyngeal swab andor bronchoalveolar lavage specimen Exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination The study protocol wasapproved by the local ethics committee P2020218Data collectionrelevantWe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport Acute respiratory distress syndrome ARDSand acute kidney injury AKI were defined accordingto standard definitions [ ]Postmortem procedureThe Belgian Public Health Institute Sciensano guidelines were integrated into our postmortem procedure[] The cadavers were kept in the refrigerator at °Cand autopsies were performed to h after death toensure the safety of the autopsy team Personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves FFP3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots In the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationAll analyses were performed at normal pressurespleen bone marrow kidney bladderUsing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking The material was biobanked by BiobanqueH´pital ErasmeULB BE_BERA1 CUB H´pital ErasmeBBMRIERIC The banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain For the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken For safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe Additional file Additional Material Formalinfixed paraffinembedded FFPEtissues underwentstandard processing to provide hematoxylin and eosinHEstained sections Special stains and immunohistochemistry IHC were used for lung Massons trichromeperiodic acidSchiff [PAS] GomoriGrocott antiCMVIHC antiHSV IHC antiPneumocystis J IHC and kidneyPAS Massons trichromeJones methenamine silversamplesMorphological analysisMorphological analysis was performed on HE stainedglass slides using the SecundOs digital platform TribVnHealth Care Chatillon Francefor digital diagnosisafter the acquisition of whole slide digital scans magnification using a Nanozoomer HT slide scanner Hamamatsu Hamamatsu City JapanSARSCoV2 detection by immunohistochemistrySince no antibody against SARSCoV2 has been validatedfor IHC on FFPE tissues we selected an antiSARSnucleocapsid protein antibody Standard IHC was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display SARSnucleocapsid protein Invitrogen PA141098dilution on Dako Omnis Agilent Technologies 0cRemmelink Critical Care Page of Santa Clara CA USA using the Envision Flexdetection system according to the manufacturersprotocol [] The sections were counterstained withhematoxylin Negative tissue controls were obtainedfrom patients who had an autopsy before the COVID pandemic Semiquantitative IHC evaluation wasperformed by two senior pathologists ND MR as follows negative between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one field SARSCoV2 detection by rRTPCRTotal nucleic acid was extracted from FFPE tissues usingthe Maxwell RSC DNA FFPE Kit reference AS1450Promega Corporation Madison WI USA and thePromega Maxwell extractor following the protocol described by the manufacturer Onestep RTPCR assaysspecific for the amplification of SARSCoV2 E envelopeprotein gene were adapted from a published protocol[] Briefly μL of RNA ng was amplified in μL reaction mixture containing μL of TaqMan FastVirus 1step master mix Life Technologies μM ofeach forward ACAGGTACGTTAATAGTTAATAGCGT and reverse ATATTGCAGCAGTACGCACACAprimers and μM of probe FAMACACTAGCCATCCTTACTGCGCTTCGBBQ The amplification condition was °C for min for reverse transcriptionfollowed by °C for s and then cycles of °C for s and °C for s A clinical sample highly positivefor SARSCoV2 was diluted and used as a positive control in each analysis A clinical sample obtainedfrom a patient who was autopsied before the COVID19pandemic was used as a negative control The quality ofthe RNA from the samples showing negative results wasassessed by amplification of the human MET RNA according to a validated ISO15189 accredited methodused as a routine diagnostic method in our laboratoryStatistical analysisData are reported as counts percentage or medians[interquartile ranges IQRs] All data were analyzedusing GraphPad Prism Version GraphPad Software San Diego CA USAResultsStudy cohortThe main characteristics of the study cohort malesout of median age [] years are given inTable The time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days All except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n None ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza A and B virusesEleven of the patients were treated with mechanicalventilation Eleven patients died in the ICU and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n Laboratory data are reported in Additional file Table S1Macroscopic findingsOne patient had had a left pneumonectomy and onepatient a right bilobectomy The lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage Thrombi were found in the large pulmonary arteries in patients and lung infarction in patientsPleural adhesions associated with pleural effusions were observed in cases We observed cardiomegaly in and hepatomegaly in patients The kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct The gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis In the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhageMicroscopic findingsfile As shown in Figs and and AdditionalTable S2the main pulmonary findings includedearlystage diffuse alveolar damage DAD which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryII pneumocytehyperplasia Microthrombi were noted in the smallpulmonary arteries in patients Ten of the patientsalso had advanced DAD lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells We observed no viralinclusions or squamous metaplasiaand typeinfiltrateAll the patients who survived more than weeksn had late DAD lesions There was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct In of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi The other patients had longer 0cRemmelink Critical Care Page of Table Characteristics of the study populationIDCT scanComorbiditiesAgeSexrRTPCRPOSTime todeathAntemorteman failureARDSAKIPOSNEGNEGMFMFMCADCVDDiabetesHypertensionCADCRFLiver cirrhosisCOPDCancerHypertensionCancerCVDCOPDCancerGGOPOSMAGGOPOSPOSTreatmentsCause of deathMechanicalventilationAntibioticsHydroxychloroquineAntibioticsCorticosteroidsMechanical ventilationHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationHydroxychloroquineAntibioticsMechanical ventilationECMORRTHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationECMOHydroxychloroquineRemdesivirCorticosteroidsAntiobioticsHydroxychloroquineAntibioticsCardiogenicshockMOFRespiratory failureRespiratory failureRespiratory failureMesentericischemiaMOFRespiratory failureRespiratory failureAntibioticsSeptic shockMOFMechanical ventilationRRTHydroxychloroquineLopinavirRitonavirAntibioticsMechanical ventilationECMORRTHydroxychloroquineOseltamivirAntibioticsHydroxychloroquineAntibioticsRespiratory failureRespiratory failureSudden deathMechanical ventilationHydroxychloroquineAntibioticsMOFHydroxychloroquineRespiratory failureHydroxychloroquineAntibioticsRespiratory failureARDSAKIHypoxichepatitisARDSAKIARDSARDSAKIHypoxichepatitisARDSAKIARDSAKIHypoxichepatitisAKIARDSAKIARDSAKIARDSARDSAKIHypoxichepatitisARDSARDSAKIHypoxichepatitisMHypertensionCRFBCPOSMNoneBCPOSMFHypertensionCADCVDCRFDiabetesHypertensionDiabetesEmphysemaPOSGGOPOSMHypertensionDiabetesGGOBCPOSMMMFDiabetesLiver cirrhosisCancerDiabetesHypertensionCADDiabetesDiabetesHypertensionDiabetesBCPOSGGOPOSGGOBCGGOBCPOSPOS 0cRemmelink Critical Care Page of Table Characteristics of the study population ContinuedIDComorbiditiesCT scanAgeSexMGGOLPPOSrRTPCRTime todeathFMHypertensionDiabetesLiver transplantHypertensionCVDGGOBCPOSGGOBCLPPOSAntemorteman failureARDSAKIPulmonaryembolismARDSAKIPulmonaryembolismARDSAKIPulmonaryembolismTreatmentsCause of deathMechanical ventilationRRTHydroxychloroquineRemdesivirAntibioticsMechanical ventilationRRTHydroxychloroquineAntibioticsMechanical ventilationECMORRTHydroxychloroquineAntibioticsSeptic shockMOFSeptic shockMOFSeptic shockMOFTime to death time from admission to death days Cause of death was reported by the attending physician M male F female rRTPCR reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test NEG negative POS positive CAD coronary artery disease CVD cerebrovascular disease LP lobarpneumonia GGO groundglass opacity MA minor abnormalities BC bilateral consolidation COPD chronic obstructive pulmonary disease CRF chronic renal failureARDS acute respiratory distress syndrome AKI acute kidney injury ECMO extracorporeal membrane oxygenation RRT renal replacement therapy MOF multiplean failuredelays between hospitalization and death daysthey had no microthrombiFifteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis Histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in In the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients Chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisFig Main histological findings Green finding present gray finding absent black unavailable 0cRemmelink Critical Care Page of Fig Pulmonary histological findings a Earlystage diffuse alveolar damage DAD hyaline membrane HE magnification with a zoom ona giant cell magnification b Fibrin thrombi in a pulmonary artery HE magnification c Latestage DAD fibroblastic proliferationHE magnification d Latestage DAD fibroblastic proliferation Trichrome staining magnification e Acute pneumonia HE magnification f AntiSARSCoV immunohistochemistry IHCpositive cells magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryLiver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate The histological changes in theabdominal ans including the esophagus stomachand colon are reported in Additional file Table S2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisBrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n We found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalSARSCoV2 detection in the lungs by IHCSARSCoV2 was identified by IHC in the lungs of ofthe patients Fig Howeverthere was largevariability in the distribution of SARSCoV2positivecells in the lung parenchymaSARSCoV2 detection by RTPCRSARSCoV2 RNA was detected in at least one anfrom every patient Fig In the lung RTPCR waspositive in patients with threshold cycle Ct valuesvarying from to Viral RNA was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples Ct valuesfor nonpulmonary ans ranged from to Eight patients had positive RTPCR in all tested ansabnormalitiesDiscussionThis postmortem study showed several histopathologicalin COVID19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though SARSCoV2 was detected in all examined ans using RTPCR We decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe This approach enabled us to 0cRemmelink Critical Care Page of Fig Detection of SARSCoV2 by immunohistochemistry IHC in FFPE post mortem lung samples of patients Semiquantitative evaluation negative result scattered positive cells between and positive cellswhole slide positive isolated cells cellswhole slide butno foci foci of positive cells more than positive cells in one field NA not availabledocument the considerable heterogeneity of histologicallesions and of SARSCoV2 spread through the bodyThe diagnosis of SARSCoV2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases In a previous autopsy study in COVID19patients [] the authors reported that DAD associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia No obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report [] Desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ARDS of many different causes especially inearlyphase ARDS [] The presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice As in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of SARSCoV2 infection [] Finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to SARSCoV2In the absence of typical postmortem viral featuresour results show that RTPCR is feasible on FFPE blocksand could be used in postmortem analyses to identifythe presence of SARSCoV2 in multiple ans and tounderstand the spread of the virus within the humanbody The discordant RTPCR and IHC results fordetection of SARSCoV2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the RTPCR whereas lowlevel viral replication might not be detected by IHC Moreover IHC wasbased on the only available antibodies which aretargeted against SARSCoV New antibodies againstSARSCoV2 need to be developed to improve theaccuracy of IHC in the analysis of tissue samples fromsuspected or confirmed COVID19 patientsMost of the previous postmortem studies in COVID19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofSARSCoV2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations As for previous coronavirusdiseases [ ] the lungs are the most affected ans inCOVID19 However DAD findings werehighly 0cRemmelink Critical Care Page of Fig Molecular detection of SARSCov2 RNA in postmortem samples Detection of SARSCoV2 by reverse transcription realtime polymerasechain reaction RTPCR in FFPE postmortem tissues of patients positive result negative result NA tissue not available NC noninformative test result due to lowquality RNAheterogeneous including both earlyonset and additionallate lesions This finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase [] As some patients died outsidethe ICU without receiving mechanical ventilation we couldnot estimate lung compliance before death The heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the DADreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections In a recent report needle postmortem biopsies suggested that COVID19 is not associated withDAD but rather with an acute fibrinous and anizingpneumonia AFOP consequently requiring corticoid treatment [] A diagnosis of AFOP is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with DAD and complete lunganalysis not just biopsies are necessary to exclude theirpresence Moreover AFOP may be a fibrinous variant ofDAD [] The limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for SARSCoV2 using RTPCR [] when compared to almost in our series In addition we did notfind specific endothelitis as previously reported in a smallcase series [] Considering the heterogeneity of postmortem COVID19 associated lesions molecular and IHCassessments are mandatory in the histological analysis ofCOVID19 tissue samplesPatients with COVID19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism PE [] In ourstudy three patients had PE already diagnosed beforedeath Four patients had pulmonary infarction In a previous study acute PE was considered as the main causeof death in four patients [] however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of PEwhen compared to our study Although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofARDS regardless of etiology [ ] As such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in COVID19 0cRemmelink Critical Care Page of patients remains to be further studied In a systematicreview of pathologicalfindings in COVID19 Polak [] identified a timeline in the histopathologicalfindings in the lung with epithelial DAD denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms Few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease Our resultsare consistent with those of Polak [] except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients We did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis The cases ofacute cardiac injury reported in COVID19 clinicalstudies [] do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia [] However using RTPCR we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theACE2 receptor which is ubiquitous but may notdirectly cause an injury As extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatSARSCoV2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in COVID19associated an dysfunction AsRTPCR mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance []just detect residual viral genomeThis study has several limitations i we only includedpatients who had had a positive RTPCR on nasopharyngeal swab andor bronchoalveolar lavage To ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic CTscan findings suggestive of COVID19 but had negativeRTPCR results This limitation in our study reflects thedifficulty of diagnosing COVID19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death This delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow SARSCoV2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related toCOVID19 Finally we did not evaluate specific mechanisms involved in the pathogenesis of an injuryConclusionThese results underline the heterogeneity of an injuriesduring COVID19 disease and the absence of specificSARSCoV2 lesions Using RTPCR SARSCoV2 couldbe detected in all ans even those without evidentmicroscopic lesionsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s13054020032185Additional file Critical careautopsyCovid Additional materialProcedure to obtain brain samplesAdditional file Critical careautopsyCovid Additional Table S1Laboratory findings on the day of admissionAdditional file Critical careautopsyCovid Additional Table S2Detailed histological findings in all patientsAbbreviationsACE2 Angiotensin converting enzyme AFOP Acute fibrinous andanizing pneumonia AKI Acute kidney injury ARDS Acute respiratorydistress syndrome COVID19 Coronavirus disease Ct Threshold cycleDAD Diffuse alveolar damage FFPE Formalinfixed paraffinembeddedHE Hematoxylin and eosin IHC Immunohistochemistry IQRs Interquartileranges MERSCoV Middle East respiratory syndrome coronavirusPAS Periodic acidSchiff PE Pulmonary embolism RTPCR Realtime reversetranscription polymerase chain reaction SARSCoV Severe acute respiratorysyndrome coronavirusAcknowledgmentsThe authors thank Nathalie Lijsen Christophe Valleys Gees LacroixBarbara Alexiou Dominique Penninck Nicole Haye and Audrey Verrellen fortechnical and logistic supports Prof Frdric Schuind and Dr DjamelEddineYahiaCherif for neurosurgical procedure Egor Zindy DIAPath ULB forproofreading the paper and Dr MariePaule Van Craynest for traineessupervisionAuthors contributionsIS had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis IS FT JLV and CD drafted the paper MRCV LL PL MLR CM ALT JCG LP RDM SD SR ND LP and OD collected thedata MR ND and RDM did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published All authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedFundingThis study received financial support from Fonds Y Bo«l Brussels BelgiumFonds Erasme pour la Recherche Mdicale Brussels Belgium and Appel projet Spcial COVID19 ULB Brussels Belgium The CMMI is supported bythe European Regional Development Fund and the Walloon Region ofBelgium Walloniabiomed grant no project CMMIULBsupport the Center for Microscopy and Molecular Imaging and its DIAPathdepartment CD is a Senior Research Associate with the FNRS BelgianNational Fund for Scientific Research 0cRemmelink Critical Care Page of Availability of data and materialsThe data that support the findings of this study are available from thecorresponding author on reasonable request Participant data without namesand identifiers will be made available after approval from the correspondingauthor and local Ethics Committee The research team will provide an emailaddress for communication once the data are approved to be shared withothers The proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data Additional materials may also be required during theprocess D'Haene N Melndez B Blanchard O De N¨ve N Lebrun L VanCampenhout C Design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas CancersBasel Corman VM Landt O Kaiser M Molenkamp R Meijer A Chu DK et alDetection of novel coronavirus 2019nCoV by realtime RTPCR EuroSurveill de Hemptinne Q Remmelink M Brimioulle S Salmon I Vincent JL ARDS aclinicopathological confrontation Chest Menter T Haslbauer JD Nienhold R Savic S Hopfer H Deigendesch N et alEthics approval and consent to participateThe study protocol was approved by the local ethics committee ErasmeHospital P2020218 The ethical committee has waived the need for writteninformed consentPostmortem examination of COVID19 patients reveals diffuse alveolardamage with severe capillary congestion and var | Thyroid_Cancer |
immunerelated genes pairs signature predict the prognosis of cervical cancer patientsHan Nie1 Fanqin Bu2 Jiasheng Xu1 Taoshen Li1 Jun Huang2To screen the key immune genes in the development of cervical cancer construct immune related gene pairs IRGPs and evaluate their influence on the prognosis of cervical cancer Tumor Genome Atlas TCGA database and geo database were downloaded as training set and validation set respectively and immune related gene data were downloaded from immport IRGPs model is established by machine learning and the model is analyzed and evaluated Using the Uclcan to analyze the immune genes expression in cervical cancer and to further explore the association with the expression level and the clinical stage and prognosis of cervical cancer According to the analysis of training set we identified IRGPs as key gene pairs and constructed the model The AUC value of the model was greater than and the model group survival rate was conspicuous different P The reliability of the model was confirmed in the validation group Our IRGPs play an important role in the occurrence and development of cervical cancer and can be used as a prognostic marker and potential new target of cervical cancerCervical cancer is one of the four most common gynecological tumors1 Every year at least women in the world are diagnosed with cervical cancer and more than people are killed2 In recent years the incidence rate of cervical cancer has decreased significantly through universal screening and health knowledge However the incidence rate of cervical cancer is still high in developing countries3 For women with low education in less developed areas the coverage rate of cervical cancer screening is still very low4 Squamous cell carcinoma is the most common type of cervical cancer accounting for of cervical cancer cases while adenocarcinoma only accounts for about In developing countries of cervical cancer patients have local infiltration or metastasis which has led to the high mortality of cervical cancer in developing countries Early cervical cancer is usually treated by radical hysterectomy When there are risk factors such as lymph node metastasis and endometriosis that may lead to recurrence they will be treated with chemotherapy5 The standard treatment for patients with locally advanced cervical cancer is conventional radiation therapyCRT6 The fiveyear survival rate of patients with locally advanced cervical cancer can be as high as after surgical resection radiotherapy chemotherapy CRT and so on7 However at present all treatment methods are not effective for patients with paraaortic lymph node metastasis and their threeyear progression free survival time PFS and total survival time OS are and respectively The fiveyear survival rate of cervical cancer patients with recurrence and metastasis was as low as Limited treatment is the main reason for this situation Now palliative chemotherapy is the most commonly used for patients with metastatic and recurrent cervical cancer10 The median survival time of patients with metastatic or recurrent cervical cancer treated with platinumtaxane chemotherapy and bevacizumab can be extended to a0months11 However these treatments are far from enough for most locally advanced and metastatic cervical cancer patients with positive lymph node metastasisIn recent years immunotherapy has been developed and increasingly used in cancer patients For example PDL1 is overexpressed in a variety of tumor cells including liver cancer cells and lung cancer cells and plays an important role in regulating the immune response of tumor cells12 Currently there are several clinical trials involving FDAapproved immunosuppressive checkpoint inhibitors which attack tumor cells expressing PDL1 by blocking the PDL1PD1 signaling pathway so as to improve the treatment and prognosis of patients From the current situation immunosuppressive therapy has achieved good results in many solid tumors16 The 1Department of Vascular Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China 2Department of Gastrointestinal Surgery The Second Affiliated Hospital of Nanchang University No Minde Road Nanchang Jiangxi Provence China email junhuangncu163comScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cresults of PD1PDL1 inhibition in cervical cancer are also satisfactory However at present immunoassay sites with a therapeutic effect are scarce and the research on tumor immunotherapy is far from sufficient In this study we screened immune genes that are significantly related to the prognosis of cervical cancer constructed an immune gene pair IRGP model based on these genes and used it to verify the unique prognostic markers of cervical cancerMethodData acquisition Gene expression profile data of patients with cervical squamous cell carcinoma were obtained from cancer and tumor gene map TCGA wwwtcga and gene expression profile data set gse4400116 was obtained from gene expression compilation GEO wwwncbinlmnihgovgeo database including samples of cervical cancer patientsAcquisition of sample immune gene expression immune related genes were downloaded from immport wwwimmpo rthome including antigen presenting cells chemokines and their receptors cytokines and their receptors interferon interleukin etc Using limma package in R we compared the gene expression data of cervical cancer samples downloaded from TCGA database and geo database as training set and verification set with immune related genes and extracted the expression amount of immune related genes in cervical cancer samplesConstruction of immune related gene pairs IRGPs In the two groups of data processed in the previous step the IRGP of the sample is calculated and the relatively high change is selected according to the standard of media absolute deviation IRGP values are calculated by comparing gene expression levels in specific samples or profiles in pairs The immune related genes are matched to compare the IRGPs If the first IRG is larger than the second IRG the output of the IRGP is otherwise the output is If the ratio of IRGP score of or in training set and verification set is higher than then remove the IRGP and retain the remaining IRGP as candidate IRGP for prognosis prediction The logistic rank test was used to screen the prognosis IRGP FDR Cox risk regression analysis and glment in R were used to perform tenfold cross validation to analyze the candidate IRGP and obtain the IRGP index We constructed the best gene pairs as immune gene pair model We use ROC to calculate the optimal cutoff value of IRGP index and use it as the basis to distinguish high and low risk groupsIRGPs model validation The single factor and multi factor Cox proportional risk analysis and survival analysis of TCGA and gse44001 cervical cancer samples were carried out with IRGPs modelInfiltration of immune cells in cervical cancer samples In order to study the infiltration of immune cells in the high and low risk groups of cervical cancer we used CIBERSORT17 to evaluate and predict the enrichment of immune cells in the samples CIBERSORT is a tool for deconvolution of the expression matrix of immune cell subtypes based on the principle of linear support vector regression RNA SEQ data can be used to estimate the infiltration of immune cells CIBERSORT can analyze the relative abundance of immune infiltrating cells in each sample including NK cells T cells B cells and macrophagesFunctional enrichment analysis of GSEA go and KEGG Gene set enrichment analysisGSEA enrichment analysis was carried out for each gene related to immune prognosis using the fgsea package in R Cluster profiler19 was used to enrich Gene ontologyGOfunction and KEGG pathway Significant enrichment criteria the absolute value of NES is greater than the nomp value is less than and the fdrq value is less than Expression of immune gene in cervical cancer Ualcan were used to analyze the expression of immune genes in cervical cancerStatistical analysis Measured data were expressed as mean ± standard deviation x ± s and data were compared using t test Kaplan Meier method was used for survival analysis The receiver operating characteristic curve ROC curve and ROC analysis were completed by survivalROC103 Single factor and multi factor analysis using Cox proportional risk regression model P was statistically significant P a0 as the difference has very significant statistical significanceEthical approval and consent to participate This article does not contain any studies with patients or animals performed by any of the authorsResultsExpression of immune related genes and construction of IRGPs in cervical cancer samples We obtained gene expression data of cervical cancer samples from TCGA database as training set cervical cancer samples from gse44001 as verification set immune related genes from immport and immune related genes from cervical cancer samples by comparing the two Through these immune related genes we constructed IRGPs We remove more than of the IRGP with a score of or from the training set and validation set leaving IRGP as candidates Combine TCGA clinical data with training set data Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cTCGA clincial dataAge ¥ ¥ GradeG1G2G3G4TT1T2T3T4MM0M1NN0N1Table TCGA clinical dataIRG1APOBEC3HARG2BTCCCL2CCL20CCL20CCL20CCL28CXCL1CXCL2DESDESDLL4FLT3LGHCKHCKHLADQA2IL1BIL1BJAK1NOD1NRP1PLXNB3PSMD7RBP7RBP7STC1TLR3VAV3Immune processesAntimicrobialsAntimicrobialsCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesCytokinesAntimicrobialsCytokinesAntimicrobialsAntimicrobialsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsAntimicrobialsAntimicrobialsCytokine_ReceptorsCytokine_ReceptorsAntigen_Processing_and_PresentationAntimicrobialsAntimicrobialsCytokinesAntimicrobialsBCRSignalingPathwayTable Model information about IRGPIImmune processesCytokinesCytokinesCytokinesCytokine_ReceptorsIRG2BTCCLCF1IL16FGFR3APOBEC3C AntimicrobialsARAFPLXNA1MAP3K14TNFSF10PTAFREPORVEGFCDESINHBASAA2STC2LTB4R2DUOX1EDN1APOBEC3C AntimicrobialsCSF2RBCD3DFGFR2SHC1CXCR3DESTNFRSF18CXCR6NRP1NaturalKiller_Cell_CytotoxicityChemokine_ReceptorsTCRsignalingPathwayTNF_Family_MembersChemokine_ReceptorsCytokine_ReceptorsCytokinesCytokinesTGFb_Family_MemberChemokinesCytokinesCytokine_ReceptorsAntimicrobialsChemokinesCytokine_ReceptorsTCRsignalingPathwayCytokine_ReceptorsNaturalKiller_Cell_CytotoxicityChemokine_ReceptorsCytokinesCytokine_ReceptorsAntimicrobialsCytokine_ReceptorsCoefficientScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A Timedependent ROC curve for IRGPI in the training cohort B Timedependent ROC curve for IRGPI in a0year C Timedependent ROC curve for IRGPI in a0year D Timedependent ROC curve for IRGPI in a0yearTable a0 prognosis related IRGPs were screened by lasso Cox proportional risk regression analysis After iterations we selected optimal IRGPs to build the immune prognosis model Table a0IRGPs model validation The immune prognosis model was applied to the training set and the patients in each training set were scored According to ROC curve analysis the optimal cutoff value for patients to be divided into high and low risk groups is Fig a01A After evaluating the model we found that AUC value of model and a0years is Fig a01B Fig a01C and Fig a01D The results show that our immune prognosis gene has a high reliability for the model The training set was divided into highrisk group Fig a02A and highrisk group Fig a02B The results showed that the overall survival rate OS of highrisk group was significantly lower than that of lowrisk group For TCGA training set data single factor and multi factor Cox risk regression analysis showed that only IRGPs model showed significant prognostic effect in single factor Cox Fig a02C while age and IRGPs could be significant independent prognostic factors in multi factor Cox Fig a02D Applying this model to the validation set of gse44001 Fig a03A Table a0 survival analysis showed that the OS of patients in the highrisk group was significantly lower than that in the lowrisk group Fig a03B In the single factor and multi factor Cox analysis IRGPs model and tumor size were significantly correlated with prognosis Fig a03CDInfiltration of immune cells in cervical cancer samples Most studies believe that the occurrence and development of tumor are closely related to immune cells so it is an ideal method to study the infiltration of immune cells in tumor We used CIBERSORT to analyze the infiltration of kinds of immune cells in patients with high and low risk groups Figure a04A shows the expression of immune cells in different risk groups Macrophage M0 Fig a04B activated mast cells Fig a04C were significantly overexpressed in the highrisk group while stationary dendritic cells Fig a04D stationary mast cells Fig a04E activated CD4T cells Fig a04F and cd8t cells Fig a04G were overexpressed in the lowrisk groupScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A The model divides the training set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Training set single factor Cox regression analysis forest map D Training set multivariate Cox regression analysis forest mapFunctional enrichment analysis of GSEA go and KEGG We analyzed the function enrichment of IRGP in the model The results of go analysis showed that IRGP in the model was mainly enriched in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the binding of fibroblast growth factors and the activity of tyrosine kinase Fig a05AB KEGG results showed that these IRGP were involved in cytokine cytokine receptor interaction chemokine signaling tumor necrosis factor signaling MAPK signaling NF kappa B signaling natural killer cellmediated cytotoxicity viral proteins and cytokines and Th1 and Th2 cell differentiation Fig a05CD The results of GSEA Fig a06A showed that these IRGP were significantly enriched in trace ribonucleoprotein complex Fig a06B neurotransmitter transporter activity Fig a06C endopeptidase activity Fig a06D fibroblast growth factor receptor binding Fig a06E hormone activity Fig a06F fibroblast cell proliferation Fig a06G and growth factor receptor binding Fig a06HExpression of immune gene in cervical cancer We explored the expression of IRGP in cervical cancer using the ualcan model Table a0 There were lowlevel expression of IRGP in cervical cancer Fig a0 and highlevel expression of IRGP Fig a0 There were differences in the expression of low expression IRGP and high expression IRGP in different age groups Fig a0 and there were differences in the expression of IRGP in different stages of cervical cancer Fig a0DiscussionCervical cancer is one of the most common gynecological malignancies HPV infection is considered to be the main cause of cervical cancer2122 although the incidence rate of cervical cancer has been significantly decreased due to the development and promotion of HPV vaccine23 But incidence rate of cervical cancer is still high in developing countries and Chinas low income countries24 At present for cervical cancer patients without invasion and lymphatic metastasis the effect of surgery combined with radiotherapy and chemotherapy is better If metastasis and infiltration occur the treatment effect of cervical cancer patients will become very unsatisfactory In recent years immunotherapy has performed well in a variety of cancers including cervical cancer25 Blocking PDL1 PD1 signaling pathway to attack tumor cells expressing PDL1 is the current mainstream method28 Although the anticancer activity of PD1 and PDL1 inhibitors is exciting such immunotherapy is not effective for all patients and a metaanalysis shows that patients who receive PD1 PDL1 inhibitors have a Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure A The model divides the validation set patients into lowrisk or highrisk groups B Kaplan Meier curve between high and low risk groups C Validation set single factor Cox regression analysis forest map D Validation set multivariate Cox regression analysis forest mapGSE44001 clincial dataStageLargest diameter cm ¥ ¥ ¥ Table GSE44001 clincial datahigher risk of rash thyroid dysfunction pruritus pneumonia and colitis29 Therefore it is of great significance for the detection and treatment of cervical cancer to predict and find more biomarkers that may be related to immune prognosisAt present most of the prognostic genes need to be standardized to reduce the errors caused by sequencing platform and samples In this study the scores of IRGPs constructed by us are calculated from the gene expression data of the same sample which can not only ignore the impact of different platforms but also do not need to standardize and scale the data This method has been used in many studies including cancer molecular classification with high reliability3233In this study we screened pairs of IRGP to construct the immune prognosis model related to the overall survival rate of cervical cancer patients The AUC values of the model in and a0years were all greater than According to these pairs of IRGP they were divided into highrisk group and lowrisk group In TCGA training group and GSE44001 verification group the OS of highrisk group was significantly lower than that of lowrisk group P These pairs of IRGP have a good effect on sample discrimination We found that macrophage Mo and activated mast cells were significantly over expressed in highrisk group by immunocyte infiltration analysis of samples The existing research shows that mast cells and macrophages play an important Scientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Immune infiltration status within IRGPI risk groups B Expression of Macrophage M0 C Expression of Mast cells activated D Expression of Dendritic cells resting E Expression of Mast cells resting F Expression of T cells CD4 memory activated G T cells CD8role in cervical cancer which can promote the development of cervical cancer by promoting lymphangiogenesis and angiogenesis34 However in the lowrisk group the expression of static dendritic cells static mast cells activated CD4T cells and cd8t cells is high Although the effect of CD4T cells on cervical cancer has not been agreed the cd8t cells are closely related to the better prognosis of cervical cancer patients37 there is evidence that dendritic cells will decrease in patients with high HPV infection which indicates that high expression of dendritic cells is beneficial to resist cervical cancer40 which is consistent with our results The enrichment analysis of go and GSEA showed that these immune genes were mainly involved in the binding of cytokines and their receptors the binding of chemokines and their receptors the binding of growth factors and their receptors the binding of epidermal growth factor receptors the activity of metalloendopeptidase the binding of fibroblast growth factors and their receptors hormone activity fibroblast proliferation and the binding process of growth Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Histogram graph of Immunerelated genes GO analysis results B Point graph of Immunerelated genes GO analysis results C Histogram graph of Immunerelated genes KEGG pathway analysis results D Point graph of Immunerelated genes KEGG pathway analysis resultsfactor receptorsAs we all know cytokines and chemokines are the key factors in the immune response for example In cervical cancer IL10 can interfere with the differentiation of dendritic cells and thus play a strong immunosuppressive effectTGFβ can inhibit T cell proliferation and attenuate immune response41 Research shows that growth factors and epidermal growth factors are closely related to the growth of cervical cancer and the survival rate of cervical cancer patients High expression of growth factors and epidermal growth factors often predict poor prognosis42 Growth of fibroblasts can stimulate angiogenesis at the early stage of tumor The proliferation and invasion of cancer cells and the remodeling of extracellular matrix promote the growth of cervical cancer4546 KEGG results showed that these immune genes were mainly enriched in chemokine signaling pathway tumor necrosis factor signaling pathway MAPK signaling pathway NF kappa B signaling pathway natural killer cellmediated cytotoxicity viral protein and cytokine and Th1 and Th2 cell differentiation Th1 and Th2 may be involved in the pathogenesis and growth of cervical cancer Th1 may be the target of predicting chemotherapy response of advanced cervical cancer47 while other pathways are classical signal pathways related to cancer Immune cytokines play an important role in cervical lesions Torres et a0al Found that IL10 is highly expressed in the cervix of women with persistent HPV which may be related to the persistence of HPV and the promotion of disease progression Further research by their team showed that copy individuals of IL4 IL6 IL10 and TGFB1 were significantly associated with cervical cancer and could be used as biomarkers for susceptibility to the disease5152These pairs of IRGP have different immune genes most of which are cytokines antimicrobial agents and natural killer cells which are involved in various stimulation reactions and play a key role In cervical cancer HPV can inhibit the apoptosis of cervical cancer cells by down regulating NOD153 In our sample we also found that the expression of NOD1 in tumor tissue is low and there are differences in different ages and stages Figs a07D 9C 10I Sang Yeon Cho et a0al Found that duox1 is highly expressed in cervical squamous cell carcinoma and can play a good prognostic role by increasing the amount of innate immune cells54 The analysis also showed that DUOX1 is highly expressed in tumor tissues and related to age and grade Figs a08B 9G 10D Stc2 can promote the proliferation of cervical cancer cells and increase the resistance to cisplatin55 while high expression of DDL4 is usually associated with low pelvic lymph node metastasis and survival rate of cervical cancer56 Therefore we believe that the IRGP constructed in this study plays an important role in the development and prognosis of cervical cancerThere are also some deficiencies in our research Although we select data samples from two databases for analysis and use more advanced methods to reduce the errors caused by platforms samples etc this is still a retrospective analysis If we can carry out a prospective study or obtain clinical samples and evaluate them with Western blot or immunohistochemistry it will be more convincingScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A GSEA analysis of immune signature genes BH In the high immune risk group of cervical cancer cancer marker genes were abundant P FDR Scientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cCLCF1DLL4INHBANOD1NRP1RBP7CXCR3DUOX1FGFR3AgeNormalvsAge4160YrsAge2140YrsvsAge4160YrsAge2140YrsvsAge6180YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsAge2140YrsvsAge81100YrsAge4160YrsvsAge6180YrsAge4160YrsvsAge81100YrsAge6180YrsvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge81100YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsHLADQA2NormalvsAge4160YrsLTB4R2STC2TNFSF10VAV3NormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge4160YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsNormalvsAge2140YrsNormalvsAge4160YrsNormalvsAge6180YrsAge2140YrsvsAge6180YrsAge4160YrsvsAge6180YrsPval777E05123E02483E04115E07209E02433E07270E06354E05281E02982E03260E03384E04322E02163E05600E04868E03106E02109E02122E04396E03281E04459E02421E04876E03441E02105E02384E04566E05218E05803E09139E06225E08111E16204E13725E10305E02422E07693E10174E05433E15162E12550E10341E02803E04159E06458E04192E02524E10415E11559E10162E12162E12934E14108E02208E03StageNormalvsStage2NormalvsStage3Stage1vsStage2Stage1vsStage3Stage1vsStage4NormalvsStage1NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage4Stage3vsStageNormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Stage1vsStage3NormalvsStage1NormalvsStage2NormalvsStage3NormalvsStage4Pval185E06220E04542E03304E02460E04696E07546E03252E03210E02439E02390E03186E02395E02317E02122E03164E03396E04984E03222E02315E06226E02387E07734E03433E01118E04173E05355E04222E02160E05259E09131E09258E04162E12199E12176E05202E04663E12390E05241E04162E12860E10479E09175E04148E02240E04435E05119E04463E02230E13297E09263E07840E04170E02162E12493E12284E12125E04Table P value of IRGPs expression in different ages and stagesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues B Expression of DLL4 in cervical cancer and normal tissues C Expression of INHBA in cervical cancer and normal tissues D Expression of NOD1 in cervical cancer and normal tissues E Expression of NRP1 in cervical cancer and normal tissues F Expression of RBP7 in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CXCR3 in cervical cancer and normal tissues B Expression of DUOX1 in cervical cancer and normal tissues C Expression of FGFR3 in cervical cancer and normal tissues D Expression of HLADQA2 in cervical cancer and normal tissues E Expression of LTB4R2 in cervical cancer and normal tissues F Expression of STC2 in cervical cancer and normal tissues G Expression of TNFSF10 in cervical cancer and normal tissues H Expression of CESC in cervical cancer and normal tissuesScientific RepoRtS 101038s41598020705005Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different ages B Expression of DLL4 in cervical cancer and normal tissues at different ages C Expression of NOD1 in cervical cancer and normal tissues at different ages D Expression of NRP1 in cervical cancer and normal tissues at different ages E Expression of RBP7 in cervical cancer and normal tissues at different ages F Expression of CXCR3 in cervical cancer and normal tissues at different ages G Expression of DUOX1 in cervical cancer and normal tissues at different ages H Expression of FGFR3 in cervical cancer and normal tissues at different ages I Expression of HLADQA2 in cervical cancer and normal tissues at different ages J Expression of LTB4R2 in cervical cancer and normal tissues at different ages K Expression of STC2 in cervical cancer and normal tissues at different ages L Expression of TNFSF10 in cervical cancer and normal tissues at different ages M Expression of VAV3 in cervical cancer and normal tissues at different agesConclusionWe constructed an immune gene pair model which is closely related to the prognosis of cervical cancer patients The model contains IRGP and immunerelated genes The biological functions of these immunerelated genes are closely related to the occurrence and development of cervical cancer Therefore we think that these IRGPs may be the target of predicting or diagnosing cervical cancer and suggest that immunotherapy can improve the prognosis of cervical cancer patients by regulating these IRGPsScientific RepoRtS 101038s41598020705005Vol0123456789wwwnaturecomscientificreports 0cFigure a0 A Expression of CLCF1 in cervical cancer and normal tissues at different stages B Expression of CXCR3 in cervical cancer and normal tissues at different stages C Expression of DLL4 in cervical cancer and normal tissues at different stages D Expression of DUOX1 in cervical cancer and normal tissues at different stages E Expression of FGFR3 in cervical cancer and normal tissues at different stages F Expression of HLADQA2 in cervical cancer and normal tissues at different stages G Expression of INHBA in cervical cancer and normal tissues at different stages H Expression of LTB4R2 in cervical cancer and normal tissues at different stages I Expression of NOD1 in cervical cancer and normal tissues at different stages J Expression of NRP1 in cervical cancer and normal tissues at different stages K Expression of RBP7 in cervical cancer and normal tissues at different stages L Expression of STC2 in cervical cancer and normal tissues at different stages M Expression of TNFSF10 in cervical cancer and normal tissues at different stages N Expression of VAV3 in cervical cancer and normal tissues at different stagesData availabilityAll data are available Please contact us to access if it is neededReceived May Accepted July References Stewart Bernard W et al Cancer prevention as part of precision medicine plenty to be done Carcinogenesis 101093carci nbgv16 Bray F et al Global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin 103322caac21492 Passos Camila M Sales Jacqueline B Maia Emanuella G Caldeira ThaÃs C M Rodrigues Roberta D Figueiredo N Claro Rafael M Trends in access to female cancer screening in Brazil J Public Health Oxf 101093pubme dfdaa0 Asrabuddhe V V Parham G P Mwanahamuntu M H Vermund S H Cervical cancer prevention in low and middleincome countries feasible affordable essential Cancer Prevent Res 10115819406207CAPR110540 Koh WuiJin AbuRustum Nadeem R Bean Sarah Bradley Kristin Campos Susana M Cho Kathleen R Chon Hye Sook Chu Christina Clark Rachel Cohn David Crispens Marta Ann Damast Shari Dorigo Oliver Eifel Patricia J Fisher Christine M Frederick Peter Gaffney David K Han Ernest Huh Warner K Lurain John R Mariani Andrea Mutch David Nagel Christa Nekhlyudov Larissa Fader Amanda Nickles Remmenga Steven W Reynolds R Kevin Tillmanns Todd Ueda Stefanie Wyse Emily Yashar Catheryn M McMillian Nicole R Scavone Jillian L2019 Cervical Cancer Version NCCN Clinical Practice Guidelines in Oncology J Natl Compr Canc Netw 106004jnccn Chen J et al Nanotechnology in the management of cervical cancer Rev Med Virol 25Suppl 101002 Varia M A et al Cervical carcinoma metastatic to paraaortic nodes extended field radiation therapy with concomitant 5fluorouracil and cisplatin chemotherapy a Gynecologic Oncology Group study Int J Radiat Oncol Biol Phys 101016s0360 Randall Leslie M Monk Bradley J Darcy Kathleen M Tian C Burger Robert A Liao SY Peters William A Stock Richard J Fruehauf John P Markers of angiogenesis in highrisk earlystage cervical cancer a Gynecologic Oncology Group study Gynecol Oncol 101016jygyno Boussios S et al Management of patients with recurrentadvanced cervical can | 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structures assigned to the products were concordant with the microanalytical andspectral data Compounds 4e18 were screened for their ability to induce the antioxidant enzyme NADPH quinone oxidoreductase NQO1 in cells a classical target for transcription factor nuclear factorerythroid268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamide showed the most potent NQO1inducer activity in vitro Compound had low toxicity in mice LD50 ¼ mgkg It also reduced thedamaging effects of gamma radiation as assessed by the levels of Nrf2 NQO1 reactive oxygen speciesROS and malondialdehyde MDA in liver tissues In addition compound showed amelioration in thecomplete blood count of irradiated mice and enhanced survival over a period of days followingirradiation Molecular docking of inside the Nrf2binding site of Kelchlike ECH associated protein Keap1 the main negative regulator of Nrf2 showed the same binding interactions as that of the cocrystallized ligand considering the binding possibilities and energy scores These ï¬ndings suggest thatcompound could be considered as a promising antioxidant and radiomodulatory agent The Authors Published by Elsevier Masson SAS This is an access under the CC BYlicense httpcreativecommonslicensesby40 IntroductionThe extensive use of radiotherapy and the damage caused to thesurrounding normal ans have provoked researchers to ï¬nd newstrategies to protect normal tissues from radiation hazards []The risk of injury from radiation can diminish the value of radiotherapy and contribute to complications for longterm cancer survivors [] Ionizing radiation interrupts cell functions throughradiolysis of water and the production of reactive oxygen speciesROS or reactive nitrogen species RNS [] Excessive productionof ROS and RNS promotes oxidative stress which can affect allcellular components including single or double DNA strand breaks Corresponding authorEmail address mmsghorabyahoocom MM Ghorab[] This ROSmediated toxicity can lead to mutations and consequently cause cardiovascular neurological toxicities and sexualdysfunction as well as cancer [7e10] In order to reduce theseradiationinduced side effects radioprotective drugs are used []Also the use of multitarget antioxidants that act as radioprotectorscan help limit normal tissue damage caused by ionizing radiation[12e14]Nuclear factor erythroid 2related factor Nrf2 is a transcription factor that regulates the expression of various antioxidantproteins to protect against oxidative damage in the cell [] Theabundance of Nrf2 is negatively regulated by Kelchlike ECH associated protein Keap1 a substrate adaptor for a Cullin3Rbx1ubiquitin ligase that binds and continuously targets Nrf2 for ubiquitination and proteasomal degradation [16e18] Under conditionsof oxidative stress redoxsensitive cysteine sensors of Keap1 aremodiï¬ed leading to loss of its ability to target Nrf2 for degradation101016jejmech2020112467 The Authors Published by Elsevier Masson SAS This is an access under the CC BY license httpcreativecommonslicensesby40 0cAM Soliman European Journal of Medicinal Chemistry consequently Nrf2 transports into the nucleus where it initiates thetranscription of its downstream target genes such as NADPHquinone oxidoreductase1 NQO1 []Quinazolinone is a strategic scaffold that has a wide range ofpharmacological activities such as antioxidant antiammatoryand anticancer activities [20e23] Sulfonamides in addition totheir use as antibiotics [24e27] have many pharmacological activities and can be used as antiviral [] antiammatory []antioxidant [] and anticancer agents [32e35] These versatilepharmacological activities make the two chemical classes excellentcandidates for developing new multitarget agents through a slightalteration in the structure that might lead to diversity in the biological activity []In addition numerous studies haverevealed iodine to be a potent antioxidant with higher potency thanthat of ascorbic acid [] Iodine can act as an electron donor that 0fquenches ROS such as OHand H2O2 [] or decreases thedamaging effects of ROS thus increasing the total antioxidant status in human serum []In this context it seemed of interest to search for new compounds with the ability to scavenge ROS and protect cells A seriesof new 68diiodoquinazolin43Hone conjugated to benzenesulfonamide was synthesized by the introduction of the sulfonamide group at the N3 of quinazolinone with the incorporation ofvarying acetamide terminal aimed at exploring the potential antioxidant and radioprotective activity The antioxidant potential ofthe target compounds was ï¬rst measured using a quantitative androbust NQO1 inducer activity bioassay in cells Acute toxicity studyfor the most active compound was then performed in vivo A nontoxic dose was subsequently selected to investigate the potentialprotective effect against wholebody gamma irradiationinducedoxidative stress in experimental mice All groups were observed days after irradiation for survival and weight changes Additionally molecular docking was performed inside the Nrf2bindingsite of Keap1 to gain insights into the molecular interactions andpossible mode of action Results and discussion Chemistry wasreactionofpreparedfrom theScheme shows the synthesis of thioacetamide quinazolinonebenzenesulfonamide derivatives 5e18 The starting material 68diiodo2mercapto4 oxoquinazolin34Hyl benzenesulfonamideisothiocyanatobenzenesulfonamide [] and 2amino35diiodobenzoic acid The coupling of with the 2chloroNsubstituted acetamide in dry acetone and anhydrous K2CO3 yieldedthe corresponding 268diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNsubstituted acetamide 5e18 IRspectra of 5e18 displayed additional NH CH2 aliphatic and CObands at their speciï¬ed regions 1H NMR spectra of 5e18 revealedthe acetamide group through the presence of two singlets one at417e431 ppm referring to the CH2and the other at966e1121 ppm attributed to the NH protons with the disappearance of SH singlet of at ppm 13C NMR of 5e18 exhibited twosignals peculiar to the CH2 and CO carbons 1H NMR spectra of 6e8displayed singlets at and ppm assigned to the CH3group at the ortho meta and parapositions of the phenyl group 13CNMR of 6e8 showed signals at and ppm for theCH3 group 1H NMR spectra of 9e11 revealed triplets at and ppm attributed to the CH3 ethyl and quartet at and ppm referring to the CH2 ethyl at the ortho meta and parapositions 13C NMR of 9e11 showed two signals at due to CH3 ethyl and due to the CH2 ethylgroups respectively 1H NMR spectra of revealed singlet at ppm attributed to the OCH3 protons while 13C NMR of showed a signal at ppm due to the OCH3 carbon 1H NMRspectra of revealed triplet at ppm and quartet at ppmdue to the ethoxy group 1H NMR spectra of revealed a singlet at ppm due to the 2OCH3 protons while revealed two singletsat and ppm due to the 3OCH3 protons IR of 16e18 showedNO2 bands Biological activityIn vitro screeningThe antioxidant activity of compounds 4e18 was screened usingthe NQO1 inducer activity assay The Concentration of the novelcompounds to Double the speciï¬c enzyme activity of NQO1 CDvalue was used as a measure of inducer potency and results obtained are presented in Fig Table Evaluation of the NQO1inducer activity showed that compounds and wereinactive whereas compounds and had activityhowever CD value was not reached Compounds CD ¼ mMand CD ¼ mM showed concentrationdependent induceractivity These diiodoquinazolinones represent a new chemicalclass of NQO1 inducers thus adding to the existing knowledge ofthe diversity of the many chemical scaffolds that have been reported to induce this antioxidant enzyme The classical NQO1 inducers are primarily oxidants and electrophiles or othercompounds that react or are metabolized to products that reactand chemically modify cysteine sensors of Keap1 [] A newgeneration of NQO1 inducers is also emerging that of noncovalentsmallmolecule modulators of the Keap1eNrf2 proteinproteininteraction [44e46] Because our diiodoquinazolinones havesome common features with the Keap1eNrf2 proteinproteininteraction inhibitors in this study we tested the potential abilityof these compounds to directly disrupt the binding of Keap1 to Nrf2by molecular modeling see section In vivo evaluation Determination of toxicity lethal dose ï¬fty LD50 of compound The most promising compound was investigatedin vivo for acute toxicity LD50 in albino mice and the value wasfound to be mgkg body weight ip Subsequently onetenthof this dose was selected as the therapeutic dose for further evaluation of the potential radioprotective effects of compound Evaluation of the radiomodulatory effect of compound inmice Four groups of mice were used the ï¬rst group served ascontrol the second group was irradiated at a dose of Gy as a singledose the third group was injected ip with compound only for consecutive days and the last group received compound thenexposed to Gy of gamma radiation After days from irradiationï¬ve mice were checked for liver and hematopoietic system toxicities The residual mice in all groups were monitored over daysto evaluate the survival rate and body weight changes The effect of compound on radiationinduced livertoxicity Gamma radiationinduced hepatic oxidative stress asshown by a signiï¬cant increase in hepatic levels of nuclear Nrf213fold NQO1 32fold ROS 15fold and the lipid peroxidation product malondialdehyde MDA 2fold as compared to nonirradiated control mice This was in agreement with other studies[]Ionizing radiation is believed to induce damage through thegeneration of ROS resulting in an imbalance in the oxidantantioxidant ratio in cells [] In the current experiment the presenceof ROSmediated damage was conï¬rmed by the increase in MDAlevels in irradiated liver in addition to the increase in the expression of the enzymatic antioxidant system Moreover these results 0cAM Soliman European Journal of Medicinal Chemistry Scheme The synthetic pathways for the development of the diiodoquinazolinone derivatives 4e18support the notion that Nrf2 is an initial regulator of cellular responses to radiation exposure [] Once Nrf2 translocates to thenucleus it induces expression of endogenous antioxidant enzymessuch as NQO1 [] a ï¬avoprotein involved in cellular protectionagainst oxidative stress []Treatment of nonirradiated mice with compound led to anincrease in NQO1 and ROS levels and a decrease in Nrf2 with nosigniï¬cant change in MDA level as compared to normal nonirradiated mice Fig A signiï¬cant increase in Nrf2 levels aswell decrease in the levels of NQO1 ROS and MDA was observed in irradiated mice livers treated with compound when compared to the group subjected to radiation aloneFig Moreover treatment with compound improved bothsurvival and body weight of the animals following irradiation 0cAM Soliman European Journal of Medicinal Chemistry Additionally it has been reported that Nrf2 modiï¬es ROS production partly by regulating NQO1 expression [] On the other handthe NQO1 levels were signiï¬cantly higher than the nonirradiatedcontrols in agreement with the cell culture results this studyNotably the increased levels of ROS in nonirradiated mice treatedwith compound are consistent with the increased levels of ROSfollowing genetic Nrf2 activation by Keap1 knockdown []Importantly however the increased ROS production that accompanies NQO1 induction does not lead to damage as evidenced bythe lack of increase in the levels of MDA this study The effect of compound on the hematopoietic systemTo examine the possible role of compound in protecting thehematopoietic system against irradiation we measured the peripheral blood cell counts of red blood cells RBCs white bloodcells WBCs hemoglobin HGB and platelets PLT The irradiatedmice exhibited a signiï¬cant decrease in RBCs WBCs HGB and PLTcompared with the control group Fig These results are mainlyattributed to the fact that irradiation causes the formation of freeradicals which initiate a chain of events leading to the decline in thelevels of hematological parameters [] Indeed it has been wellestablished that gamma irradiation induces RBC injury includingmorphological and quantitative changes of RBCs These alternations may be partly attributed to radiationinduced oxidative stressin RBCs Exposure to radiation results in the formation of reactiveoxygen species ROS and reactive nitrogen species RNS as well asDNA damage which can then lead to severe injury to the hematopoietic system [] This is in harmony with Wang []who stated that injury to the hematopoietic system is the mostcommon injury induced by irradiation This was attributed to theeffect of ionizing radiation on hematopoietic stem cells and hematopoietic progenitor cells which are principally responsible forhematopoietic recovery Treatment of irradiated mice with compound ameliorated the decrease in peripheral blood cellsparticularly RBCs HGB and PLT Hence the antioxidant propertiesof compound may contribute to the amelioration of RBC countsand HGB in irradiated mice This is consistent with other studies forantioxidants effects on the hematopoietic system [] Thismight be explained through the promotion effect of radioprotectorsto proliferate hematopoietic stem cells and they also could increasethe levels of leukocyte growth factors [] Besides severalpotent radioprotectors protect various membrane systems as wellas hematopoietic stem cells from peroxidative damages thatFig Concentration dependence of the NQO1 inducer activity of compounds 4e18Fig without affecting the liver weight Fig as compared toirradiated mice The present results indicate that compound hasan antioxidant capacity as the treatment of irradiated mice with prevents oxidative stress reducing the increase in lipid peroxidation markers and maintaining the expression of Nrf2 comparedwith the irradiated group suggesting improved hepatic antioxidantcapacity Hence compound validated its radiomodulatory andantioxidant effect through its main structure quinazolinone andsulfonamide that goes in line with Soliman [] Also thisï¬nding was reinforced by Cuadrado and his colleagues whoemphasized the importance of therapeutic targeting for Nrf2because of its resourceful cytoprotective roles against a plethora ofdiseases that are associated with oxidative stress []At the same time it was found that NQO1 expression levels ofirradiated mice treated with were signiï¬cantly lower ascompared to vehicletreated irradiated ones but still signiï¬cantlyhigher than normal levels Interestingly the levels of NQO1 in allexperimental groups correlate with the levels of ROS suggestingROS involvement in the NQO1 induction The lower levels of NQO1and ROS in the irradiated group that also received could be theresults of increased antioxidant capacity due to Nrf2 activation []Table NQO1 inducer activity and CD values of compounds 4e18Conc mMCompound noCDaNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNRNR means not recordeda CD values are the averages of three independent experiments each with eight replicate wells of cells and SD for each data point was within of the value 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A Nrf2 B NQO1 C ROS and D MDA levels in liver of nonirradiated control and irradiated mice after days of irradiation The results wereexpressed as mean ± SE Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different from control group signiï¬cantly different from irradiated group at p n ¼ happened after irradiation so it could protect blood componentsagainst irradiation [] Taken all together these results demonstrate the protective effect of compound against gammaradiation Molecular dockingMolecular docking was performed to assess the ability ofcompound to block the Kelch domain of Keap1 Through its Kelchdomain Keap1 binds to Nrf2 promoting its degradation resultingin low cytoprotective gene levels [] The PDB ï¬le 4IQK was obtained from the Protein Data Bank The binding site of Kelch domainhas been reported to have ï¬ve subpockets P1 P2 P3 P4 and P5[] P1 and P2 are positively charged pockets that contain thearginine triad Arg Arg and Arg This triad is crucialfor the selectivity of the molecular recognition together with a 0cAM Soliman European Journal of Medicinal Chemistry Fig A Survival percent and B Body weight changes of control irradiated compound and compound þ irradiated mice through days after irradiation Theresults were expressed as mean ± SE n ¼ Statistical analysis was carried out byKaplanMeier method followed by the ManteleCox test for survival analysis Bodyweight changes between groups were analyzed by twoway ANOVA followed byBonferronis post test signiï¬cantly different from control group signiï¬cantlydifferent from irradiated group at p group of hydrophobic residues contributes to the stability of thecomplex P1 is formed by residues Arg Ile Gly Phe Arg and Ser P2 is formed by Ser Arg Asn andAsn P3 is a neutrally charged pocket composed of Gly Ser Ala Gly Ser and Gly P4 is formed by Tyr Gln and Tyr whereas P5 is formed by Tyr and Phe The main interactions observed by the cocrystallized ligand NN0naphthalene14diylbis4methoxybenzenesulfonamidearetwo cationpi interaction with Arg piepi interaction with Tyr and two hydrogen bonds with Ser and Ser with S ¼ kcalmol Fig Compound showed the same key interactions exhibited by the cocrystallized ligand Compound S ¼ kcalmol RMSD ¼ has adopted a conformationallowing the presence of two cationpi interaction between Arg and the aromatic rings in addition to a hydrogen bond with themethoxy group Fig three hydrogen bonds made by ser andArg towards the methoxy groups and another hydrogen bondbetween Leu and NH2 group of the sulfonamide Superimposition between compound and the cocrystallized ligand showedthat they adopt the same orientation inside the binding site Fig Finally compound possessing the highest NQO1 inducer activityCD ¼ mM in this series showed the same interactions and thesame orientation of the native ligand inside the receptor indicatinga possible correlation between those multiple interactions and thenoted higher potency Based on the abovementioned resultscompound could possibly bind to Keap1 and disrupt its interaction with Nrf2The results from this study complement previous reportsshowing that the classical electrophilic Nrf2 activator sulforaphaneprotects cells including human retinal pigment epithelial cellskeratinocytes and mouse leukemia cells against oxidative damageFig Effect of compound on relative liver weight in nonirradiated control andirradiated mice after days of irradiation The results were expressed as mean ± SEn ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test There were no signiï¬cant differences between groupscaused by oxidative stressors of four different types namelymenadione tertbutyl hydroperoxide 4hydroxynonenal and peroxynitrite as well as by exposure to ultraviolet radiation []Furthermore unlike the effects of most direct antioxidants theindirect antioxidant effect of sulforaphane which results from Nrf2activation persists for several days after sulforaphane is no longerpresent in the cell culture medium This is because direct antioxidants such as ascorbic acid tocopherols carotenoids and polyphenols which neutralize ROS and other chemical oxidants areconsumed in these reactions whereas Nrf2 activation results intranscriptional upregulation of antioxidant defences which aremediated by proteins with long halflives often several days Thenew compounds generated in the current study have an additionaladvantage in that they are nonelectrophilic and are therefore expected to have a broader therapeutic window compared to electrophilic Nrf2 activators This is supported by the very low toxicityof compound in mice Taken together these results demonstratethe powerful effect of Nrf2 activation and induction of NQO1 inprotecting cells and animals against high levels of ROS and preventing ROSmediated damage This is of particular relevance toprotecting the hematopoietic system which is highly sensitive toROS Conclusiontheacetamide268diiodo4oxo34sulfamoylphenyl3In summary a hybridization strategy was adopted using theiodinated quinazolinone scaffold and sulfonamide moiety to producedihydroquinazolin2ylthioNsubstitutedderivatives 5e18 Different substitutions were introduced to theacetamide group to study the structureactivity relationship All thecompounds were screened for their antioxidant potential using theNQO1 inducer activity assay The 345trimethoxyphenyl derivative showed the highestinducer activity in this seriesCD ¼ mM and had low toxicity LD50 ¼ mgkg Treatmentof gammairradiated mice with compound lowered oxidativestress as evidenced by the lower levels of MDA ROS and NQO1 inliver Furthermore compound ameliorated the complete bloodpicture of irradiated mice as well as enhanced the survival of mice 0cAM Soliman European Journal of Medicinal Chemistry Fig Effect of compound on A RBCs B WBCs C HGB concentration and D PLT counts in nonirradiated control and irradiated mice after days of irradiation The resultswere expressed as mean ± SE n ¼ Statistical analysis was carried out by oneway ANOVA followed by Bonferronis multiple comparison test signiï¬cantly different fromcontrol group signiï¬cantly different from irradiated groupover a period of days postirradiation Molecular docking of inside the active site of Keap1 conï¬rmed that it binds in the samemanner as that of the cocrystallized ligands The inducer activity ofcompound in upregulating NQO1 strongly suggests that it couldbe used as a lead antioxidant and radiomodulatory agent for furtheroptimization of the quinazolinone scaffold Materials and methods ChemistryAll chemicals were purchased from SigmaAldrich and are of ARgrade Melting points were determined in capillary on aGallen Kamp melting point apparatus Sanyo Gallen Kamp UKThin layer chromatography using precoated silica gel plates Kieselgel mm F254 Merck Germany was performed with asolvent system of chloroformmethanol to detect the spots byIR spectra KBr disc were recorded using an FTIRUV lightspectrophotometer Perkin Elmer USA NMR spectra were scannedon NMR spectrophotometer Bruker AXS Inc Switzerland operating at MHz for 1H and MHz for 13C Mass spectra wererecorded on the ISQ LT Thermo Scientiï¬c GCMS model Massachusetts USA Chemical shifts are expressed in dvalues ppmrelative to TMS as an internal standard using DMSOd6 as a solventElemental analyses were done on a model CHNSO analyserPerkin Elmer USA All the values were within ± of thetheoretical values 8diiodo2mercapto4 oxoquinazolin34Hylbenzenesulfonamide A mixture of 2amino35diiodobenzoic acid g mol and isothiocyanatobenzenesulfonamide g mol in absolute ethanol mL containing drops of triethylamine was reï¬uxed for h The solid product formed wascollected by ï¬ltration and crystallized from ethanol to give 00 NH2 Yield mp 0eC IR KBr Ê cm 0cAM Soliman European Journal of Medicinal Chemistry the NN0naphthalene14diylbis4Fig 2D and 3D interaction poses ofmethoxybenzenesulfonamide showing cationp pp interaction and hydrogenbonds with the key amino acids inside the binding pocket arom CO CN SO2 1H NMRDMSOd6 d ppm s 1H d 2H J ¼ Hz AB d2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s2H 13C NMR DMSOd6 d ppm Anal Calcd for C14H9I2N3O3S2 C H N Found C H N 34Dihydroquinazolinsulfonamide derivatives General procedure A mixture of g mol and chloroNsubstituted acetamide derivatives mol in dryacetone mL and anhydrous K2CO3 g mol was stirredat room temperature for h ï¬ltered and the solid product formedwas crystallized from dioxane to give 5e18 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNphenylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 2H 703e730 m 3H760e783 m 4H s 2H d 2H J ¼ Hz AB d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm þ ] [Mþ1 MS mz [] [M] [] Anal Calcd for C22H16I2N4O4S2 C Fig 2D and 3D interaction pose of compound showing cationp pp interactionsinside the binding pocket of 4IQKH N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioNotolylacetamide Yield 00 NH NH2 mp 0eC IR KBr Ê cmarom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H ddd 1H J ¼ Hz 730e755 m 3H d 2H J ¼ HzAB s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl34 Yielddihydroquinazolin2ylthioNmtolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H 0cAM Soliman European Journal of Medicinal Chemistry Hz 721e748 m 2H 770e804 m 5H s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6 dppm Anal CalcdforC24H20I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz 780e805 m 4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm Anal Calcd forC24H20I2N4O4S2 C H N Found C H N 268Diiodo 4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioN 4methoxyphenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d2H J ¼ Hz AB d 2H J ¼ Hz s 2H d 1HJ ¼ Hz d 1H J ¼ Hz s 1H 13C NMR DMSOd6d ppm Anal Calcdfor C23H18I2N4O5S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN4ethoxyphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB d 2H J ¼ Hz AB 803e810 m4H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal Calcdfor C24H20I2N4O5S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34acetamidedihydroquinazolin2ylthioN35dimethoxyphenyl 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s6H s 2H dd 1H J ¼ Hz dd 2H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB s2H d 1H J ¼ Hz d 1H J ¼ Hz s 1H 13CNMR DMSOd6 d ppm Anal CalcdforC24H20I2N4O6S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN345trimethoxyphenyl acetamideFig Superimposition of compound magenta and the cocrystallized ligand redshowed that they adopt the same orientation inside the receptor For interpretation ofthe references to color in this ï¬gure legend the reader is referred to the Web version ofthis m 1H 731e756 m 3H d 2H J ¼ Hz AB d2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H18I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34Yielddihydroquinazolin2ylthioNptolylacetamide 00 NH NH2 mp 0eC IR KBr Ê cm arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 3H s 2H d 2H J ¼ Hz AB m 2H d 2H J ¼ Hz AB d 2H J ¼ Hz AB s 2H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm þ MS mz [] [M ] [] Anal Calcd forC23H18I2N4O4S2 C H N Found C H N acetamide 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2ethylphenyl 00 NH Yield mp 0eC IR KBr Ê cmNH2 arom aliph 2CO1619 CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H dd 1H J ¼ Hz m 1H ddd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz AB d 2H J ¼ Hz AB m 2H d1H J ¼ Hz d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz []þ ] [Mþ1 ] [] Anal Calcd for [MC24H20I2N4O4S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN3ethylphenyl 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm t 3H J ¼ Hz q 2H J ¼ Hz s 2H ddd 1H J ¼ acetamide 0c 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN SO2 1H NMR DMSOd6 d ppm s 6H s 3H s 2H d 2H J ¼ Hz d 2HJ ¼ Hz AB s 2H d 2H J ¼ Hz d 1HJ ¼ Hz AB d 1H J ¼ Hz s 1H 13C NMRDMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C25H22I2N4O7S2 C H N Found C H N 268Diiodo4oxo34sulfamoylphenyl34dihydroquinazolin2ylthioN2methyl4nitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 3H s 2H d 1H J ¼ Hz d 2HJ ¼ Hz AB 790e805 m 6H d 1H J ¼ Hz d 1HJ ¼ Hz s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C23H17I2N5O6S2 C H N Found C H N N2methyl6nitrophenyl 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthioacet 00 amide Yield mp 0eC IR KBr Ê cm NH NH2 arom aliph 2CO CN NO2 SO2 1H NMRDMSOd6 d ppm s 3H s 2H dd 1H J ¼ Hz dd 1H J ¼ Hz d 2H J ¼ Hz 801e810m 5H d 1H J ¼ Hz d 1H J ¼ Hz s 1H13C NMR DMSOd6 d ppm MS mz [] þ[M ] [] Anal Calcd for C23H17I2N5O6S2 C H N Found C H N 8Diiodo4oxo34sulfamoylphenyl3 dihydroquinazolin2ylthio N24dinitrophenyl acetamide 00 Yield mp 0eC IR KBr Ê cmNH NH2 arom aliph 2CO CN NO2 SO2 1H NMR DMSOd6 dppm s 2H d 2H J ¼ Hz AB d 1H J ¼ Hz800e804 m 2H d 2H J ¼ Hz AB d 1H J ¼ Hz830e834 m 3H s 1H 13C NMR DMSOd6 d ppm Anal Calcd for C22H14I2N6O8S2 C H N Found C H N Biological evaluation NQO1 in vitro inducer activityHepa1c1c7 murine hepatoma cells were grown in a humidiï¬edatmosphere at 0eC CO2 The cells were tested routinely toensure that they were mycoplasmafree The aminimum essentialmedium aMEM supplemented with vv heat andcharcoalinactivated g100 mL min at 0eC fetal bovineserum was used For evaluation of the potential NQO1 inducer activity cells 104well were grown in transparent ï¬atbottomplastic 96well plates for h after which the cell culture medium was replaced with fresh medium containing each inducerdissolved in DMSO and diluted in the medium and theAM Soliman European Journal of Medicinal Chemistry cells were grown for further h Three replicates of each treatment of eight serial dilutions of inducers were used The ï¬nal DMSOconcentration in the cell culture medium was maintained at vv in all wells Cell lysates were prepared in digitonin and thespeciï¬c activity of NQO1 was determined using menadione as asubstrate as described [] Brieï¬y the cell culture medium wasremoved from each well and the cells were washed three timeswith mL of phosphate buffered saline PBS and subsequentlylysed in mL of digitonin suspension in the presence of EDTA for min with shaking Of the cell lysate mL was transferred to t | Thyroid_Cancer |
"Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1 mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2 in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9To this end the central premise put forth in this study is that focused ultrasound FUSa safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissuescan synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17 colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbeccos Modified Eagles Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1 mice were obtained from The Jackson Laboratory 4T1 or E0771 cells à were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume lengthÃwidth22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a sham treatment consisting of exposure to the °C degassed water bath exposure for min Following sham or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to sham or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of vehicle treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day early αPD1 or day delayed αPD1T cell depletionsT cell depletion antibodiesanti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cellwere diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1BC The exceptionally small focus of this system rendered a low ablation fraction of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCsand CD86 DCs in particularsuggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C Sham mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test FH or two way analysis of variance followed by Tukey multiple comparison correction IK nsnot significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2BCBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly à and à reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2DE In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytesin particular CD8 and CD4 T cellsplay an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3BC and EF Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3BC and EF From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6AB Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cellsa reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells BC Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to EF Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to IK Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6 FUS monotherapy n4 GEM monotherapy n9 and combinatorial FUSGEM therapy n6 groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fishers least significant difference LSD without multiple comparisons correction for IK Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fishers LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6CD These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantationthat is days subsequent to final GEM administrationrevealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4BC Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4DE However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1 model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1 mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1 mice relative to their WT counterparts with average 4T1 tumor volume in Rag1 mice being over fivefold higher than that of WT mice on terminati | Thyroid_Cancer |
"variability around prevalence estimates of multimorbidity due to poorconsensus regarding its definition and measurement Medicationbased measures of morbidity may be valuableresources in the primarycare setting where access to medical data can be limited We compare the agreementbetween patient selfreported and medicationbased morbidity and examine potential patientlevel predictors ofdiscordance between these two measures of morbidity in an older ¥ years communitybased populationMethods A retrospective cohort study was performed using national pharmacy claims data linked to The IrishLongituDinal study on Ageing TILDA Morbidity was measured by patient selfreport TILDA and two medicationbased measures the RxRisk years and RxRiskV ¥ years which classify drug claims into chronic diseaseclasses The kappa statistic measured agreement between selfreported and medicationbased morbidity at theindividual patientlevel Multivariate logistic regression was used to examine patientlevel characteristics associatedwith discordance between measures of morbidityResults Two thousand nine hundred twentyfive patients were included years N and ¥ years N Hypertension and high cholesterol were the most prevalent selfreported morbidities inboth age cohorts Agreement was good or very good κ for diabetes osteoporosis and glaucoma andmoderate for high cholesterol asthma Parkinsons and angina κ All other conditions had fair or pooragreement Age gender marital status education poordelayed recall depression and polypharmacy weresignificantly associated with discordance between morbidity measuresConclusions Most conditions achieved only moderate or fair agreement between selfreported and medicationbased morbidity In order to improve the accuracy in prevalence estimates of multimorbidity multiple measures ofmultimorbidity may be necessary Future research should update the current RxRisk algorithms inline with currenttreatment guidelines and reassess the feasibility of using these indices alone or in combination with othermethods to yield more accurate estimates of multimorbidityKeywords Agreement Selfreport Rxrisk RxriskV Morbidity Polypharmacy Older people Correspondence caitrionacahirrcsiie Clionadh Mannion and John Hughes are joint first authors2Division of Population Health Sciences Royal College of Surgeons in IrelandDublin IrelandFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMannion BMC Geriatrics Page of Key pointsKey findings and implications Agreement between patient selfreported morbidityand medicationbased measures of morbidity RxRisk and RxRiskV was mainly moderate or fairDiabetes was the only condition for which the levelof agreement was found to be very good The results of our study indicate that neithermeasure of morbidity is completely reliable and wesuggest that researchers may require multiplemeasures selfreport and medicationbased measures of morbidity to fully capture accurate prevalence estimates of multimorbidity Our study identified several limitations of thecurrent versions of the RxRisk indices which require updating if medicationbased measures ofmorbidity are to be used to assess the epidemiologyof chronic conditions and multimorbiditytheofIndeedattentionBackgroundMultimorbidity is commonly defined as the presence oftwo or more chronic medical conditions and its prevalence has been shown to increase with age [] As theworlds older population continues to grow multimorbidity has become an important public health issue caphealthcareturingresearchersprofessionals as well as policy makersforhealthcare systems to effectively adapt and manage thedelivery of healthcare to our growing older populationan accurate description of the epidemiology of chronicconditions is required However to date studies in theliterature reveal wide disparities in prevalence estimatesof multimorbidity ranging from to [ ] Thislarge variability is thought to be due to the lack of standards defining multimorbidity and validated methods forhow it should be measured [] A recent systematic review reported definitions of multimorbidity involving differenttheappropriateness of different measures of multimorbidityis also variable depending on both the outcome of interest as well as the type of data that is available []In additioncriteria[]Measures of multimorbidity include diagnosisbasedmeasures eg Charlson Index based on hospital diagnosis codes ICD codes [] medicationbased measureseg RxRisk and RxRiskV for those aged ¥ yearsbased on pharmacy data [] and patient selfreportDiagnosisbased measures of multimorbidity are themost common measures and are generally based on hospital or physician records [] Medicationbased measures of multimorbidity include the RxRisk and RxRiskV two algorithms which determine an individualscurrent comorbidities based on their dispensed medication The RxRisk indexes only include morbidities forwhich a medicine could be prescribed and include categories of morbidities based on the World Health anisation WHO Anatomical Therapeutic Classification ATC system [] The RxRisk and RxRiskVhave good reliability and criterion validity against ICD9diagnoses and have been shown to predict costs of caremortality and health care utilisation [] Previous studies have reported medicationbased measures of morbidity such as the Medicines Disease Burden Index MDBIand RxRiskV to be useful in epidemiological studieswhen adjusting for comorbidity [] However there arefew studies describing the use of these indices to directlymeasure chronic conditions Patient selfreport is also avalid method of identifying disease categories A study ofolder patients with multimorbidity reported good agreement between patient selfreport and general practitioner GP report for a wide range of diseases []A number of studies have compared the differentmeasures of multimorbidity with differing results [ ] A study of older primary care patients inIreland found that medicationbased measures ofmultimorbidity such as RxRiskV performed betterthan diagnosisbased measures of multimorbidity inpredicting emergency and ambulatory care sensitiveACS admissions [] Studies comparing patientselfreport and diagnosisbased measures of multimorbidity have reported a stronger association between selfreport measures of multimorbidity andqualitythandiagnosisbased measures [ ] However no previous research has compared selfreported morbidityin the primary care or community setting with theRxRisk measures of morbidity Comparison betweenselfreported morbidity data and pharmacy records isimportant in order to understand the relative meritsof each measure of morbidity and the potential formisclassification particularly in the community setting where access to medical or clinical data can belimitedfunctionaloutcomesandlifeofStudies have also indicated that agreement betweenselfreport measures and other measures of morbiditymight be influenced by patient recall bias [] Patientrecall has been reported to be influenced by age maritalstatus and education [] There is also some evidencethat cognition and memory influence patient recall []The impact of these factors needs to be explored furtherwhen assessing and comparing measures of morbidityThe aim of this study was to compare the agreementbetween patient selfreported morbidity and medicationbased morbidity RxRisk and RxRiskV and examine potential patientlevel predictors of discordance between theincludingdemographic cognitive and mental health factors in anolder community based populationtwo measures of morbidity 0cMannion BMC Geriatrics Page of MethodsThe STrengthening the Reporting of ObservationalStudies in Epidemiology STROBE guidelines were usedin the reporting of this study []Study populationThis was a retrospective cohort study using data froma national pharmacy claims database the Health Service ExecutivePrimary Care Reimbursement ServiceHSEPCRS General Medical Services GMS schemelinked to the first wave of The Irish LongituDinalstudy on Ageing TILDA TILDA is a nationally representative sample of community dwelling individualsaged ¥ years in Ireland The sampling framework isbased on the Irish Geodirectory a comprehensive anduptodate listing and mapping ofresidential addresses in Ireland compiled by the Ordinance SurveyOffice and participants aged ¥ years were randomlyselected using the RANSAM sampling procedureThis meant that each residential address in Irelandhad an equal probability of selection and thus ensured that the TILDA sample was representative ofthe Irish population aged ¥ years The first wave ofdata collection began in October through toFebruary N participants aged ¥ yearswhere participants completed a computeraided personal interview CAPI and a health assessment measuring their health economic and social circumstancesFurther information on TILDAs study design andsampling framework is described in detail elsewhere[]The HSEPCRS GMS scheme is the largest pharmacy claims dataset in Ireland covering more than of the general Irish population [] It is meanstested and provides free health servicesincludingmedications to eligible persons in Ireland Qualification for the GMS scheme is on the basis of incomerelated meanstesting Automaticforthose aged ¥ years occurred between July andDecembercurrent study period meanstesting was introducedbut with a higher income threshold than the generalpopulation As of of men and ofwomen in the general population aged ¥ yearswere eligible [] The HSEPCRS GMS pharmacyclaims data were available for consenting TILDAparticipants aged ¥ years with GMS eligibility N entitlementhoweversinceJanuaryWithin the HSEPCRSGMS pharmacy claims dataprescriptions are coded using the WHO ATC classification system and prescriber information defineddaily doses strength quantity method and unit ofadministration of each drug dispensed are all available Pharmacy claims data was extracted for yearprior to each participants TILDA interview GMSpatientstypically receive their medications on amonthly basis []ifthey had any ofSelfreported morbidityAs part of the TILDA interview participants wereasked to reportthe followingdoctordiagnosed chronic diseases high blood pressure or hypertension high cholesterol angina congestive heart failure heart attack diabetes stroke orministroke abnormal heart rhythm arthritis osteoporosis cancer Parkinsons disease emotional nervous or psychiatric problems alcohol or substanceabuse dementia serious memory impairment stomach ulcers glaucoma incontinence or chronic painParticipants were also asked to selfreport urinaryincontinence in the past months as well as painmoderate or severe and if they were taking medication for pain management If participants reportedthat they had arthritisthey were asked to clarifythe type of arthritis eg osteoarthritis rheumatoidarthritis some other kind of arthritis Similarlyifparticipants reported emotional nervous or psychiatric problems they were asked to clarify from a listof conditions eg anxiety depression emotionalproblems psychosis manic depressionfillsthatclassify prescription drugMedicationbased measures of morbidity Rxrisk andRxriskVThe RxRisk and RxRiskV indices were applied tothe HSEPCRS pharmacy claims data The RxRiskindex was applied to the population aged yearswhile the RxRiskV was applied to the populationaged ¥ years The RxRisk and RxRiskV are algorithmsintochronic disease classes for older populations basedon the WHO ATC classification system []Within the RxRiskV cardiac disease is separatedinto a number of categories anticoagulation antiplatelet agents arrhythmias congestive heart failureCHFhypertension hypertensionischaemic heartdisease IHDangina and ischaemic heart diseaseIHDhypertension [] For a medication to be eligible as a measure of morbidity per RxRisk and RxRiskV chronic disease classes a patient was required to have been dispensed two or more consecutive prescriptions of the medication in question egdonepezil was required to be dispensed on ¥ consecutive prescriptions to link this medication withthe RxRiskV condition dementia This definitionhas previously been used by other pharmacoepidemiological studies [] 0cMannion BMC Geriatrics Page of Comparison of selfreported morbidity with Rxrisk andRxriskVEach selfreported condition in TILDA was matched tothe equivalent RxRisk and RxRiskV condition at theindividual patient level for those aged years and ¥ years respectively This was performed by consensusbetween two pharmacists FM CM For some selfreported conditions the ATC classes of medicationsspecific to these conditions eg antiwere notthrombotic agents B01AC04 B01AC30 were matchedto the selfreported condition of a heart attack and alsoto stroke There were four selfreported TILDA conditions which could not be matched to an RxRisk or RxRiskV condition but the prevalence was low Appendix in Tables and The RxRisk and RxRiskV alsoreported conditions which patients had not been askedabout during their TILDA interview Appendix in Tables and Patientlevel characteristics associated with discordancebetween the two measures of morbidityPatient characteristics were assessed to determine discordance patient recall bias between selfreported morbidity TILDA and the RxRisk years and RxRiskV ¥ years medicationbased measures of morbidity These characteristics were age gender maritalstatus education poor delayed recall depression andpolypharmacy Marital status was subcategorised intomarried never married separated or divorced Educationwas categorised into primarynone secondary or thirdhigher level education Delayed recall based on participants being presented with words during the interview and being later asked to recall as many as possiblewas defined as poor where or fewer words wererecalled Depression was defined as scoring or greateron the Centre for Epidemiologic Studies DepressionScale CESD [] Polypharmacy was defined as reporting regular use of five or more prescription medications[]Statistical methodsAgreement between selfreported morbidity TILDAand the RxRisk and RxRiskV measures of morbiditypharmacy claims was assessed using Cohens Kappastatistic as neither source was considered to be a goldstandard for reporting morbidity Interpretation of thevalue of Kappa was as follows poor fair moderate good and verygood []Multivariate logistic regression was used to examinethe association between the patientlevel characteristicsand discordance between the two measures of morbidityAdjusted odds ratios OR and confidence intervalsCIare presented Discordance was defined asparticipants reporting to have the condition in the absence of any dispensed medication for the condition perRxRisk years or per RxRiskV ¥ years andparticipants reporting to not have the condition butmedication was found to be dispensed for the conditionper RxRisk years or RxRiskV ¥ years Allsignificance tests were twotailed Statistical significancewas set at P after adjustment for a false discoveryrate of [] Analyses were performed using Stata SEVersion statistical package StataCorp College Station TXResultsStudy populationIn total patients were included in this cohortstudy patients were aged years and were aged ¥ years Characteristics ofthe study participants are presented in Table On average patients aged years had SD conditionsper the RxRisk and patients aged ¥ years had SD conditions per the RxRiskV The proportion ofpatients with thirdhigher level education was relatively years N low across both age ¥ years N Poor delayed recall years N ¥ years N years N ¥ years N were significantlymore prevalent in the older cohort compared to theyounger cohort p polypharmacygroupsandAgreement between selfreported morbidity andmedicationbased measures of morbidity Rxrisk and RxriskVTables and present a comparison between the number and percentage of patients selfreported morbiditiescompared to the RxRisk Table aged years andRxRiskV Table aged ¥ years measures of morbidity High blood pressure or hypertension yearsN ¥ years N and highcholesterol years N ¥ years N were the most prevalent selfreportedmorbidities in both age cohorts in the TILDA datasetHigh cholesterol was also found to be highly prevalentin the RxRisk N and RxRiskV N measures of morbidity Other prevalentRxRisk and RxRiskV conditions included arthritisRxRisk N stomach ulcers RxRiskN RxRiskV N strokeRxRiskV N heart attack RxRiskVN and other heart trouble RxRiskVN There was very good agreement between the selfreported TILDA measure of diabetes and the RxRiskand RxRiskV measures κ There was also good 0cMannion BMC Geriatrics Page of Table Characteristics of study participants by age years and ¥ years years N Age¥ years N GenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimarynoneSecondaryThirdHigher LevelPoor delayed recall YesDepression YesPolypharmacy Yes Data presented as N or mean CI unless otherwise statedagreement between selfreported measures of osteoporosis κ and glaucoma κ and the RxRiskV measure of these morbidities in the older cohort Despite the high prevalence of high cholesterolin both measures of morbidity there was only moderate agreement κ RxRisk κ RxRiskVbetween the two measures There was moderateagreement also for asthma κ RxRisk Parkinsons κ RxRiskV and angina κ RxRisk V Agreement was fair for selfreported highblood pressure or hypertension RxRisk and RxRiskV heart attack RxRisk stroke RxRisk abnormalheart rhythm RxRiskV cancer RxRisk depression RxRisk and RxRiskV and pain RxRiskVand RxRisk measures of these conditions κ All other conditions had poor agreement κ including arthritis RxRisk chronic lungdisease and incontinence RxRiskV and emotionalnervous psychiatric problems anxiety and stomach ulcers RxRisk and RxRiskV Tables Patientlevel characteristics associated with discordancebetween the two measures of morbidityAge gender marital status education poor delayedrecall depression and polypharmacy were all associated with discordance between the two measures ofmorbidity Table Females were five times morelikely to have discordance in reporting osteoporosisOR Confidence Intervals CI P Females were also more likely to have discordance in reporting anxiety OR CI emotional problems OR CI and depression OR CI as well as use of pain medication OR CI and incontinence OR CI They were less likely to have discordance in reporting stroke and high cholesterol TablePatients who were never married were less likely tohave discordance in reporting a heart attack OR CI and stroke OR CI Patients with third level educationwere lesslikely to have discordance in reportinghypertension OR CI comparedto those with primary level education Table Patients with poor delayed recall and depression weremore likely to have discordance in reporting anxietyand depression In general discordance was higher inpatients with polypharmacy Table found thatagreement between patientDiscussionWithin a population based study of ageing in Irelandweselfreported morbidity and medicationbased measures ofmorbidity RxRisk and RxRiskV was generally notgood with most conditions achieving only moderateor fair agreement There was very good agreementκ between selfreported diabetes and pharmacy dispensing records across both age cohortsThis was the only morbidity common to both age cohorts for which the level of agreement was found tobe very good Many research studies confirm this 0cGlaucomaHigh CholesterolAsthmaHigh blood pressure orHypertensionCancer or a malignant tumourDepressionStroke cerebral vascular diseaseParkinsonHeart attack including myocardialinfarction or coronary thrombosisManic depressionEmotional nervous or psychiatricproblem such as depression oranxietyCirrhosis or serious liver damageStomach ulcersArthritis including osteoarthritis orrheumatismN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX Hyperlipidaemia C10AA01C10BX17 Asthma R03AAR03AL R03BAR03BX R03CAR03CC R03DAR03DX Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02C09BA09 C09DA01C09DA07 C02AB01C02AC05 C02DB02C02KX01 Malignancies L01AA01L01XX31 Depression N06AA01N06AG02 N06AXAntiplatelet therapy B01AC04B01AC30Parkinsons disease N04AA01N04BX02Antiplatelet therapy B01AC04B01AC30Bipolar disorder N05AN01 Anxiety N05BA01N05BA12Anxiety N05BA01N05BA12Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11 GORD Peptic ulcer A02B A02BB A02BC Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01L04AB01L04AB05 L04AD01 L04AX03Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Anxiety Mannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRisk algorithm yearsTILDAStandardErrorSelfreported morbidityDiabetes or high blood sugarRxRisk Pharmacy ClaimsMedicationbased Morbidity ATCKappaκNAny other heart troubleRheumatoid arthritis only Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01Ministroke or TIAL04AB01L04AB05 L04AD01 L04AX03Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06ATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient coprescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm []same level of agreement for diabetes [ ] Thiswas expected given that previous research has demonstrated the reliability of reporting to be better inmorbidities for which there are clear diagnostic criteria eg diabetes [] Furthermore with many educational resources promoting selfmanagement of thiscondition patients with diabetes are more likely toplay an active role in managing their condition egregular selfmonitoring of blood glucose levels dietarymanagement recognising and dealing with symptomssuch as hypo and hyperglycaemia andor medication taking and are therefore more likely to selfreport accurately []There was good agreement between both measures ofmorbidity for osteoporosis and for glaucoma in the olderage group A MultiCare cohort study of primary carepatients in Germany found only moderate agreement between patientreported and GPreported osteoporosis[] A retrospective cohort study of older patients in asecondarycare setting in Canada also found moderateagreement for glaucoma between physician and patientreports [] Similar to diabetes patients are required toplay an active role in the management of osteoporosiswhile glaucoma is very often a comorbidity of diabetes[]There was moderate agreement between the measures of morbidity for asthma in the younger age cohort years Similar results have been reported for agreement between selfreported asthma and medical recorddata in older hospitalised patients [] There was alsomoderate agreement for high cholesterol in both agecohorts and for angina and Parkinsons disease in the 0cMannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRiskV algorithm ¥ yearsTILDASelfreported morbidityDiabetes or high blood sugarRxRiskV Pharmacy claimsMedicationbased Morbidity ATC KappaκNStandardErrorN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX OsteoporosisPagets disease M05BA01M05BB09 M05BX03Pain taking pain medication Pain Opioids N02AA01N02AX02 GlaucomaOsteoporosisParkinsonAnginaHigh CholesterolManic depressionHigh blood pressure orHypertensionG03XC01 A12AX92Parkinsons disease N04AA01N04BX02 Angina C01DA02C01DA14 C01DX16 C01EB17C01EB18 Hyperlipidaemia C10AA01C10BX17 Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02Bipolar disorder N05AN01C09BA09 C09DA01C09DA09 C02AB01C02AC05 C02DB02C02KX01PainAbnormal Heart RhythmDepressionDementiaChronic lung disease such aschronic bronchitis or emphysemaCancer or a malignant tumourEmotional nervous or psychiatricproblem such as depression oranxietyPain Inflammation M01AB01 M01AH06 Pain Opioids N02AA01N02AX02Pain Inflammation M01AB01 M01AH06 Arrhythmia C01AA05 C01BA01C01BD01 C01BD07 Depression N06AA01N06AG02 N06AX Dementia N06DA02 N06DA01Chronic airways disease R03AC02R03DC03 Malignancies L01AA01L01XX31 Anxiety N05BA01 N05BA12Congestive heart failureCirrhosis or serious liver damageHeart attack including myocardialinfarction or coronary thrombosis Chronic heart failure C03CA01C03CC01 C09AA01C09AA10C09CA01 C09CA03 C09CA06C09CA07Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11Antiplatelet therapy B01AC04B01AC30AnxietyStomach ulcersAlcohol or substance abuseAnxiety N05BA01N05BA12 GORD Peptic ulcer A02BA A02BCAny other heart trouble Stroke cerebral vascular diseaseMinistroke or TIA Alcohol dependence N07BB01 N07BB04Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Antiplatelet therapy B01AC04B01AC30Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06 B01AB10Incontinence Neurogenic Bladder Urinary Incontinence V07ANPsychotic illness N05AA01 N05AX17PsychosisATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient prescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm [] 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskVAge yearsGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecall YesDepression YesPolypharmacyYesAgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyHypertension HeartAttack StrokeTIAHigh Cholesterol HeartTrouble Cancer EmotionalProblems Depressiononly Stomachulcers Asthma Arthritisgeneral RheumatoidArthritis only Angina Congestive HeartFailure Abnormal HeartRhythm Anxiety LungDisease 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskV ContinuedHypertension HeartAttackOsteoporosis Psychosisonly StrokeTIAHigh CholesterolHeartTroubleCancerEmotionalProblemsAnxietyIncontinence PainPain meds AgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyExcluded diabetes Parkinsons disease manic depression cirrhosis glaucoma alcohol or substance abuse and dementia as number of patients misreporting wassmall N p older age cohort Other studies have reported loweragreement for high cholesterol and higher agreement forangina and Parkinsons diseases [ ] Discordancehere may be explained by patients managing their cholesterol using nonpharmacological means eg lifestylemodifications[]Interestingly the prevalence of selfreported angina inTILDA was higher than the prevalence reported by RxRiskV This may reflect poor patient adherence if prescribed medications were not dispensedincluding cardioprotective dietThere was only fair agreement between both measures of morbidity for hypertension despite hypertensionbeing the most prevalentselfreported morbidityacross both age cohorts Higher agreement betweenselfreported antihypertensive drug use and pharmacyrecords has been reported in a populationbasedstudy and a cohort study of older people in theNetherlands [ ] The discordance observed hereis likely attributable to the omission of a major group[]increasingantihypertensivesofcalciumchannelblockersCCBs in the current version of the RxRisk and RxRiskV algorithms [ ] This is significant giventhat CCBs are recommended as firstline therapy inpatients aged years [] Equally since hypertension is considered to be a condition without symptomsthis may influence patient adherence toantihypertensive medications and their proclivity tofill a prescription for these medications There wasalso fair agreement for pain in the older age groupwith agreementsomewhat when selfreported pain specified taking pain medication Theprevalence of selfreported pain was higher than themedicationbased RxRiskV prevalenceand thismay be due to patients managing their pain throughnonpharmacological or lifestyle interventions such asphysiotherapy and cognitive behavioural therapy []In both age cohorts there was poor to fair agreement between selfreporting of emotional problems 0cMannion BMC Geriatrics Page of poorfoundagreementeg depression anxiety and medicationbased measures These findings are consistent with previous research whichbetweenphysician diagnosis and patient selfreports of anxiety and depression [] This low level of agreementmay be due to a potential stigmatisation bias as only of patients regularly dispensed antidepressants selfreported as having depression in theolder age cohort [ ] Equallyit may be thatcertain antidepressants eg amitriptyline are beingused for other indications such as neuropathic pain[ ] There was also poor agreement in bothage cohorts for stomach ulcers and for incontinenceand chronic airways disease COPD in the older cohort Like depression poor agreement here may bedue to gastrointestinal medications being used by patients for other indications such as preventative orsymptomatic reasons [] The poor agreementforchronic airways disease may reflect the nonspecificquestion used in TILDA to measure this selfreportedmorbidity as there is evidence in the literature thatquestionnaire design is an important determinant ofpatient recall In a US study the prevalence of selfreported COPD was found to increase when more explicit questions were asked about emphysema chronicbronchitis and COPD in combination [] The pooragreement between the two measures for incontinenceis most likely reflective of the current version of theRxRiskV which compares selfreported urinary incontinence with dispensed diapers and pads supplies []agepoordelayedincreasingA number of factors were associated with discordance between the two measures of morbidity particularlyrecalldepression and polypharmacy A study determiningthe agreement between selfreported and diagnosisbased multimorbidity in older community dwellingwomen reported similar findings where agreementwas found to decrease with decreasing cognition andeducation increasing age and fo | Thyroid_Cancer |
"pharmacological therapies and treatments targeting pancreatic neuroendocrine tumorsPNETs have proven ineffective far too often Therefore there is an urgent need for alternative therapeuticapproaches Zyflamend a combination of antiinflammatory herbal extracts that has proven to be effective invarious in vitro and in vivo cancer platforms shows promise However its effects on pancreatic cancer in particularremain largely unexploredMethods In the current study we investigated the effects of Zyflamend on the survival of betaTC6 pancreaticinsulinoma cells TC6 and conducted a detailed analysis of the underlying molecular mechanismsResults Herein we demonstrate that Zyflamend treatment decreased cell proliferation in a dosedependent mannerconcomitant with increased apoptotic cell death and cell cycle arrest at the G2M phase At the molecular level treatmentwith Zyflamend led to the induction of ER stress autophagy and the activation of cJun Nterminal kinase JNK pathwayNotably pharmacological inhibition of JNK abrogated the proapoptotic effects of Zyflamend Furthermore Zyflamendexacerbated the effects of streptozotocin and adriamycininduced ER stress autophagy and apoptosisConclusion The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer viamodulation of the JNK pathwayKeywords Pancreatic neuroendocrine tumor cells Zyflamend JNK Apoptosis Autophagy ER stressPlain English summaryThrough investigating the effects of treating an experimental model of pancreatic neuroendocrine tumor cells withZyflamend we discovered a novel therapeutic potential ofthis polyherbal blend Findings from this study could helppioneer future advancements in our understanding of howphytochemicals and natural compounds could synergistically prove effective against pancreatic cancer by alteringcancer cell survival and proliferation Furthermore the evidence presented within promotes Zyflamend as an adjuvantprospect where it could enhance the effectiveness of standard cancer therapies In addition we believe that thesenovel findings will be of major interest to a broad spectrumof scientists and may pave the way towards more effectiveand translatable therapies Correspondence abettaieutkedu1Department of Nutrition University of Tennessee Knoxville CumberlandAvenue Jessie Harris Building Knoxville TN USA3Graduate School of Genome Science and Technology University ofTennessee Knoxville TN USAFull list of author information is available at the end of the BackgroundPancreatic cancer remains one of the deadliest types ofcancer in the United States with over new casesand deaths in accounting for of allcancer deaths [] Recent epidemiological studies predict The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cPuckett Cell Communication and Signaling Page of that in pancreatic cancer will be the third leadingcause of cancerrelated death [] Although advancements in science and health care have led to decreasedmortality from numerous forms of cancer pancreaticcancer survival rates have not improved significantlyover the past several decades leaving a desperate needfor more effective treatment options The risk of developing pancreatic cancer has been associated with numerous biological environmental pathological andgenetic factors These factors include variables such asfamilial history chronic pancreatitis smoking obesityand diabetes reviewed in [ ] In addition hereditaryfamilial factors and germline mutations could contributeto increased risk of cancer onset However the survivaloutcomehighlydependent upon the time of diagnosis While pancreaticductal adenocarcinoma PDAC is the most commonlycontracted and investigated subtype the pathological nature ofthe rarer pancreatic neuroendocrine tumorsPNETSs remains elusive []effectivenesstreatmentandarePNETs account for less than of all pancreatic cancers and are often diagnosed at a late stage in patientswith advanced metastasis making surgery a nonviabletreatment option [ ] Additionally because of theirheterogeneous clinical presentation and responses tochemotherapeutic agents current pharmacological therapies and treatment options targeting PNETs have toooften proven ineffective [] PNETs treatment optionsoften include the use of chemotherapeutic compoundssuch as streptozocin 5fluorouracil doxorubicin andcisplatin both alone or in combination reviewed in [] The effectiveness of these compounds often increases at higher doses but this directly exacerbates therisk for cytotoxicity and collateral side effects [] Inaddition adjunct therapy involving the combination ofvarious treatment approaches such as surgery and radiotherapy is often implemented [] In pursuit of survivaland improved quality of life patients often seek to enhancetherapiesthrough dietary and supplemental means [ ]effectiveness ofconventionaltheNew Chapter Brattleboro VT first launched Zyflamend based on the idea of combining extracts of ten different herbs to effectively reduce inflammation throughcyclooxygenase COX inhibition [] A large volume ofresearch has emerged over the last two decades that supports the antiinflammatory properties of Zyflamend andits ability to inhibit COX in various types of cancer including prostate [] melanoma [] and oral cancer[] Individually many of the extracted components ofZyflamend have proven to exhibit anticancer activity[ ] However the high doses required to optimizeeffectiveness against cancer could prove infeasible forthe majority In theory the combined effects generatedthrough integrating these unique and powerful herbscould grant superior benefit over their isolated form[] Additionally Zyflamend has shown the capabilityto interact with a variety of integral cellular signalingpathways beyond COX These signaling pathways andmechanisms of interaction include AMPactivated protein kinase AMPK [ ] nuclear factor kappalightchainenhancer of activated B cells NFκB [ ]mammalian target of rapamycin mTORC1 [] apoptosis cell growth [ ] endoplasmic reticulum ERstress [ ] and finally autophagy [] Whilethese studies show that Zyflamend could exhibit profound potential in the therapeutic application more research is required to elucidate the molecular basisunderlying its anticancer effects In the current studywe investigated the effects of Zyflamend on the survivalof betaTC6 pancreatic insulinoma cells TC6 anddeciphered the underlying molecular mechanismsMethodsChemicals and reagentsMedia sera and trypsin for cell culture were purchasedfrom Gibco Thermo Fisher Scientific Waltham MAPrimary antibodies and secondary antibodies were acquired from varying sources Supplementary Table General caspases inhibitor ZVADfmk was obtainedfrom Calbiochem La Jolla CA Zyflamend¢whole bodywas purchased from New Chapter New Chapter IncBrattleboro VT Zyflamend composition is indicated inSupplementary Table Quality assurance is in full compliance with Good Manufacturing Practicing Standardsas mandated by CRF Part Additionally full description and characterization of Zyflamend and itspreparation have been previously described in detail[] Chemical reagents such as dithiothreitol DTTpercoll digitonin phenylmethylsulfonyl fluoride PMSFprotease inhibitors cocktail sodium deoxycholate Tritonglycolbis2aminoethylNNN²NX100²tetraacetic acid EGTA sodium fluoride NaF sodium phenylbutyrate 4PBA Hoechst propidium iodide streptozotocin STZ adriamycin ADRautophagy inhibitor 3methyladenine 3MA and JNKinhibitor SP600125 were acquired from MilliporeSigma Burlington MA Finally AMPK inhibitor BML aka compound C was purchased from Santa CruzBiotechnology Santa Cruz CAethyleneCell cultureMouse betaTC6 pancreatic insulinoma TC6 ATCC®CRL11506¢ and rat pancreatic insulinoma RIN5FATCC® CRL2058¢ cells were cultured as monolayersin Eagles modified Dulbecco medium plus Lglutamine mM sodium pyruvate mM and fetal bovineserum FBS GibcoThermo Fisher Scientific WalthamMA Cells were maintained in tissue culture plates 0cPuckett Cell Communication and Signaling Page of Thermo Fisher Scientific Waltham MA at °C in ahumidified atmosphere of CO2 Medium was replaced with fresh medium h before experimentsZyflamend treatmentZyflamend was dissolved in dimethyl sulfoxide DMSOat a concentration of mgml Cells were treated withZyflamend at the indicated concentrations and for theindicated durations Treatments were terminated by twowashes with icecold phosphate buffer saline PBSPlates were then flashfrozen in liquid nitrogen andstored at °C until further analysesProliferation assayCell proliferation assay was performed using the sulforhodamine B SRB MilliporeSigma method as previously described [] with modification Briefly an equalnumber of TC6 cells X cells were seeded in well plates Six h later cells were treated with the indicated concentrations of Zyflamend and incubated at °C in an atmosphere of CO2 for the indicatedtime Treatment was stopped by two washes with icecold PBS and cells were fixed with trichloroaceticacid in PBS Intracellular proteins were stained for min at room temperature using SRB dissolved in acetic acid Excess SRB stain was removed by rinsingthe plates thoroughly with running tap water Plateswere airdried for at least h prior to dissolving the stainin mM Tris pH Intracellular proteins werequantified using the Synergy¢ HTX MultiMode microplate reader BioTek Instruments Inc Winooski VT ata wavelength of nm The relative survival rates ofcells were determined by dividing the absorbance observed for a given treatment by the absorbance detectedin control cells treated with DMSO and expressed as afold changeCytotoxicity assayThe MTT [45dimethylthiazol2yl]25diphenyltetrazolium bromide cytotoxicity assay was performed aspreviously described with modification [] Briefly cells were plated in a 96well plate for h Then afreshly prepared solution of Zyflamend alone or in combination with ZVADfmk 10uM 4PBA 250uMSP600125 10uM STZ mM or adriamycin μMfor an additional h The experiment was terminatedby adding μl of the MTT solution mgml to eachwell for h then the cell culture medium was removedand the dye was dissolved in μl SDS solution overnight at °C Relative cytotoxicity was determinedby measuring the absorbance at nm using the Synergy¢ HTX MultiMode microplate readerColognic testColonie formation assay was performed as previously described [ ] with modification Cells were seeded inthe presence of DMSO control Zyflamend alone or incombination with ZVADfmk uM 4PBA uM SP600125 uM STZ mM or adriamycin μM After h media was replaced with a freshlyprepared new cell culture media and plates were incubated for days at °C in an atmosphere of CO2After incubation the colonies were washed with icecoldPBS fixed and stained with a mixture of glutaraldehyde and crystal violet for min The plates werewashed with water dried and colonies with morethan cellscolony were counted The relative number of colonies in each condition was determined bydividing the number of colonies for a given treatmentby the total number of colonies in DMSO treatedcells control and expressed as a percentage relativeto DMSOtreated cells CtrlWestern blotting analysisCells were lysed in radioimmunoprecipitation assayRIPA buffer as previously described [] Lysates wereclarified by centrifugation at g for min andprotein concentrations was determined using bicinchoninic acid assay kit Pierce Chemical Dallas TX Proteins μg were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresisSDSPAGE andtransferred to polyvinylidene fluoride PVDF membranes Immunoblotting of lysates was performed withprimary antibodies Supplementary Table and afterincubation with secondary antibodies proteins were visualized using Luminata¢ Forte Western Chemiluminescent HRP Substrate MilliporeSigma Pixel intensitiesofusingFluorChem Q Imaging software Alpha Innotech CorpSan Leandro CA Data for phosphorylated proteins arepresented as the intensity of phosphorylation normalizedto total protein expression while total protein expression was normalized to the loading control actinimmunoreactivebands werequantifiedMorphological analysis of apoptosisTC6 cells were exposed to Zyflamend for the indicated duration then washed with PBS and labeledwith Hoechst μgmlbluegreenfluorescence Hoechst binds to condensed nuclearchromatin [] and was used to visualize apoptoticcells green fluorescence by fluorescence microscopyLeica DMI8 Leica Microsystems Inc Buffalo GroveIL For each condition atleast cells werecounted Percentages of apoptotic cells were calculated relative to total cellsin PBS 0cPuckett Cell Communication and Signaling Page of MilliporeSigmaAnnexin V stainingQuantification of externalized phosphatidylserine anearly event in the apoptotic cascade was performedusing flow cytometry as previously described [] withmodification Briefly confluent TC6 cellswere exposed to Zyflamend for h then washed withPBS and resuspended in μl of PBS containing FBS Immediately after an equal volume ofthe 2XGuava Nexin reagentcontainingAnnexin V Fluorescein isothiocyanate FITC and aminoactinomycin D 7AAD was added to each treatment and incubated for min at room temperatureunder lightprotected conditions Intensities of fluorescence emitted by Annexin V FITC and 7AAD weremeasured using the Guava® easyCyte Flow CytometerMilliporeSigma on PM1 and PM2 channels respectively Viable negative for both Annexin V and 7AADstaining and apoptotic cells both at early Annexin Vpositive 7AAD negative and late positive for bothAnnexin V and 7AAD stages were quantified using theInCyte¢ and GuavaSuite Software package LuminexCorp Austin TXCell cycle analysisCell cycle analysis was conducted through assessing theDNA content of cells stained with propidium iodide aspreviously described [] with modification Briefly confluent TC6 cells were starved in serummedia for h then complete growth media was addedto the cells along with various freshly prepared concentrations of Zyflamend h later cells were harvestedwashed twice with icecold with PBS and fixed overnight in ethanol at °C Next cells were washedtwice with icecold PBS and incubated in a freshly prepared RNase solution [ mM TrisHCl pH containing Uml of DNasefree RNase A AppliedBiosystems Austin TX] for min at °C Cells werewashed twice with icecold PBS and incubated in a solution of propidium iodide PI μgml in PBS overnightat °C under light protected conditions Fluorescenceintensity of PI was measured using the Guava® easyCyteflow cytometer on PM2 channel DNA histogram analysis was performed on cells using the InCyte¢ andGuavaSuite Software package and the proportions ofcells with one or two copies of their chromosomal DNAwere calculatedStatistical analysisData were analyzed using JMP Pro program SASInstitute NC and presented as means standard errorof the mean SEM Unpaired heteroscedastic twotailStudents t test was used for all statistical analyses anddifferences were considered significant at p Singlesymbol such as or was used to indicate a p valuethat is less than while double symbol such as or corresponds to a p value that is less than ResultsZyflamend decreases cell proliferation causes G2M cellcycle arrest and induces apoptotic cell death inpancreatic cancer cellsWe first examined the effects of varying doses of Zyflamend on the proliferation of pancreatic insulinoma TC6 cells Zyflamend caused a significant dose andtimedependent decrease in cell growth Fig 1a Additionally a Zyflamend dose of μgml was sufficient toinhibit cell proliferation by after h of treatmentwhile a dose of μgml completely abolished cell proliferation Fig 1a In line with these findings cell cycleanalysis demonstrated that Zyflamend alters cell cycledistribution in a dosedependent manner Indeed Zyflamend treatment resulted in the enrichment of the G2Mfraction with N DNA content which was accompaniedby a reduction in cell cycle progression through the G0G1 and S phases Fig 1bc These results suggest thatZyflamendinduced inhibition of cell proliferation is mediated at least in part through cell cycle arrest in theG2M phaseIn order to determine whether Zyflamendinduced inhibition of cell proliferation was associated with apoptotic cell death we determined changes in apoptosis inTC6 cells treated with increasing doses of Zyflamend and μgml for h usingtwo approaches the Guava Nexin Annexin V assay andHoechst stain Using the Annexin V assay thepercentages of both Annexin V positive7AAD negativecells reflective of early apoptotic cells and Annexin Vpositive7AAD positive cells reflective oflate apoptosis exhibited a dosedependent and significant increase in response to Zyflamend treatment Fig 1deConsistent with this observationthe number ofHoechstpositive cells was also higher in Zyflamendtreated cells compared to control cells Fig 1fgHoechst is a nucleic acid dye that binds to condensed chromatin in the nucleus of apoptotic cells thusgiving an assessment of overall apoptotic cell death []At a dose of and μgml the percentages ofapoptotic cells were ± ± and ± respectively further emphasizing the proapoptotic effects of Zyflamend on these cells Similarfindings were obtained using the MTT assay Fig 1hA human equivalent dose of Zyflamend induces apoptoticcell death in TC6 cellsTo further characterize the proapoptotic properties ofZyflamend we conducted a time course analysis using aphysiological relevant fixed dose of Zyflamend μgml [] This dose is representative of the maximum 0cPuckett Cell Communication and Signaling Page of Fig Zyflamend Reduces Cell Survival and Induces Cell Death of Pancreatic Cancer Cells in a Dose Dependent Manner a Effects of Zyflamendon cell survival and proliferation cells were treated with increasing doses of Zyflamend for h Line graphs represent the intensity of SRBstaining reflective of the cell number and presented as means SEM bc Cell cycle analysis and assessment of DNA content in TC6 cellstreated with DMSO control or the indicated concentration of Zyflamend for h Representative histogram distributions for each treatment areshown c Bar graphs represent the percentages of cells in each phase of the cell cycle which were estimated using the GuavaSuite Softwarepackage and are presented as means SEM from three independent experiments p p indicate significant difference betweenthe indicated concentration and control cells treated with the vehicle DMSO de Zyflamend treatment induces apoptosis in TC6 Cells confluent cells were treated with increasing concentrations of Zyflamend and then labeled with Annexin VFITC and 7AAD Representative dotplots are shown Annexin V positive and 7AAD negative cells lower right quadrants represent early stages of apoptosis whereas cells that arepositive for both Annexin V and 7AAD upper right quadrants are in late stages of apoptosis e Bar graphs represent live early and lateapoptotic cells are presented as means SEM of at least three independent experiments p p indicate significant differencebetween the indicated concentration of Zyflamend and control cells treated with the vehicle DMSO fg Chromatin condensation in cells treatedwith increasing doses of Zyflamend for h Representative images are shown Scale bar μm g Bar graphs represent the number of apoptoticcells Hoechst positive as means SEM of at least three independent experiments h Cell toxicity assay using the MTT method Bar graphsrepresent the intensity of formazan produced from MTT by viable cells staining reflective of the cell number and presented as means SEM ofat least three independent experiments In g and h p p indicate a significant difference between cells treated with Zyflamendand nontreated cellstreatmentplasma concentration of a primary ingredient of Zyflamend curcumin that was reported in humans after oraladministration [] At this dose a marked increase inchromatin condensation and apoptotic cell number wasobserved after h ofFig 2ab Subsequently markers of apoptosis and cell survival were investigated using Western blotting Zyflamend inducedcleavage of caspase3 and its downstream target polyADPribose polymerase PARP Fig 2cd In additionwe examined changes in the mitogenactivated proteinkinases MAP kinases pathways in response to Zyflamend Our data revealed that TC6 cells treated withZyflamend exhibited a marked decrease in the phosphorylation of protein kinase B AKT and extracellularsignalregulated kinases ERK particularly after h oftreatmentTo determine whether Zyflamendinduced cell deathwas associated with the caspase dependent pathwaysof apoptosis we tested whether blocking caspasesactivity usingcarbobenzoxyvalylalanylaspartyl[Omethyl]fluoromethylketone ZVADfmk could inhibit Zyflamendinduced chromatin condensation andapoptosis ZVADfmk is a potent cell permeable pancaspase inhibitor which acts by irreversibly bindingto the catalytic site of the caspase proteases and thusinhibiting their activities Our study shows that pretreatment with ZVADfmk caused a significant decreased in the levels of chromatin condensation inZyflamendtreated cells Fig 2ef Additionally ZVADfmk treatment alleviated Zyflamendinduced celltoxicity as judged by the MTT Fig 2g and the colony formation Fig 2hi assays Taken together ourfindings indicate that Zyflamend treatment reducescell viability and induces cell death through the induction of the apoptotic machinery in TC6 cellsZyflamend induces ER stress apoptosis and autophagyresponses in TC6 cellsA plethora of intrinsic and extrinsic pathways can leadto apoptosis in response to stressors including ER stressand autophagy among many more Therefore in orderto dissect the precise molecular mechanism mediatingthe proapoptotic effects of Zyflamend we examined theactivation of key signaling molecules related to thesepathways Zyflamend μgml significantly inducedER stressactivation of ERjudged byasthe 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Apoptotic Cell Death in TC6 Cells ab Effects of Zyflamend on chromatin condensation Cells were treated withZyflamend μgml for the indicated time and chromatin condensation was evaluated by fluorescence microscopy using Hoechst Representative images are shown Scale bar μm b Bar graphs represent the number of apoptotic cells Hoechst positive as means SEMp indicates a significant difference between cells treated with Zyflamend and nontreated cells cd Immunoblots of key proteins in cellsurvival and apoptosis markers in cells treated with μgml of Zyflamend for the indicated time d Bar graphs represent cleaved caspase3 CCasp 3actin cleaved PARP CPARPactin pAKTAKT and pERKERK as means SEM p p indicates a significant differencebetween cells treated with Zyflamend and nontreated cells ef Chromatin condensation in TC6 cells treated with μgml Zyflamend withand without the pancaspase inhibitor ZVADfmk Representative images are shown Scale bar μm f Bar graphs represent the number ofapoptotic cells Hoechst positive as means SEM g Cell toxicity assay using the MTT method Bar graphs represent the intensity of formazanstaining reflective of the cell number and presented as means SEM hi Colony formation assay i Bar graphs represent the relative number ofcolonies in each condition determined by dividing the number of colonies for a given treatment by the total number of colonies in DMSOtreated cells Ctrl and expressed as a percentage In g and i p p indicate a significant difference between cells treated withZyflamend and nontreated cells p p indicate a significant difference between cells treated with ZVADfmk and nontreatedproteinCHOPby Westerntransmembrane sensors protein kinase RNAlike endoplasmic reticulum kinase PERK and inositolrequiringtransmembrane kinaseendoribonuclease 1α IRE1αalong with downstream targets such as eukaryotic translation initiation factor alpha EIF2α and CEBP homologousblottingZyflamend induced ER stress as evidenced by increasedPERK Thr980 EIF2α Ser51 and IRE1α Ser724 phosphorylation Fig 3a Furthermore the level of CHOPexpression was elevated a direct downstream target ofboth the PERK and IRE1 pathways The activation ofCHOP a potent inducer of apoptotic cell death [ ]in response to ER stress Fig 3ab strengthens our conclusions of Zyflamendinduced apoptosis in these cellsMoreover Zyflamend has been shown to activateAMPK and our results recapitulate these previous findings []The AMPK signaling pathway has been shown toinregulate autophagy and cell death [] Thereforeorder to assess whether Zyflamend induces autophagy inTC6 cells we immunoblotted for autophagyrelatedproteins We observed a time dependent increase inbeclin microtubuleassociated proteins 1A1B lightchain LC3I II and autophagyrelated proteins and ATG57 Fig 3cd The increase in the expressionof these proteins is indicative of elevated autophagy inthese cells Because ER stress inflammation and autophagy can all lead to apoptosis we used the pancaspaseinhibitor ZVADfmk to determine which pathway mightbe responsible for the proapoptotic effects of Zyflamend Our data shows that while there was a significantattenuation of Zyflamendinduced cleavage of caspase3and its downstream target PARP treatment with ZVADfmk had no effects on Zyflamendinduced activation of the AMPK autophagy and ER stress signalingcascades Fig 3ef These findings suggestthat ERstress autophagy and MAP kinases pathways are upstream of the apoptotic signaling cascade that might bemediating the proapoptotic effects of Zyflamend in TC6 cellsZyflamendinduced cell death is mediated through the ERstressJNKautophagy pathwayThe exact molecular mechanisms leading to apoptosisby Zyflamend in cancer cells hashave not been revealedyet although recent studies have supported the role ofAMPK in the regulation of cancer cell growth bioenergetics autophagy and cell death To investigate the potential role of AMPK in Zyflamendinduced apoptosiswe pretreated cells with the AMPK inhibitor compoundC CC μM for h prior to Zyflamend treatment foran additional h The dose and duration of exposurewere determined based on the ability of compound C toreverse AMPKdependent inhibitory phosphorylation ofacetylCoA carboxylase ACC data not shown Cellswere then examined for AMPK activation as well as activation of inflammation ER stress autophagy and celldeath Fig 4a While the level of phosphorylated AMPKwas reduced in cotreated cells pretreatment with compound C had no effects on Zyflamendinduced JNKphosphorylation ER stress autophagy or cell death Fig4ab These data suggest that Zyflamendinduced apoptosis in TC6 cells is independent of AMPK activationNext we sought to examine whether blocking autophagyusing 3MA could protect cells against Zyflamendinduced apoptosis 3MA inhibits autophagy by blockingautophagosome formation via the inhibition of class Iand class III phosphatidylinositol 3kinases PI3K []Cells were preincubated with 3MA nM for hprior to Zyflamend treatment Cotreatment with Zyflamend and 3MA significantly decreased the expressionof beclin LC3 and cleaved caspase3 but had no effects on ER stress markers nor on AMPK phosphorylation Fig 4cd These data suggest that autophagymediates Zyflamendinduced apoptosis and that bothAMPK and ER stress activation by Zyflamend occur upstream of autophagy and apoptosisThe relationship between these two fundamental processes ER stress and autophagy is complex and poorlyunderstood Recent literature demonstrates that bothpathways display dual roles in cell survival in multiple 0cPuckett Cell Communication and Signaling Page of Fig See legend on next page 0cPuckett Cell Communication and Signaling Page of See figure on previous pageFig Zyflamend Induces Inflammatory ER Stress and Autophagy Responses in TC6 Cells ab Total cell lysates from control and Zyflamendtreated cells for and h were immunoblotted for ER stress markers pPERK pEIF2α pIRE1α their respective unphosphorylatedproteins sXBP1 CHOP and actin as a loading control Representative immunoblots are shown b Bar graphs represent pPERKPERK pEIF2αEIF2α pIRE1IRE1 sXBP1actin and CHOPactin as means SEM p p indicate a significant difference between cells treatedwith Zyflamend and nontreated cells cd Markers of autophagy were examined in the same lysates using antibodies against Beclin LC3 IIIATG5 ATG7 and actin as a loading control d Bar graphs represent Beclin 1actin LC3actin ATG5actin and ATG7actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells ef Immunoblots of keyproteins in autophagy AMPK ER stress and apoptosis signaling in TC6 cells treated with μgml Zyflamend with and without the pancaspase inhibitor ZVADfmk Representative immunoblots are shown f Bar graphs represent pAMPKAMPK pPERKPERK pEIF2αEIF2α pIRE1αIRE1α sXBP1actin CHOPactin pJNKJNK Beclin 1actin LC3IIIactin and cleaved caspase3actin as means SEM p p indicate a significant difference between cells treated with Zyflamend and nontreated cells p p indicate a significant differencebetween cells treated with ZVADfmk and nontreated cellscancer celllines Similar to ER stress autophagy hasbeen shown to promote cell survival by clearing unwanted components from the cells Nonetheless a considerable body of evidence also indicates that bothautophagy and ER stress can lead to apoptosis in tumorcells In addition to this a growing body of literaturesupports existing crosstalk between the two pathways[ ] However which pathway is upstream of theother is yet to be determined Our data suggest thatZyflamendinduced autophagy is likely to be downstream of ER stress To test this hypothesis we pretreated TC6 cells with an ER stress inhibitor phenylbutyrate 4PBA mM for h prior to Zyflamend treatment and we examined changes in inflammation ER stress autophagy and cell death Fig 4ef PBA is a cell permeant chemical chaperone that hasbeen shown to inhibit ER stress and ER stressinducedapoptosis in many cancer cell types including pancreaticcancer cells [ ] Our findings show a profound decrease in ER stress autophagy and cell death markers inresponse to Zyflamend Fig 4ef when cells were pretreated with 4PBA Additionally 4PBA treatment alleviated the decrease in cell proliferation Fig 4g and colonycaused by ZyflamendFurthermore pretreatment of TC6 cells with 4PBAreduced Zyflamendinduced chromatin condensationFig 4jk Conversely 4PBA did not alter the activationof AMPK by Zyflamend Fig 4ef suggesting that ERstress occurs upstream of autophagy and apoptotic celldeathformation Fig 4hiPrevious studies have shown that the ER stress sensorIRE1 may promote autophagy through the TRAF2ASK1JNK pathway [ ] To test this hypothesis wetreated TC6 cells with SP600125 a selective JNK inhibitor and investigated changes in inflammation ERstress and proliferation in response to Zyflamend treatmentFig As expected cells pretreated withSP600125 exhibited a significant reduction in the phosphorylation of JNK and reduced expression of autophagyand cell death markers in response to Zyflamend Fig5ab Likewise JNK inhibition protected TC6 cellsfrom Zyflamendinduced reduction in cell survival Fig5c colo | Thyroid_Cancer |
Epidemiologic and clinical features of patients with COVID19 in BrazilCaractersticas epidemiolgicas e clnicas dos pacientes com COVID19 no BrasilVanessa Damazio Teich1 Sidney Klajner1 Felipe Augusto Santiago de Almeida1 Anna Carolina Batista Dantas1 Claudia Regina Laselva1 Mariana Galvani Torritesi1 Tatiane Ramos Canero1 Ot¡vio Berwanger1 Luiz Vicente Rizzo1 Eduardo Pontes Reis1 Miguel Cendoroglo Neto1 Hospital Israelita Albert Einstein S£o Paulo SP Brazil 1031744einstein_journal2020AO6022 Objective This study describes epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil Methods In this retrospective singlecenter study we included all laboratory confirmed COVID19 cases at Hospital Israelita Albert Einstein S£o Paulo Brazil from February until March Demographic clinical laboratory and radiological data were analyzed Results A total of patients with a confirmed diagnosis of COVID19 were included in this study Most patients were male with a mean age of years A history of a close contact with a positivesuspected case was reported by of patients and had a history of recent international travel The most common symptoms upon presentation were fever nasal congestion cough and myalgiaarthralgia Chest computed tomography was performed in patients and of those showed abnormal results Hospitalization was required for patients and were admitted to the Intensive Care Unit Regarding clinical treatment the most often used medicines were intravenous antibiotics chloroquine and oseltamivir Invasive mechanical ventilation was required by of Intensive Care Unit patients The mean length of stay was days for all patients and days for patients requiring or not intensive care respectively Only one patient died during followup Conclusion These results may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemicKeywords Communicable diseases Lung diseasesepidemiology SARSCoV2 COVID19 Coronavirus infections Epidemiology RESUMOObjetivo Descrever as caractersticas epidemiolgicas e clnicas de pacientes com infec§£o confirmada pelo SARSCoV2 diagnosticados e tratados no Hospital Israelita Albert Einstein que admitiu os primeiros pacientes com essa condi§£o no Brasil Mtodos Neste estudo retrospectivo de centro ºnico inclumos todos os casos com confirma§£o laboratorial de COVID19 no Hospital Israelita Albert Einstein em S£o Paulo SP de fevereiro a mar§o de Foram analisados dados demogr¡ficos clnicos laboratoriais e radiolgicos Resultados Foram includos pacientes com diagnstico confirmado de COVID19 A maioria dos pacientes era do sexo masculino com mdia de idade de anos Foi relatada histria de contato prximo com um caso positivosuspeito por dos pacientes e tinham histria de viagens internacionais recentes Os sintomas mais comuns foram febre congest£o nasal tosse e mialgiaartralgia A tomografia computadorizada de trax foi realizada em pacientes e deles apresentaram How to cite this Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Epidemiologic and clinical features of patients with COVID19 in Brazil einstein S£o Paulo 202018eAO6022 httpdx1031744einstein_journal2020AO6022Corresponding authorVanessa Damazio Teich Avenida Albert Einstein MorumbiZip code S£o Paulo SP Brazil Phone Email vanessateicheinsteinbrReceived onJuly Accepted onJuly Conflict of interest noneCopyright This content is licensed under a Creative Commons Attribution International LicenseORIGINAL ISSN eISSN 23176385Official Publication of the Instituto Israelita de Ensino e Pesquisa Albert Einsteineinstein S£o Paulo 0cresultados anormais A hospitaliza§£o foi necess¡ria para pacientes e foram admitidos na Unidade de Terapia Intensiva Quanto ao tratamento clnico os medicamentos mais utilizados foram antibiticos intravenosos cloroquina e oseltamivir A ventila§£o mec¢nica invasiva foi necess¡ria em dos pacientes na Unidade de Terapia Intensiva O tempo mdio de interna§£o foi dias para todos os pacientes e dias para pacientes que necessitaram ou n£o de cuidados intensivos respectivamente Apenas um paciente morreu durante o acompanhamento Conclus£o Estes resultados podem ser relevantes para o Brasil e outros pases com caractersticas semelhantes que come§aram a lidar com essa pandemiaDescritores Doen§as transmissveis Pneumopatiasepidemiologia SARSCoV2 COVID19 Infec§µes por coronavrus Epidemiologia INTRODUCTIONSince December several cases of pneumonia of unknown origin have been reported in Wuhan China1 The pathogen was further identified as a novel RNA coronavirus currently named as severe acute respiratory syndrome coronavirus SARSCoV22 Huang et al reported the first cases in China with a common clinical presentation of fever cough myalgia fatigue and dyspnea with an dysfunction eg acute respiratory distress syndrome ARDS shock acute cardiac and kidney injuries and death in severe cases3Afterwards in January the World Health anization WHO declared the outbreak a Public Health Emergency of International Concern PHEIC and next in March it was characterized as a pandemic4 As of April a total of cases had been reported in countries and regions across all five continents with deaths worldwide5 More recently the Chinese Center for Disease Control and Prevention published data on patients with classified as confirmed cases of coronavirus disease COVID19 Most patients were aged to years with mild clinical presentation ie nonpneumonia and mild pneumonia and overall casefatality rate of increased in elderly population with casefatality rate of in those aged years and older6On February the first Brazilian patient had a confirmed diagnosis of COVID19 at Hospital Israelita Albert Einstein HIAE Hospital Israelita Albert Einstein is a philanthropic hospital in the city of S£o Paulo SP Brazil with twelve health care units including a quaternary hospital with beds and four outpatient emergency care units By the end of this study on March of patients with confirmed COVID19 in Brazil had been diagnosed at HIAE Given the rapid spread of the COVID19 clinical and epidemiological data of several countries are being published on a daily basis79 However no studies have been reported to date presenting the characteristics of COVID19 patients diagnosed in Brazil OBJECTIVETo describe epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil METHODSStudy design and oversightThis was a retrospective observational singlecenter study which included all consecutive patients with a confirmed diagnosis of COVID19 at HIAE between February and March The study was supported by an internal grant from HIAE and designed by the investigators The study was approved by the Research Ethics Committee of the anization protocol number CAAE and the National Commission for Research Ethics PatientsThe diagnosis of the COVID19 disease was performed according to the WHO interim guidance10 A confirmed case of COVID19 was defined as a positive result of realtime reverse transcriptase polymerase chain reaction RTPCR assay of nasal and pharyngeal swab specimens11 All cases included in the current analysis had laboratory confirmationData sourcesThe data were obtained from patients electronic medical records EMR including inpatients and outpatients with laboratoryconfirmed COVID19 Data collected included demographic clinical laboratorial and radiological information and was anonymized so that patients could not be identified Demographic characteristics included age sex tobacco smoking weight and body mass index BMI Clinical information included medical travel Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cand exposure history signs symptoms underlying comorbidities continuous medication use and treatment measures ie antiviral therapy steroid therapy respiratory support and kidney replacement therapy Laboratory assessment consisted of complete blood count assessment of renal and liver function and measurements of electrolytes Ddimer procalcitonin lactate dehydrogenase Creactive protein and creatine kinase Radiologic abnormality was defined based on the medical report documented in the EMR Disease duration from onset of symptoms hospital and Intensive Care Unit ICU length of stay LOS were also documented Statistical analysis Continuous variables were expressed as means with standard deviations medians minimum and maximum values Categorical variables were summarized as counts and percentages No imputation was made for missing data All statistics are deemed to be descriptive only considering that the cohort of patients in our study was not derived from random selection All analyses were performed using Microsoft Excel RESULTSDemographic and clinical characteristicsBetween February and March a total of patients were diagnosed with COVID19 at HIAE This study included patients for whom data regarding demographics clinical symptoms laboratory and imaging findings were available in the EMR The remaining patients had only used the hospital laboratory facilities and were followedup by physicians not working in our service network Patients demographic and clinical characteristics are shown in table A total of had a recent international travel history and had been at the same marriage celebration in Bahia a state in the Northeast region of Brazil patients had a history of close contact either with a positive or suspected case of COVID19 Most patients were male and the mean age was years Only of patients were younger than years old and were older than yearsFever was present in only of patients upon admission but had a reported history of fever followed by nasal congestion cough Table Clinical and epidemiological characteristicsCharacteristicAge years Mean±SDMedianMinimumMaximumNumber of patientsAge distribution years ¥Sex MaleFemaleTravel historyTotal patients n510Total patientsNonhospitalized patients n438Hospitalized patients n72±±± European Union and United Kingdom United States of America and Canada Middle East and IranChina and Japan Latin America Other countries Bahia Brazilian stateNo travel history Exposure source of transmission contact with confirmed or suspected casesExposureNo exposureHealthcare professionalYesNoSmoking historyCurrent smokerFormer smokerNever smokedFever on admission YesNoMedianTemperature distribution on admission°C °C°C°CSymptomsNasal congestionHeadacheCoughSore throatSputum production continueEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cContinuationTable Clinical and epidemiological characteristicsContinuationTable Clinical and epidemiological characteristicsCharacteristicFatigueDyspneaNausea or vomitingDiarrheaMyalgia or arthralgiaChillsFeverConjunctival congestionOther symptomsNo symptomsSymptoms duration daysMean±SDMedianMinimumMaximumSigns of infectionThroat congestionTonsil swellingSkin rashOther alterationsNo alterationsCoexisting disordersAny coexisting disorderAsthma or chronic pulmonary obstructive disorderDiabetesHypertensionCoronary heart disease or other heart conditionsCerebrovascular diseaseHepatitis B C HIV or other immunodeficiencyCancerChronic renal diseasean transplantPregnancy Other coexisting disordersNo coexisting disordersMean BMI±SDChronicuse medicationsAny medicationStatinMultivitaminAntidepressantAntihypertensiveAntiplatelet or anticoagulantThyroid hormonesTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 ± ± ± ±± ± continueCharacteristicAntidiabeticPain killersAntibioticsCorticosteroidInhaled medicationsOther medicationsNo use of medicationsTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 Chronicuse medications number of medications distributionOnly type of medication types of medications types of medications or more types of medications polypharmacy ESI on arrival Destiny after first evaluation Discharge to homeAdmission on general wardAdmission on ICU Return to the emergency room after first evaluation SimN£oResults expressed by total nn if not otherwise indicatedSD standard deviation BMI body mass index ESI Emergency Severity Index ICU intensive care unitand myalgia or arthralgia The mean duration of symptoms was days which was the same for patients hospitalized or not Upon admission the majority of patients had no significant changes on physical examination Considering all included patients had at least one comorbidity This rate however was far higher in the hospitalized group when compared with the nonhospitalized group the most common comorbidities were hypertension and diabetes The distribution of patients in the Emergency Severity Index ESI differs between the two groups analyzed with the hospitalized group showing a higher rate of ESI indicating that the initial severity was greater in this group since the onset of symptoms Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cRadiologic and laboratory findingsTable demonstrates the radiologic and laboratory findings upon admission Only of patients were initially evaluated with chest radiographs whereas were submitted to computed tomography CT Of the radiographs performed had some abnormality while of CT scans showed abnormal results The most common patterns on chest CT were groundglass opacity and bilateral patchy shadowing Table Radiologic and laboratory findingsCharacteristicsRadiologic findings in chest radiographChest radiograph performedAbnormalities on chest radiograph Groundglass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalities Radiologic findings in chest CT Chest CT performed Abnormalities on chest CT Ground glass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalitiesLaboratory findingsMedian PaO2FiO2 ratio IQRWhite blood cell countMedian per mm3 Distribution per mm3 Lymphocyte countMedian per mm3 Distribution per mm3 Platelet countTotal number of patients n510 Total number of patientsNonhospitalized patients n438Hospitalized patients n72 Median per mm3Distribution per mm3 Median hemoglobin gdLDistribution of other findings Creactive protein 5mgL Procalcitonin 05ngmLLactate dehydrogenase 214UL Aspartate aminotransferase 40UL Alanine aminotransferase 40UL Total bilirubin 12mgdL Creatine kinase UL Creatinine 1mgdLDdimer 500ngmL Mean sodium mmolLMean potassium mmolL Results expressed by total nn if not otherwise indicatedCT computer tomography PaO2FiO2 oxygen partial pressurefractional inspired oxygen IQR interquartile rangeleukopenia Upon admission lymphocytopenia was identified in of patients thrombocytopenia in and in Most patients had elevated levels of both Creactive protein and lactate dehydrogenase Less common findings were elevated levels of Ddimer aspartate aminotransferase and alanine aminotransferase The hospitalized group had more patients with higher levels of Creactive protein procalcitonin and lactate dehydrogenase The other results do not show any major difference between groups A viral panel was collected in patients and it was positive for rhinovirus in nine cases influenza B in two cases and influenza A in one caseTreatment and complicationsAs shown in table patients had been hospitalized at HIAE by the time of the analysis Among Table Treatments complications and clinical outcomesTotal number of patients n510 CharacteristicDisease severitySevereNot severeIntensive care use during hospital stayYesNoHospital treatments medicationsIntravenous antibioticsOseltamivirLopinavir and ritonavirChloroquineCorticosteroidsHospital treatments support treatmentsOxygen therapyMechanical ventilationInvasiveNoninvasiveExtracorporeal membrane oxygenationContinuous renal replacement therapyComplicationsSeptic shockAcute respiratory distress syndromeAcute kidney injuryPneumoniaMean length of stay daysLOS all patientsPatients requiring ICU daysICUInpatients unitsPatients not requiring ICU inpatients units daysResults expressed by total nn if not otherwise indicated LOS length of stay ICU intensive care unitEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cthose patients required intensive care during their hospital stay in that were referred from the emergency room to the ICU and eight presented worsening of the clinical condition at inpatients units and were transferred to the ICU The majority of patients received intravenous antibiotic therapy received chloroquine and oseltamivir Oxygen therapy was necessary in of hospitalized patients required mechanical ventilation invasive and noninvasive and extracorporeal membrane oxygenation ECMO was used in only one case Considering patients admitted to the ICU invasive mechanical ventilation was required by of them During hospital admission most patients were diagnosed with pneumonia followed by acute kidney injury and ARDS The mean LOS was days considering only patients requiring intensive care the mean ICU LOS was days and the mean total LOS was days whereas for patients not admitted to the ICU the mean LOS was days Only one patient died in this series that is mortality rate DISCUSSION It took months from the first diagnosed case of COVID19 in China until diagnosis of patient zero in Brazil on February at HIAE During days after the first diagnosis all cases had a history of recent international travels On March the first case of local transmission was confirmed also at HIAE A relevant proportion of all patients with confirmed COVID19 infection had been diagnosed at HIAE by the time of the analysisThe patients in our series had a mean age of years and were mostly male The studies describing demographic characteristics in the infected general population showed a median age of years712 and the proportion of males was in the Chinese report7 and in the Singapore report The respiratory symptoms were similar to those of patients described in reports from China United States and Europe7913 However the mean days of symptoms was far lower in our series days versus days in Singapore12 days in the United States13 and days in China3 Although fever was reported by the majority of patients it was only present in of patients at the initial assessment at hospital suggesting not only it might not be considered to determine severity of illness but also that diagnostic algorithms using fever for testing may mask the total number of cases and delay diagnosis The prevalence of chronic diseases was far higher in the hospitalized group as compared to nonhospitalized group This prevalence was even higher in the subgroup admitted to the ICU The mean age of hospitalized patients was higher than nonhospitalized patients versus years and the required hospitalization increased with age for patients aged to years for to years and for patients older than years In this Brazilian case series hospitalization was required for patients and of them demanded critical care accounting for of total admissions a number far greater than the Chinese series in which only required ICU7The majority of patients were admitted to the ICU because of acute hypoxemic respiratory failure that required ventilatory support Invasive mechanical ventilation was needed in of ICU patients of total hospitalizations whereas were managed with noninvasive mechanical ventilation The necessity of invasive mechanical ventilation was similar to an ICU series reported from the United States of Washington13 lower than that reported in an Italian publication of Lombardy9 but higher than the Chinese reports and of Wuhan half of these treated with extracorporeal membrane oxygenation31415 Considering the use of noninvasive ventilation the rate was again similar to that reported in Washington and lower than the rates in China and of Wuhan including patients receiving highflow nasal cannula31415 A total of three patients of patients admitted to the ICU developed acute kidney injury and required continuous renal replacement therapy Among those only one patient had chronic kidney disease The prevalence of chronic kidney disease was among hospitalized patients in the Chinese report14 and among patients admitted to the ICU in the series from the United States This study has important limitations First part of the cases had incomplete information documented in the medical records and patient clinical history documentation was not homogeneous among all patients This is a common limitation in retrospective observational studies taking into account that data Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cgeneration was clinically driven and not in systematic fashion Second since many patients remained at the hospital and the outcomes were unknown at the time of data collection we censored the data regarding their clinical outcomes as of the time of the analysis Third only patients hospitalized at HIAE were included in the hospitalization group and there is no documentation of hospital admissions outside of our service network Finally this study only included patients attended as outpatients or inpatients at HIAE therefore asymptomatic and mild cases who did not seek medical care were not considered Hence our study cohort may represent more severe COVID19 cases CONCLUSIONTo date there is no study in Brazil reporting the characteristics of patients diagnosed with COVID19 Brazil is the country in the south hemisphere with the highest number of confirmed cases this disease and Hospital Israelita Albert Einstein is the center where the first patient was diagnosed with a representative sample of all confirmed COVID19 cases in the country The results presented in this study may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemic CONTRIBUTION OF AUTHORSData were analyzed and interpreted by the authors All authors reviewed the manuscript and checked the exactness and completeness of data AUTHORS INFORMATIONTeich VD httporcid0000000285396037Klajner S httporcid0000000341201047 Almeida FA httporcid0000000171310039 Dantas AC httporcid0000000195056784 Laselva CR httporcid0000000182859633 Torritesi MG httporcid0000000236236475 Canero TR httporcid0000000273994718Berwanger O httporcid0000000249722958Rizzo LV httporcid0000000199499849 Reis EP httporcid000000015110457X Cendoroglo Neto M httporcid0000000281634392 REFERENCES Lu H Stratton CW Tang YW Outbreak of pneumonia of unknown etiology in Wuhan China The mystery and the miracle J Med Virol Zhu N Zhang D Wang W Li X Yang B Song J Zhao X Huang B Shi W Lu R Niu P Zhan F Ma X Wang D Xu W Wu G Gao GF Tan W China Novel Coronavirus Investigating and Research Team A novel coronavirus from patients with pneumonia in China N Engl J Med Huang C Wang Y Li X Ren L Zhao J Hu Y et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Erratum in Lancet Jan World Health anization WHO Coronavirus disease COVID19 outbreak [Internet] Geneva WHO [cited July ] Available from httpswwwwhointwesternpacificemergenciescovid19 The Johns Hopkins Coronavirus Resource Center CRC COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University [Internet] CRC USA [cited July ] Available from httpscoronavirusjhuedumaphtml Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China Summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA Feb 101001jama20202648 Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical characteristics of coronavirus disease in China N Engl J Med Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H Spitters C Ericson K Wilkerson S Tural A Diaz G Cohn A Fox L Patel A Gerber SI Kim L Tong S Lu X Lindstrom S Pallansch MA Weldon WC Biggs HM Uyeki TM Pillai SK Washington State 2019nCoV Case Investigation Team First case of novel coronavirus in the United States N Engl J Med Grasselli G Zangrillo A Zanella A Antonelli M Cabrini L Castelli A Baseline characteristics and outcomes of patients infected with SARSCoV2 admitted to ICUs of the Lombardy region Italy JAMA World Health anization WHO Clinical management of severe acute respiratory infection when novel coronavirus 2019nCoV infection is suspected interim guidance [Internet] Geneva WHO [cited July ] Available from httpswwwwhointdocsdefaultsourcecoronaviruseclinicalmanagementofnovelcovpdf Brasil Ministrio da Saºde Centro de Opera§µes de Emergªncias em Saºde Pºblica Coronavirus Covid19 Boletim Di¡rio [Internet] Braslia DF Ministrio da Saºde [citado Jul ] Disponvel em httpswwwsaudegovbrimagespdf2020marco2929COVIDpdf Young BE Ong SW Kalimuddin S Low JG Tan SY Loh J Ng OT Marimuthu K Ang LW Mak TM Lau SK Anderson DE Chan KS Tan TY Ng TY Cui L Said Z Kurupatham L Chen MI Chan M Vasoo S Wang LF Tan BH Lin RT Lee VJ Leo YS Lye DC Singapore Novel Coronavirus Outbreak Research Team Epidemiologic features and clinical course of patients infected with SARSCoV2 in Singapore JAMA Mar 101001jama20203204 Bhatraju PK Ghassemieh BJ Nichols M Kim R Jerome KR Nalla AK Covid19 in critically Ill patients in the Seattle region Case series N Engl J Med Wang D Hu B Hu C Zhu F Liu X Zhang J Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA Feb 101001jama20201585 Yang X Yu Y Xu J Shu H Xia J Liu H Clinical course and outcomes of critically ill patients with SARSCoV2 pneumonia in Wuhan China a singlecentered retrospective observational study Lancet Respir Med Erratum in Lancet Respir Med 202084e26Epidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0c' | Thyroid_Cancer |
"Oral cancer is one of the most common noncommunicable diseases worldwide This paper presentsan evaluation of the trends and geographical distributions of oral cancers in the Saudi Arabian populationMethods Data from Saudi Cancer Registry reports were used in this analysis which assessed the period between and All cancer cases are recorded in these reports as well as the age gender region and histologicalcancer sites for each patient Agestandardised and agespecific incidence rates were calculated in these reportsFor the purposes of this paper only cancers of the lips tongue and mouth were considered oral cancersResults Between and the Saudi Cancer Registry identified cancer cases in total Of these were oral cancer The mean agestandardised rate of oral cancer for the study period was per peoplefor females it was and for males it was The incidence of oral cancer varied by region with Jazan displayingthe highest agestandardised rate and Hail displaying the lowest A positive correlation was observed between oralcancer incidence and ageConclusion The overall trend of the agestandardised rate for both sexes remained constant from to However the oral cancer incidence in Saudi Arabia varies by region Studying this variation in more detail will helpto guide awareness programmes in the regions that are most in needKeywords Cancer epidemiology Cancer prevention and control Oral neoplasmBackgroundCancer is an intractable global health problem and theleading cause of death in the developed world in the developing worldit is the secondleading cause [] In the most recent year for which information fromthe International Agency for Research on Cancer IARCis available approximately million new cancer caseswere diagnosed and million people died from cancerworldwide [] In this same year new cases oflip and oral cavity cancers were reported representing of all cancer casesA review of the global prevalence of oral cancer revealsa wide variation in distribution among countries []Correspondence bmalshehrinuedusaDepartment of Clinical Laboratory Faculty of applied Medical SciencesNajran University PO Box Najran Kingdom of Saudi ArabiaTwothirds of the estimated incidence of oral cancer occurred in developing countries with up to of allnew oral cancer cases in Sri Lanka India Pakistan andBangladesh [] Converselyin France which has thehighest rate of oral cancer incidence in the EuropeanUnion only oral cancer cases were reported in representing just of all cancer cases [] Inthe USA the American Cancer Society estimated that in approximately people were diagnosed withoral cavity or oropharyngeal cancer and will dieof these cancers [] In Arab countries the prevalence oforal cancer is concentrated between western and southeast Asia [] While this type of cancer is relatively uncommon across Arab gulf countries Saudi Arabia andYemen are notable exceptions [] No studies have beenpublished discussing the epidemiological parameters andgeographic distribution of oral cancer cases or any of its The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cAlshehri World Journal of Surgical Oncology Page of subtypes in the Saudi Arabian population Thereforethis study analysed and discussed oral cancer trends inthe Saudi population by using the most recent dataavailableAccording to the International Classification of Diseases 10th revision ICD10 oral cancer is classifiedinto six sites mucosal lip ICD10 C00 tongue ICD C02 gum ICD10 C03 mouth floor ICD10C04 palate ICD10 C05 and mouth ICD10 C06However examining trends in oral cancer incidencerates that include all oral sites can be misleading Thedata analysed in this study only include cancers of thelip tongue and mouth ICD10C00C06 which formthe majority of oral cancers moreover they have severalrisk factors in common and share a similar biology []Thus those accounting for a minority of oral cancercases were excludedMaterials and methodsDataThis retrospective descriptive epidemiological study analysed oral cancer cases in a Saudi population that hadbeen diagnosed from January through December The study used a method of analysis similar tothat used by Alshehri [] Their analyses incorporated male and female data on lip tongue and mouthICD10C00C06 cancer cases to evaluate disease patterns in the Saudi population Data for the present studywere obtained from the Saudi Cancer Registry SCR apopulationbased registry established in by theMinistry of Health in Saudi Arabia This data can onlybe obtained from the reports published by the SCRSince the SCR has been publishing reports oncancer in Saudi Arabia with the primary objective of defining populationbased cancer incidences The presentstudy was conducted using these reports to derive a descriptive epidemiology of oral cancer in Saudi Arabia InSCR reports agestandardised ASR and agespecificAIR incidence rates were calculated with a focus ongenderspecific and regional differencesThe analysis included cases recorded in the SCR filesfrom January to December totalling cancer cases overall approximately of which wereoral cancerData analysisThe GraphPad Prism6 software was used to analyse thedata Descriptive analyses of epidemiological data wereconducted by calculating the mean of the percentagesand ASR stratified by age sex region and year of diagnosis The ASR was calculated in the SCR reports byadjusting all Saudi regions populations mathematicallyto have the same age structure On the other hand theAIR was calculated by summation of the number ofcancer cases occurring during the year in a regionspopulation among specific age and sex groups dividedby the midyear population of these age and sex groupsUsing these two standardised rates is important because age is a basic element of the risk of developingcancer globally [] Using summary measure tools suchas the ASR and AIR which represent the schedule ofagespecific rates in different regions and across timewill give us a more representative picture of the characteristics in question and enable comparisons of cancerincidences between several populations of Saudi regionsthat differ with respect to ageResultsIncrease in the number of oral cancer casesThe total number of cancer cases identified by the SCRfrom to was with males and females Of this total cases were oral cancer The number of registeredoral cancer cases increased gradually from MF in to a peak of MF in however only cases were reported in MF Table Table Number of oral cancer cases in Saudi Arabia for theperiod from to YearNumber of female casesNumber of male casesTotal 0cAlshehri World Journal of Surgical Oncology Page of The percentage of cases representing oral cancers was for females and for males Fig in These percentages decreased to for females and for males in Fig The percentage curve fororal cancer out of all cancer types for males and femalescorrelated with increases and decreases over the studyperiod apart from the years and Fig ASR of oral cancer fluctuated over the study periodBetween and the ASR per male casesfluctuated in it was trending downwards to alow of in and peaking at in beforedropping again to in Fig The female ASRper increased from in to a peak of in decreasing again to in Fig For bothsexes ASR curves like oral cancer percentages correlateto increases and decreases over the study period apartfrom the years and Fig and generally remained constant from to ASR of oral cancer varies by regionThe ASR data for oral cancer cases of all persons demonstrated a wide variation across Saudi regions TheASR means per people for the period from to ranged from in Hail to in Jazan with anational average of per Fig The Jazan region had the highest male ASR mean at followed by the Najran and Tabuk regions at each Fig Conversely Qassim Baha Hail and theNorthern province reported the lowest ASR averages at and per respectively Fig Male and female ASR data were generally equivalentin terms of region rankings with the Jazan region posting the highest overall ASR of as an average valueof both genders followed by the Makkah region at and the Najran region at Fig Similarly the HailBaha Qassim and Madinah regions posted the lowestASR averages at and respectively FigAIR of oral cancer increases with ageThe AIR data from to showed a positive correlation between oral cancer incidence and age withmost cancer cases occurring in the older age groups Figure shows the AIR of oral cancer increasing noticeablywith age up until age More than of cases werediagnosed after the age of Some AIR differences were found between the sexesacross age groups From ages to rates of oral cancer were higher in females than in males however thistrend had reversed to favour males in the 75andoverage group The overall AIR per showed onlyslight differences between the sexes at for femalesand for males Fig DiscussionA review of oral cancer data in Saudi Arabia for theperiod from to showed an overall increasingtrend in the numbers of oral cancer patients Despitethis rise ASR data trends for oral cancer remained constant from to Fig This curve stabilised inthe face of a substantial Saudi Arabian population increase from million in to million in [] Many accumulative factors could be contributed tothis stability First the significant increased access tohealth services in Saudi regions has contributed to thedissemination of oral health awareness and early diagnosis of some cases of metaplasia that were discovered before they could develop into cancerous tumours SecondFig Consistency in percentage curves for oral cancer out of all cancer types from to The percentage curve for oral cancer out of allcancer types for males and females are correlated with overall increases and decreases over the period from to with the exception ofyears and 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer fluctuated over the study period Between and the male ASR was per in and dropped to in The female ASR fluctuated between and per the increased level of public health in the Kingdom isusually linked to an increase in the economic level of thecountry and individuals may have contributed to thisconstancy as many infectious factors such as virusesand fungi have been linked to oral cancers Third SaudiArabia is a majority Islamic country wherein many oralcancer risk factors such as alcohol consumption andcigarette smoking are forbidden by Islamic law Islamiclaw may thus mediate the lower number of oral cancercases in Saudi Arabia compared to the rest of the world[] Thus based on the IARC data for eight ofthe nine world regions whose ASR of oral cancer isabove the global rate [ ] were located in nonMuslimcountries [] with Melanesian regions having the highest rate [] In contrast most of the regions locatedwithin Muslim countries were ranked below the globalASR [] Further investigation of this aspect could therefore be valuable to cancer prevention effortsThe ASR data revealed that more females than maleswere diagnosed with oral cancer in Saudi Arabia at for men and for women This finding is in contrastwith global data showing that men are more likely to develop oral cancer than women [] In the most recent year for which IARC information is available theglobal ASR of oral cancer was for men and forwomen [] While these rates do not match the globalFig Agespecific incidence rates AIR of oral cancer increases with age The total AIR of oral cancer increased noticeably with up until patientswere and over More than of the cases were diagnosed after the age of 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer varies by region in Saudi Arabia For all persons the ASR means per peoplefor the period from to ranged from in the Hail region to in the Jazan regionsex distribution oral cancer in Saudi Arabia has a relatively low overall ASR when compared to the globalaverage as discussed aboveResults also revealed consistency in the ASR oral cancer curve for both sexes Fig potentially due to thepresence of common risk factors for oral cancer in malesand females This finding could be used as a startingthreshold for studying the risk factors of oral cancer inthe Saudi population through studying the common factors between the sexesAs with many other types of cancer the present studyfound a correlation between the occurrence of oral cancer and age with of cases diagnosed after the age of years In the USA the average age at diagnosis of oralcancer is years and twothirds of individuals with thisdisease are over the age of [] Ageing is accompaniedby increased susceptibility to cancercausing geneticmaturations due to accumulated exposures to environmental and behavioural risk factors Avoiding these riskfactors could greatly reduce the role that ageing plays incancerThis study found a wide variation in the incidence oforal cancers among Saudi regions Such differencescould indicate that regional environmental factors andlifestyle habits affect oral cancer incidence The resultsreviewed above found that the Jazan region possessedthe highest ASR of people with oral cancer In contrastthe Northern province presented the lowest ASR Severalstudies have focused on investigating why the Jazan region has such a high incidence of oral cancer []Ibrahim and others focused on the association ofcertain eating habits and lifestyle behaviours with the development of oral cancer especially the abuse ofshamma a form of smokeless tobacco and the chewingof khat Catha edulis leaves These substances havebeen classified as carcinogens especially in relation tooral cancer Studies by these researchers found that consuming shamma increased the odds of developing oralcancer 29fold suggesting a strong link between oralcancer and diet and lifestyle choicesAccording to the above poor dietary habits related totobacco use and its derivatives are one of the main reasons for the high incidence of oral cancer in some citiesand not others Other factors such as variations in thegenetic background of the Saudi regions citizens cannotbe excluded especially because most of the populationin the Kingdoms regions is tribal so consanguineousmarriages are highly common Thus genomic sequencing can provide information on genetic variants thatmay be present in citizens of these regions and that maybe linked with increased or decreased rates of oral cancer development Populationbased genetic testing issuggestedConclusionDespite the presence of yeartoyear changes in the incidence of oral cancer in the Saudi population there wasoverall no noticeable change in the incidence of oralcancer in the Saudi Arabian population for the periodbetween and In contrast to some international findings females were somewhat more likelythan males to be diagnosed with oral cancer in SaudiArabia The positive correlation between ageing and theincidence of oral cancer for both males and femalesdemonstrates that oral cancer is mainly a disease of theelderly both in Saudi Arabia and across the globe Thewide variation in the incidence rates among Saudi regions raises an important research question concerning 0cAlshehri World Journal of Surgical Oncology Page of World Bank World Development Indicators Washington DC WorldBank Access online via httpwwwirieduarpublicaciones_irianuariocd_anuario_2014Economia4bpdf Albar MA Islamic teachings and cancer prevention J Family CommunityMed Ibrahim EM Satti MB Al Idrissi HY Higazi MM Magbool GM Al QA Oralcancer in Saudi Arabia the role of alqat and alshammah Cancer DetectPrev Alsanosy RM Mahfouz MS Gaffar AM Khat chewing among students ofhigher education in Jazan region Saudi Arabia prevalence pattern andrelated factors Biomed Res Int Quadri MFA Alharbi F Bajonaid AMS Moafa IHY Al Sharwani A AlamirAHA Oral squamous cell carcinoma and associated risk factors in JazanSaudi Arabia a hospital based case control study Asian Pacific J CancerPrev Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspotential causes that need to be investigated furtherThe knowledge produced by this study must be translated into interventions by performing indepth analysesof regional differences This will contribute to the effortsof preventing oral cancer in Saudi ArabiaAbbreviationsAIR Agestandardised incidence rates ASR Agestandardised specific ratesIARC International Agency for Research on Cancer SCR Saudi CancerRegistry ICD10 International Classification of Diseases 10th revisionAcknowledgementsI express my thanks and gratitude to the Saudi Ministry of Health forproviding me with the Saudi Cancer Registry reportsAuthors contributionsI certify that I have participated sufficiently in the intellectual contentconception and design of this work analysis and interpretation of the dataas well as the writing of the manuscript to take public responsibility for itand have agreed to have my name listed as a contributor The author readand approved the final manuscriptFundingThis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsAvailability of data and materialsThe data that support the findings of this study are available from SaudiMinistry of Health but restrictions apply to the availability of these datawhich were used under authorization for the current studyEthics approval and consent to participateThis study was approved by the Research Ethics Committee at NajranUniversity The ethical document reference No ETConsent for publicationA secondary data analysis was conducted in this retrospective study byusing a published dataCompeting interestsThe author declares that he is the only author for this work No other authorcontributed to this work He is also in agreement with the content of themanuscript He declares no conflict of interestReceived June Accepted August ReferencesJemal A Bray F Center MM Ferlay J Ward E Forman D Global cancerstatistics CA Cancer J Clin Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Warnakulasuriya S GloWarnakulasuriya S Global epidemiology of oral andoropharyngeal cancer Oral Oncology httpsdoi101016joraloncology200806002bal epidemiology of oral andoropharyngeal cancer Oral Oncol Rick A Afsaneh B Cancer Facts Figures American Cancer Society Access online via httpswwwcancercontentdamcancerresearchcancerfactsandstatisticsannualcancerfactsandfigures2017cancerfactsandfigures2017pdfAlJaber A AlNasser L ElMetwally A Epidemiology of oral cancer in Arabcountries Saudi Med J Ariyawardana A Johnson NW Trends of lip oral cavity and oropharyngealcancers in Australia overall good news but with rising rates inthe oropharynx BMC Cancer Alshehri B Descriptive epidemiological analysis of thyroid cancer in the Saudipopulation Asian Pacific J Cancer Prev Armitage P Doll R The age distribution of cancer and a multistage theoryof carcinogenesis Br J Cancer 0c" | Thyroid_Cancer |
obesity and ethnicity alter gene expression in skinJeanne M Walker18 Sandra Garcet28 Jose O Aleman34 Christopher E Mason5 David Danko5 Simone Zuffa6 Jonathan R Swann67 James Krueger2 Jan L Breslow3 peter R Holt3Obesity is accompanied by dysfunction of many ans but effects on the skin have received little attention We studied differences in epithelial thickness by histology and gene expression by Affymetrix gene arrays and PCR in the skin of obese BMI and normal weight BMI postmenopausal women paired by age and ethnicity Epidermal thickness did not differ with obesity but the expression of genes encoding proteins associated with skin blood supply and wound healing were altered In the obese many gene expression pathways were broadly downregulated and subdermal fat showed pronounced inflammation There were no changes in skin microbiota or metabolites African American subjects differed from European Americans with a trend to increased epidermal thickening In obese African Americans compared to obese European Americans we observed altered gene expression that may explain known differences in water content and stress response African Americans showed markedly lower expression of the gene encoding the cystic fibrosis transmembrane regulator characteristic of the disease cystic fibrosis The results from this preliminary study may explain the functional changes found in the skin of obese subjects and African AmericansObesity defined as a body mass index BMI greater than a0kgm21 has become a major epidemic in industrial and emerging countries The prevalence of obesity has doubled since the 1980s and it is now estimated that million adults worldwide are obese2 Obesity affects many ans of the body and it is this an dysfunction that leads to excess mortality and morbidity3 Much attention has focused on the consequences of obesity in the heart liver and pancreas and other ans in which increased inflammation and oncogenesis become apparent4 Less attention has been paid to the effects of obesity on the skinObesity increases psoriasis5 which can be ameliorated with weight loss and cutaneous infections6 Since diabetes is common in obesity disorders such as fibroepithelial polyps and acanthosis nigricans also occur in the skin of obese subjects78 Moreover physiologic changes found in obese skin include increased transepidermal water loss with lower capacitance dry rough textured skin with pronounced erythema and reduced microvascular reactivity Altered collagen formation and increased delayedtype hypersensitivity have also been reported9Adipocyte depots that exist adjacent to the epidermis have distinct morphology and physiologic characteristics and are termed dermal or subdermal adipose tissue In addition to the principal role for dermal adipocytes in lipid storage and thermal insulation10 they also promote skin immunity11 wound healing and hair follicle cycling12 Obesity is accompanied by inflammatory immune changes in subcutaneous and visceral adipose tissues13 but the role of inflammatory changes within the adipose layer of the skin has received little attention Furthermore obesity is associated with increased circulating leptin levels which appear to independently affect dermal cell proliferation and hair growth14 In addition the microanisms that live on the skin surface also affect skin immunity11 so that it is important to analyse the skin microbiome comparing obese and normal individuals1The Rockefeller University Hospital New York NY USA 2Laboratory of Investigational Dermatology The Rockefeller University New York NY USA 3Laboratory of Biochemical Genetics and Metabolism The Rockefeller University New York NY USA 4Laboratory of Translational Obesity Research New York University Langone Health New York NY USA 5Weill Cornell Medical College New York NY USA 6Department of Metabolism Digestion and Reproduction Imperial College London London UK 7School of Human Development and Health Faculty of Medicine University of Southampton Southampton UK 8These authors contributed equally Jeanne M Walker and Sandra Garcet email walkerjrockefelleredu holtprockefellereduScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cIn view of the profound clinical and physiologic changes described in the skin in obesity it would not be surprising also to find biologically important molecular changes The present study was designed to compare gene expression in skin of healthy obese and nonobese subjects and to evaluate the potential importance of parallel changes in the microbiome and metabolites found on the adjacent skin surface and in the adipose tissue immediately below the skinMaterials and methodsSubjects Participants were recruited from the surrounding community through advertisements and from the Rockefeller University subject repository Eligible were healthy obese BMI a0kgm2 and nonobese BMI a0kgm2 postmenopausal women between the ages of and a0years The two groups were matched by age ± a0years and defined by selfreported ethnicity and by skin colour Exclusions were unstable weight change within the past three months HIV infection weight loss surgery inflammatory bowel disease history of malignancy other than nonmelanoma skin cancer in the previous a0years generalizable or systemic skin diseases history of a bleeding disorder current anticoagulant therapy or regular NSAID use current weight control medication or hypoglycaemic therapy individuals taking oestrogenprogesterone hormones and current tobacco smokers Also excluded were candidates with fasting blood glucose a0mgdl liver function tests ALT AST alkaline phosphatase greater than times the upper limit of normal ULN abnormal thyroid function test or serum creatinine ULNFourteen obese subjects were screened two refused skin biopsies one was withdrawn due to an intercurrent inflammatory illness and one was not postmenopausal by our criteria Ten obese subjects met our inclusion criteria and underwent skin swab collections and punch biopsy Twenty nonobese subjects were screened Two subjects refused to undergo punch biopsy one was withdrawn due to an intercurrent illness two with a BMI outside the required range one withdrew consent one was excluded with a history of keloid formation one with a low platelet count one with uncontrolled hypertension One nonobese subject who underwent skin punch biopsy was not included in the analysis because we were unable to find an age and ethnicitymatched obese subject These obese and agematched ethnicitymatched nonobese postmenopausal women completed all aspects of the study Fig a0 Six participants were European American and four were African Americans in each group Postmenopausal women were chosen to exclude effects of the menstrual cycle upon study end points and to exclude gender effectsBased on preliminary data from a previous study comparing skin from seven obese and six nonobese postmenopausal women there was a variation of in a set of RTPCR genes unpublished data Assuming the same variation and proportion of differentially expressed genes to be we calculated that a sample size of n subjects per group matched by age and ethnicity would provide power at a falsediscovery rate to detect the expected number of differentially expressed genes based on a threshold of twofold changesDesign and setting This was an label comparison of a group of postmenopausal obese women and postmenopausal nonobese women who were agematched ± a0years and racematched Screening comprised a complete history and physical examination and fasting blood testing for complete blood count sedimentation rate comprehensive chemistry panel lipid panel thyroid function tests hepatitis C antibody uric acid and haemoglobin A1C Observing Good Clinical Practice guidelines all participants read and signed an informed consent document approved by the Institutional Review Board and the Advisory Committee for Clinical and Translational Science at The Rockefeller University Protocol JWA0921Procedure methods Anthropometric measurements Body weight was measured daily to the nearest a0kg using a ScaleTronix scale Welch Allyn Skaneateles Falls NY with precision of ± a0kg Subjects were weighed in a hospital gown after an overnight fast and postvoiding Height was measured to the nearest a0cm at baseline with a Seca216 stadiometer Hamburg Germany in a0cm increments Body mass index BMI was calculated as kgm2 using the NIH Standard Metric BMI calculatorBlood collection and analysis Fasting blood samples were analysed in the Clinical Pathology Laboratory of the Memorial SloanKettering Cancer Center for complete blood count electrolytes glucose creatinine blood urea nitrogen liver function Creactive protein sedimentation rate and uric acid Research serum samples were drawn pre and post intervention aliquoted and stored at a0°C for subsequent analysisSkin swabbing Subjects were permitted to shower but did not wash the planned biopsy area over the midlower abdomen with soap for a0days before the biopsyFor microbiome analysis two areas of skin approximately a0cm were swabbed using the eSwab collection and preservation system for aerobic anaerobic and fastidious bacteria Copan Diagnostics Marietta CA Swabs were labelled sealed separately in the provided tubes and immediately stored at a0°C For metabolome analysis two different areas of skin approximately a0cm were swabbed using salinemoistened sterile cottontipped applicators The tips were cut sealed in separate sterile collection tubes and immediately stored at a0°CSkin microbiome The DNA extraction protocol was adapted from the Maxwell RSC Buccal Swab DNA kit Catalogue number AS1640 Promega Corporation Madison WI Briefly a0μl of lysis buffer and a0μl of Proteinase K was mixed and added to each swab tube Swab tubes were then incubated for a0min at C using a Thermo Fisher water bath removed from the tubes and fluid was transferred to well of the Maxwell RSC Cartridge The swab head was centrifuged using a ClickFit Microtube Cat V4741 and extracted fluid was added to the corresponding well of Maxwell Cartridge and eluted in a0μl of provided elution bufferScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Consort flow chart of eligible subjectsExtracted DNA was taken forward to the Nextera Flex protocol by Illumina Briefly a0μl of extracted DNA was taken into library prep protocol and run with cycles of PCR Libraries were cleaned up with a left sided size selection using a bead ratio of 08x The right sided size selection was omitted Libraries were then quantified using a Thermo Fisher Qubit Fluorometer and an Advanced Analytical Fragment Analyzer Libraries were sequenced on an Illumina HiSeqPE at the Weill Cornell Epigenomics CoreAll bioinformatic analysis was performed on Weill Cornell Medicines Athena compute cluster a highperformance grid compute system Secondary analysis was performed on a Linux and MacOS systems Unless otherwise noted programs were run with default settingsRaw sequence data were processed with AdapterRemoval v217 to remove low quality reads and reads with ambiguous bases15 Subsequently reads were aligned to the human genome hg38 including alternate contigs using Bowtie2 v230 fast preset16 Read pairs where one or both ends mapped to the human genome were separated from read pairs where neither mate mapped Read pairs where only one mate mapped were discarded Hereafter we refer to the read sets as human reads and nonhuman readsTaxonomic profiles were generated by processing nonhuman reads with KrakenUniq v032 with a database based on all draft and reference genomes in RefSeq Microbial bacteria fungi virus and archaea ca March KrakenUniq identifies kmers that are unique to taxa in a database Reads are broken into kmers and searched against this database Finally the taxonomic makeup of each sample was given by taking the proportion reads which were assigned to each clade KrakenUniq counts the number of unique marker kmers assigned to each taxon and we filtered taxa with fewer than unique markers17We performed differential abundance testing over microbial species using the ALDEx2 R package ALDEx2 performs variance stabilization read counts using a centred log ratio transformation that models samples as Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cDirichletMultinomial distributions over taxa then compares taxonomic abundances across groups18 Comparison of abundances across groups was done with a Wilcoxon rank sum test and Benjamini Hochberg Correction for multiple hypothesis testingDimensionality reduction of taxonomic profiles was performed with Principal Coordinates Analysis PCA based on a matrix of JensenShannon Divergences JSD between samples Analysis of intersample beta diversity was performed using the same matrix of JSD Intrasample alpha diversity was measured by finding Shannons Entropy of the taxonomic profile and by counting the total number of species identified in each sample richness Shannons entropy accounts for the relative size of each group in diversity estimation and is defined as H cid31 ai log2ai where ai is the relative abundance of taxa i in the sampleWe generated profiles of antimicrobial resistance genes using MegaRes v10119 To generate profiles from MegaRes we mapped nonhuman reads to the database using Bowtie2 v230 very sensitive presets Subsequently alignments were analysed using Resistome Analyzer commit 15a52dd and normalized by total reads per sample and gene length to give RPKMs MegaRes includes an ontology grouping resistance genes into gene classes AMR mechanisms and gene groupsSkin metabolome The skin was swabbed using two sterile salinemoistened culture swabs and immediately frozen at a0°C Swab heads were removed and placed in a0ml methanol water Following sonication a0min a0ml of isopropanol was added and the solution was spun at a0g for a0min The swab was removed and the samples were dried using a vacuum concentrator operating at a0°C Prior to UPLCMS analysis samples were reconstituted in a0μl of HPLCgrade water sonicated for a0min and transferred to vials for analysisA Waters 2777C sample manager Waters Corp Milford MA USA was used for sample handling This was equipped with a a0μl Hamilton syringe a a0μl loop used for fullloop injections of prepared sample and a 3drawer sample chamber maintained at a0°C with a constant flow of dry nitrogen gas to prevent the buildup of condensation The LC component was an ACQUITY UPLC Waters Corp Milford MA USA composed of a binary solvent manager and column heatercooler module Metabolic profiles were acquired using reversedphase chromatography Water and acetonitrile each supplemented with formic acid mobile phases A and B respectively were selected for the mobile phase A a0mm HSS T3 column was used at a0°C with a mobile phase flow rate of a0mlmin This generated a maximum pressure of psi in a wateracetonitrile gradient After a a0min isocratic separation at initial conditions A a linear gradient elution A to A in a0min proceeded followed by a quicker gradient A to A in a0min to final conditions The mobile phase flow rate was simultaneously increased to a0mlmin in the latter stage to facilitate faster column washing The MS component comprised a Xevo G2S QToF MS Waters Corp Manchester UK coupled to the UPLC via a Zspray electrospray ionization ESI source The cone gas flow was set to a0lh to protect the cone from residue accumulation during operation Both positive and negative ion modes RPC and RPC respectively were used Raw spectra were converted into mzML files using MSConvert20 and processed with XCMS in R21 Peak picking and peak grouping were performed using inhouse scripts in R and matrices were normalized using a median fold change approach Log transformation scaling and data analysis was performed in SIMCA Umetrics Umea SwedenSkin biopsy After the skin swabbing the abdominal site was cleansed with Chloraprep swabs chlorhexidine and isopropyl alcohol Becton Dickinson Canaan CT Using sterile technique local anaesthesia was induced by infiltration of the area with a0ml of lidocaine Hospira Inc Lake Forest IL mixed with a0ml sodium bicarbonate The skin biopsy was performed using a a0mm punch Miltex Instruments York PA Fat tissue was carefully removed from the skin core of the biopsy The dermis and epidermis were divided into two halves one half placed in a cryomold for OTC flash freezing Agar Scientific Essex UK and stored at a0°C and the other half was placed in RNAlater Stabilization Solution Thermo Fisher Scientific Fair Lawn NJ refrigerated for a0h and then frozen at a0°C The fat tissue was removed from the biopsy divided between RNAlater refrigerated for a0h then frozen at a0°C and a dry Sarstedt tube that was flash frozen in liquid nitrogen and placed in a0°C The biopsy site was sutured closed and a dry sterile dressing was applied Subjects were discharged and scheduled to return for suture removalGenearray and quantitative realtime PCR analysis RNA was extracted followed by hybridization to Affymetrix Human U133 Plus gene arrays Santa Clara CA or quantitative RTPCR as previously described2223All statistical analyses were carried out in R Limma Log 2transformed qRTPCR measurements hARP normalized and microarray expression values were assessed with a mixedeffect The fixed factors were condition obese vs nonobese race African American vs Caucasian with random intercept for each subject Quality control of microarray chips was carried out using standard QC metrics and R package microarray quality control Images were scrutinized for spatial artefacts using Harshlight24 Expression measures were obtained using the GCRMA algorithm25 A batch effect corresponding to the hybridization date was detected by PCA and adjusted using the ComBat function from the SVA package Probe sets with at least samples with expression values were kept for further analysis Fold changes for the comparisons of interest were estimated and hypothesis testing was conducted with contrasts under the general framework for linear models with the limma package P values from the moderated paired ttests were adjusted for multiple hypotheses using the BenjaminiHochberg procedure Hierarchical clustering was performed with Euclidean distance and a McQuitty agglomeration scheme26Data was deposited into Gene Expression Omnibus GEO repository GSE151839All study methods and procedures were carried out in accordance with Good Clinical Practice Guidelines by trained practitioners The protocol and informed consent were evaluated and approved by the Institutional Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression between the skin of obese and nonobese subjects A Heat map of the most differentially expressed genes in the skin of obese and nonobese subjects with an FCH fold change fdr false discovery rate B PCA principal component analysis plot of differentially expressed genes in the skin of obese and nonobese subjects with an FCH fdr Review Board and the Advisory Committee for Clinical and Translational Science at Rockefeller University prior to initiation of the study and annually thereafter Protocol JWA0921ResultsThis study was performed in ten healthy obese and ten healthy nonobese postmenopausal women matched for age and ethnicity Obese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Nonobese subjects had a mean weight of a0kg BMI of a0kgm2 and waist circumference of a0cm Supplemental Table a0S1 The skin thickness for subjects with obesity was not significantly different from that of nonobese subjects Supplemental Fig a0S1Gene expression analysis of the skin The most differentially expressed genes in the skin between obese and nonobese subjects are displayed in the heat map in Fig a02a Comparing gene expression in obese versus nonobese skin showed greater gene expression of S100A7A encoding a calcium binding protein involved in psoriasis and CORIN encoding a natriuretic peptide converting enzyme which is expressed in the dermis and is involved in specifying skin colour However the expression of CREB3LA encoding a cyclic AMP response element was lower in the skin of obese subjectsA PCA model constructed on of the most differentially expressed genes between obese and nonobese subjects showed partial separation of the groups This difference was seen in PC1 which accounted for of the variation in the included genes Fig a02bThe complete list of skin genes whose expression significantly differed between the two groups are shown with fold changes in Supplemental Tables a0S2 and S3 Again the gene expression of S100A7A was 344fold higher in the obese skin compared to the nonobese skin Similarly the expression of DEFB4A Defensin B4A which encodes an antimicrobial peptide part of the betadefensive system and SPRR2C which encodes a proline rich protein strongly induced during differentiation of human epidermal keratinocytes was also significantly higher in the obese skin being and 17fold higher respectively Genes with lower expression profiles in obese subjects than nonobese included AOP that encodes aquaporin involved in water channels present in the skin PROM1 prominin involved in cell differentiation and proliferation and Keratin and important for fibrogenesis in the epidermis Also of interest was the significantly higher expression 282fold of CFTR the cystic fibrosis transmembrane conductance receptor in nonobese subjects compared to the obese groupQTPCR analysis of genes selected from the total list of significantly differentially expressed genes in skin confirmed increased expression of the S100A 373fold DEFB4A defensin B4A 329fold and CORIN fold in the skin of obese subjects Fig a0 Significantly lower gene expression in the skin of obese subjects was found with CFTR 36fold PROM1 556fold and GABRP gamma aminobutyric acid receptor 29foldScientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cGene expression pathway analysis showed a broad downregulation of many pathways in obesity with only out of of the most highly differentially expressed pathways higher in the obese Supplemental Fig a0S2 The pathways most downregulated included cardiac beta adrenergic signalling which appears to function in skin cyclin dependent Kinase CDK5 a mutation which is important in melanoma formation and functions in skin healing and gonadotrophic releasing hormone GNRH signalling which has many extra pituitary functionsGene expression analysis in skin fat We next examined differences in gene expression between the groups of subjects in subdermal fat removed from immediately below the skin portion of the biopsy A heat map of gene expression shows a markedly different pattern between the two groups Fig a04a The expression of many of the genes upregulated in obese subdermal fat are involved with inflammation and immune function including platelet activating factor PLA2G7 ILIRN involved in IL1 activation SPPI a cytokine that can increase interferon gamma and IL12 activity and several serpins mediators involved in inflammation and immune functionThe PCA plot of genes whose expression differed significantly in subdermal fat of obese and nonobese subjects Fig a04b clearly shows separation between the two groups Most of the difference was seen in PC1 which includes of the genes whose expression was determinedSupplemental Tables a0S4 and S5 show a list of genes whose expression was relatively greater in the subdermal fat of obese subjects The expression of SPPI that encodes osteopontin which can act as a cytokine augmenting the action of interferon gamma and interleukin was approximately tenfold higher in the obese subjects EGFL6 expression which encodes an epidermal growth factor found to be enhanced in obesity and alters insulin action was increased by 85fold MMP9 which encodes metalloproteinase and ILTRN was increased by sevenfold in obesity Genes significantly downregulated in obese subdermal fat included SLC27A2 acetylCoAsynthase tenfold and C6complement fivefoldBy QTPCR in subdermal fat from obese subjects the increased expression of genes encoding proteins important in inflammation and immune function was confirmed Fig a0 This includes genes encoding proteins that determine accumulation of immune cells in adipose tissues such as CD52 the high affinity immunoglobulin gamma FC receptor FCGR1β CCL3 CZXCL8 interleukin and CLEC7A a pattern recognition receptor found in monocytes and other myeloid cells IL17F a member of the IL17 family also was specifically increased in subdermal fat from the obese as compared to nonobese individualsExpression pathway analysis Supplemental Fig a0S3 showed upregulation of several inflammatory immune pathways including the Thelper dendritic cell maturation and inflammatory signalling pathways further indicating profound effects of obesity on inflammation in subdermal fat Dramatically lower in the obese subdermal fat was the LXRRXR activation pathwayGene expression analysis by race Two subgroups were observed in the gene expression profiles of the skin based on the subjects race Using selfreported data and skin colour as criteria the data from African Americans was analysed separately from the data from European Americans This analysis showed striking differences in this very small group of subjects A heatmap of the most differentially expressed genes in skin from obese subjects divided by ethincity is shown in Fig a06a No clear differences in gene expression in the skin by ethnicity were found in nonobese subjects In contrast gene expression clearly differed between obese African American and obese European American subjects Fig a06a and also is illustrated in the PCA plot Fig a06b with PC1 responsible for the greatest variation The pattern of differences between obese and non obese skin is further illustrated in Fig a06cA list of genes whose expression significantly differed between the obese African Americans and the obese European Americans is shown in Supplemental Tables a0S6 and S7 The expression of SLC6A4 a serotonin transporter CORIN and COL8AI a collagen gene encoding a protein that is dysregulated in atopic eczema was higher in African Americans while the expression of SCCB2A2 the secretoglobin expressed in skin sweat glands and CFTR was expressed higher in European AmericansComparing the skin of obese African Americans to obese European Americans by QTPCR the former showed significantly lower expression of MYBCPI 516fold and PROM1 43fold and CFTR Fig a0 In contrast there was a small increase in the expression of CORIN 22fold a gene encoding the atrial naturalistic peptide converting enzyme and BMP2 fold also present in the skin compared to obese European AmericansPathway analysis found no racerelated differences in the nonobese samples However in the obese Supplemental Fig a0S4 there was markedly reduced expression of oestrogen mediated sphase entry pathway aryl hydrolase receptor signalling pathway and cell cycle regulation through cyclins pathways in the African Americans compared to the skin of the European American groupSubdermal fat in African Americans exhibited few differences from that found in European Americans Fig a08A Figure a08bc show the differences by weight and by ethnicity respectively illustrating the impact of obesity in the two racial groups The expression of numerous inflammatoryimmune genes was upregulated in the fat of both groups of obese subjects Fig a0 Tables a0S5 and S6 Microbiota analysis We next examined whether the microbiota collected from skin swabs around the biopsy site differed between the obese and nonobese subjects We generated taxonomic profiles for each sample using KrakenUniq and a database built from all available microbial species in RefSeq We measured the total number of AMR genes detected in each sample by aligning reads to MegaRESOverall differences between groups were minor No significant differences were noted in average taxonomic alpha diversity as measured by either Shannons entropy or richness between the groups A PCA plot of the taxonomic profiles showed slight separation between obese and lean samples and slightly higher beta diversity for obese samples however these differences were minor Fig a010a The number of antimicrobial resistant AMR Scientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the subdermal fat of obese and nonobese subjects A Heat map of the most differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr B PCA plot of differentially expressed genes in subdermal fat of obese and nonobese subjects with an FCH fdr Scientific RepoRtS 101038s41598020702442Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RTPCR between the subdermal fat of obese and nonobese subjects LS means of gene expression by RTPCR showing significant differences as p p p Figure a0 Differences in gene expression between the skin of African American and European American subjects A Heat map of the most differentially expressed genes in skin of African American and European American subjects with an FCH fdr B PCA plot of differentially expressed genes in skin of obese and nonobese African American and European American subjects with an FCH Left side of plot indicates differences in gene expression by ethnicity in nonobese subjects Right side of plot indicates differences in gene expression by ethnicity in obese subjects C PCA plot of differentially expressed genes in skin of obese and nonobese subjects by ethnicity with an FCH Left side of plot indicates differences in gene expression between obese and nonobese African American subjects Right side of plot indicates differences in gene expression between obese and nonobese European American subjectsScientific RepoRtS 101038s41598020702442Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Differences in gene expression by RT PCR between the skin of obese and nonobese African American and European American subjects LS means of gene expression by RTPCR showing significant differences as p p p genes identified was higher on average from obese subjects but this difference did not reach significance p Wilcox test Fig a010bDifferentially abundant taxa At the given sample size n no taxa were identified as significantly differentially abundant after BenjaminiHochberg correction Before correction five taxa were significantly differentially abundant at p Wilcox test These five taxa were Corynebacterium aurimucosum Corynebacterium jeikeium Corynebacterium urealyticum Streptococcus salivarius and Streptococcus sp A12 All five taxa were more abundant in samples from obese subjects on averageMetabolome analysis As highlighted by the PCA analysis no variation in the metabolites analysed by liquid chromatographymass spectrometry from the skin swabs was observed between the obese and nonobese individuals PCA Supplemental Fig a0S6 Similarly no ethnicityrelated metabolic variation was observed These results were further confirmed by the poor predictive ability of the orthogonal projection to latent structuresdiscriminant analysis OPLSDA models comparing the two different ethnic groupsDiscussionFew comprehensive reviews of skin changes occurring with obesity have been conducted despite over of the US population being obese27 A broad review of the physiologic and clinical consequences and associations was published by Hirt et a0al in The authors include a discussion of circulatory and lymphatic changes which may enhance the frequency and severity of skin ulceration and provide a comprehensive review of skin disorders that can be associated with obesity expanding on previous reviews29 and studies in rodents30In our study the thickness of | Thyroid_Cancer |
"Despite the biological link between thyroid hormones and breast cancer cell proliferation shown inexperimental studies little is known about the association between hyperthyroidism and breast cancer as well asits association with the most common mammographic and genetic risk predictors for breast cancerMethods This study estimates the incidence rate ratios IRRs of breast cancer among women diagnosed withhyperthyroidism compared to those who are not using two cohorts a Swedish national cohort of the generalfemale population n and the Karolinska Mammography Project for Risk Prediction of BreastCancer KARMA n We used logistic regression to estimate the odds ratios ORs ofhyperthyroidism according to the mammographic and genetic risk predictors for breast cancerResults An increased risk of breast cancer was observed in patients in the national cohort with hyperthyroidismIRR CI particularly for toxic nodular goiter IRR CI Hyperthyroidismwas associated with higher body mass index early age at first birth and lower breastfeeding duration Highermammographic density was observed in women with toxic nodular goiter compared to women withouthyperthyroidism Additionally among genotyped women without breast cancer in the KARMA cohort N hyperthyroidism was associated with a high polygenic risk score PRS for breast cancer overall OR CI and for estrogen receptorpositive specific PRS OR CI Conclusion Hyperthyroidism is associated with an increased risk of breast cancer particularly for patients withtoxic nodular goiter The association could be explained by higher mammographic density among these womenas well as pleiotropic genetic variants determining shared hormonalendocrine factors leading to the pathology ofboth diseasesKeywords Breast cancer Hyperthyroidism Mammographic density Genetic pleiotropy Correspondence haominyangkise1Department of Epidemiology and Health Statistics School of Public HealthFujian Medical University Xuefu North Road University Town Fuzhou China2Department of Medical Epidemiology and Biostatistics Karolinska InstitutetSE17177 Stockholm SwedenFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYang BMC Medicine Page of BackgroundBreast cancer is the most common cancer diagnosed amongwomen worldwide and the leading cause of cancer deathsamong women [] Breast cancer is usually regarded as ahormonerelated cancer with approximately ofcases being estrogen receptorpositive [] Experimental studies show that thyroid hormones can stimulate cell proliferation in breast tissue [ ] At high serum concentrationsthyroid hormones can have estrogenlike effects [] whichinduce the expression of progesterone receptors [] and canenhance estradiolmediated effects on cell proliferation [ ]Several populationbased studies have reported hyperthyroidism excessive production of thyroid hormones tobe associated with breast cancer [] while others havefound no association [] The disparity of findingsmay be explained by differences in study design smallsample sizes or using a specific subgroup of womenMoreover no study to date has examined the impact ofdifferent subtypes of hyperthyroidism on breast cancerrisk which may differ in their pathological processAlthough it is shown that thyroid hormones are associated with breast tissue proliferation and a subsequent increase in breast cancer risk [ ] the association betweenhyperthyroidism and mammographic features of breasttissue is less studied [] Mammographic density refers tothe percentage of radiologically dense epithelium stromaand connective tissues identified in a mammogram Mammographic density is a widely used mammographic featurefor breast cancer risk prediction and is considered an intermediate phenotype for breast cancer [ ] It can therefore be a powerful proxy when investigating the associationbetween hyperthyroidism and breast cancerBesides the direct effect of thyroid hormones on breastcancer risk the association between hyperthyroidism andbreast cancer could also be explained by genetic pleiotropicpathways leading to both hyperthyroidism and breastcancer [] A recent study has shown an associationbetween thyroidrelated single nucleotide polymorphismsSNPs and breast cancer risk [] Howeverit is stillunclear whether a genetic predisposition to breast cancer isassociated with hyperthyroidism which is important forexamining a potential pleiotropic genetic effectIn this study we investigated mechanisms behind the association between hyperthyroidism and breast cancer Specifically whether a genetic susceptibility to breast cancermeasured using polygenic risk score and mammographicdensity could explain some of this association while beingable to account for important factors such as menopausalstatus body mass index BMI and reproductive healthMethodsStudy populationsTo study the association between hyperthyroidism andbreast cancer risk we used two cohorts a Swedishnational cohort of the general female population and a mammographic screeningbased cohort KarolinskaMammography Projectfor Risk Prediction of BreastCancer KARMA Additional file Fig S1The national cohort included all women above age years who lived in Sweden between and N Having a main diagnosis of hyperthyroidismwas retrieved from the Swedish Patient Register andclassified according to the International Classification ofDiseases ICD code E05 Types of hyperthyroidism weredefined by the ICD codes E050 Graves disease E051E052 toxic nodular goiter and E053E059 other orunspecified types Followup of the cohort ended on thedate of breast cancer diagnosis date of death date ofemigration or end of followup December whichever came first Information on breast cancer diagnosis death and emigration was obtained by usingunique personal identification numbers to link the cohort to the Swedish Cancer Register Swedish Cause ofDeath Register and the Swedish Migration Register TheSwedish Cancer Register was founded in The completeness and accuracy of this register are estimated at and respectively [] The ICD7 code wasused to identify breast cancer diagnoses in the cancerregister and to exclude breast cancer cases before includingquestionnaireKARMA is a screeningbased cohort formed throughan invitation to all women participating in a mammographic screening or clinical mammography in one offour hospitals in Sweden between January andMarch [] During the recruitment period women consented to join the study with a participationrate of Aside from mammographic imaging andblood sample collection participants also answered awebbaseddemographicanthropometric reproductive lifestyle and familial riskfactors related to breast cancer This cohort was linkedto the Swedish Patient Register to retrieve diagnoses ofhyperthyroidism Breast cancer cases in the cohort wereidentified through linkage to the Swedish Cancer Register For consistency with the national cohort followupof the KARMA cohort also started from and endedwith the same criteria as the national cohort except foran extension of the followup time until Dec We therefore excluded women with hyperthyroidism orbreast cancer before ending with the final studypopulation of women Characteristics of womenin the national and the KARMA cohort are shown inAdditional file Table S1Polygenic risk scoreBlood samples from a subset of women who didnot have breast cancer when they joined the KARMAcohort were genotyped These women were part of theBreast Cancer Association Consortium and were 0cYang BMC Medicine Page of iSelectcustom Illuminarandomly selected as controls for the genomewideassociation studies GWAS [ ] Genotyping wasperformed using aarrayiCOGS comprising SNPs [] or OncoArraycomprising of SNPs [] Details of the arraydesign sample handling quality control processes andimputation of nongenotyped variants are described elsewhere [ ] Hyperthyroidism cases were defined aswomen who were diagnosed with hyperthyroidism from to while the controls were women without adiagnosis of hyperthyroidism To assess whether hyperthyroidism is associated with a genetic predisposition ofbreast cancer we selected genomewide significantSNPs reported in a recent metaanalysis of breast cancerGWAS for constructing polygenic risk scores for breastcancer overall and by estrogen receptor ER status []For all individuals a weighted polygenic risk score PRSwas calculated using the following formulaPRS ¼ βx1 þ βx2 þ ¦Î²kxk þ βnxnwhere β is the perallele log odds ratio OR of breastcancerassociated risk allele for SNP k xk is the numberof alleles for the same SNP and n is the totalnumber of the breast cancer SNPs included in the profile The SNPs and corresponding betas used to derivethe PRS are summarized in Additional file Table S2For the analyses PRS was categorized into quartilesMammographic densityFullfield digital mammograms from mediolateral oblique views of the left and right breasts in the most recent screening before were used For the KARMAcohort we used the areabased STRATUS method tomeasure mammographic density [] Percent densitywas calculated by dividing the dense area by the totalbreast area in the mammogram and further categorizedinto quartiles For this part of the study we excludedwomen with any cancer diagnosis as well as those whohad a breast enlargement reduction or surgery resulting in a final study population of Only womendiagnosed with hyperthyroidism before the latest screening were considered as casesThe study was approved by the Regional Ethical Review Board in Stockholm Sweden Dnr and Statistical analysisFor both cohorts the incidence rate ratio IRR of breastcancer among hyperthyroidism patients was calculatedusing Poisson regression using attained age as the timescale and adjusting for calendar period 3year categories For these analyses hyperthyroidism was treated as atimevarying exposurein which the exposed persontime was counted from months after the hyperthyroidism diagnosis index date and the unexposed persontimewas counted from Jan and ended on the date ofbreast cancer diagnosis date of death date of emigrationindex date or end of followup whichever came first Theanalysis was further stratified by menopausal status ageand time since hyperthyroidism diagnosis and type ofhyperthyroidism In the analyses Model adjusted forcalendar period and Model further adjusted for BMIage at menarche number of births family history of breastcancer oral contraceptive use hormone replacementtreatment and having had a benign breast diseaseThe association between hyperthyroidism and variouslifestyle risk factors for breast cancer were assessed amongKARMA women using logistic regression models Theserisk factors included having had a benign breast diseasenumber of births age at first birth breastfeeding durationBMI family history of breast cancer oral contraceptiveuse hormone replacement therapy use age at menarcheand menopausal status Two models were conducted forthis analysis a univariable model and a multivariablemodel adjusting for all these risk factors simultaneouslyGiven that mammographic density is causally relatedto breast cancer risk we also tested the associationbetween having a prior diagnosis of hyperthyroidism andmammographic density among KARMA women usinglogistic regression models Model was the univariablemodel Model adjusted for age at mammogram andBMI and Model further adjusted for age at menarchenumber of births family history of breast cancer oralcontraceptive use hormone replacement treatment andhaving had a benign breast disease Linear regressionmodels were also used to test the effect of hyperthyroidism on mammographic density square roottransformed as a continuous variableTo test the association between hyperthyroidism andhaving a genetic predisposition to breast cancer we usedlogistic regression models to calculate the ORs of hyperthyroidism among KARMA women who had beengenotyped by quartiles of PRS for breast cancer overallfor ERpositive cancers and for ERnegative cancersThese associations were adjusted for age the first threeprinciple components and for genotyping array We further adjusted for age at menarche number of birthsfamily history of breast cancer BMI menopausal statusoral contraceptive use hormone replacement treatmentand having had a benign breast diseaseStatistical analyses were performed using SAS version SAS Institute Inc Cary NC USA and Stata softwareversion Stata Corporation College Station TXResultsIn the national cohort cases of breast cancerpatients were observed during person years 0cYang BMC Medicine Page of following a diagnosis of hyperthyroidism correspondingto an incidence rate of person years Patientswith hyperthyroidism experienced a increased riskof breast cancer IRR CI In theKARMA cohort there were cases of breast cancerafter hyperthyroidism diagnosis during a followup timeof person years corresponding to an incidence rateof person years KARMA women with adiagnosis of hyperthyroidism also had a increasedrisk of breast cancer although the association was notstatistically significantin the multivariable adjustedmodel IRR CI In both cohortswomen diagnosed with hyperthyroidism aged youngerthan years had a significantly increased risk of breastcancer while there was no strong difference for the riskof breaststatusTable Analyses by type of hyperthyroidism in thenational cohort indicated a stronger association betweenbreast cancer and toxic nodular goiter IRR CI according to menopausalcancerAmong the major risk factors for breast cancer a diagnosis of hyperthyroidism was significantly associatedwith obesity BMI OR CI which was predominantly driven by toxic nodular goitersAdditional file Table S3 Other reproductive factorsincluding early age at first birth breastfeeding for lessthan a month and menopausal status were also associated with hyperthyroidism Table When investigating the association between hyperthyroidism and mammographic density no overall statistically significant association was observed Table However a prior diagnosis of toxic nodular goiter wassignificantly associated with high mammographic densityOR for the highest quartile CI p for continuous even after adjustingfor all other potential risk factors for breast cancerAdditional file Table S4Among KARMA women with genotyped data a higherPRS for breast cancer was associated with hyperthyroidism OR for the highest quartile CI This association was mainly driven by the association between hyperthyroidism and ERpositive breastcancer OR for the highest quartile CI Table There was no association betweenhyperthyroidism and PRS for ERnegative breast cancerDiscussionWomen diagnosed with hyperthyroidism had an increased risk of breast cancer compared to the generalpopulation This finding was more pronounced amongTable Risk of breast cancer in women in the national and KARMA cohorts with a diagnosis of hyperthyroidism compared towomen without hyperthyroidismSwedish national cohort N IRR CI Model No HT No BCKARMA cohort N No HTNo BCIRR CI Model IRR CI Model OverallPremenopausalPostmenopausalBy age years By years sincehyperthyroidismdiagnosisBy types of hyperthyroidismGraves diseaseToxic nodular goiterOthers or unspecified Hyperthyroidism patients were identified by the main diagnosis given in the inpatient and outpatient registers IRRs were calculated by comparinghyperthyroidism patients to women without hyperthyroidism using Poisson regression using age as the underlying time scale In the analyses stratified bymenopausal status women with age younger than in the national cohort were considered as premenopausal women while in the KARMA cohort the exactage at menopause was used Model adjusted for calendar period and Model further adjusted for BMI age at menarche number of births family history ofbreast cancer hormone replacement therapy use oral contraceptive use and benign breast disease Statistically significant results are boldedHT hyperthyroidism BC breast cancer IRR incidence rate ratio CI confidence interval 0cYang BMC Medicine Page of Table The association between hyperthyroidism and major breast cancer risk predictors in KARMA cohort N Variable namesBenign breast diseaseNo of nonhyperthyroidismOR2 CINo of hyperthyroidismOR1 CINoYesNumber of births Age at first birthyears ¥ Breastfeeding duration months BMI kgcm2 category Family history of breast cancerNoYesOral contraceptive useNoYesHormone replacement therapy useNoYesAge at menarche years Menopausal statusPremenopausalPerimenopausalPostmenopausalHistory of irregular menstrual periodsNoYes REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF REF OR1 was calculated using a univariate model while OR2 was calculated with a multivariate model including all risk predictors simultaneously The analysis wasrestricted to KARMA women who had not had a breast cancer diagnosis when they entered the studyOR odds radio BMI body mass indexAnalyses for age at first birth and breastfeeding duration were limited to parous women 0cYang BMC Medicine Page of Table The association between hyperthyroidism and mammographic density among KARMA women without a cancer diagnosisn Mammographic densityQ1Q2Q3Q4P for square root continuousNoYesOR CIModel REF Model Model REF REF Model is the univariate model Model adjusted for age and BMI Model further adjusted for age at menarche number of births family history of breastcancer hormone replacement therapy use oral contraceptive use and benign breast diseasethose diagnosed at a younger age and with a toxic nodular goiter Hyperthyroidism was also associated withbreast cancer risk predictors such as BMI age at firstbirth and duration of breastfeeding The association between mammographic density and hyperthyroidism wasonly observed in women with a diagnosis of toxic nodular goiter Additionally having hyperthyroidism wasassociated with a high PRS for breast cancer particularlyfor ERpositive breast cancerOur estimates for an increased risk of breast canceramong women diagnosed with hyperthyroidism were thesame when analyzing data from the national andKARMA cohorts which is also consistent with previousin Denmark and Taiwan [ ] ThisestimatesTable The association between hyperthyroidism and breast cancer polygenic risk scores PRS among women in the KARMAcohort N PRS for BC overallQ1Q2Q3Q4P for trendStandardized continuousPRS for ER BCQ1Q2Q3Q4P for trendStandardized continuousPRS for ER BCQ1Q2Q3Q4P for trendStandardized continuousNoNo caseOR1 CIOR2 CI REF REF REF REF REF REF Analysis was performed among KARMA women without breast cancer and who had genotyped data OR1s were adjusted for age array of genotyping andprinciple components OR2s were additionally adjusted for BMI menopausal status age at menarche number of births family history of breast cancer HRT useoral contraceptive use and benign breast disease Statistically significant results are boldedNo case the number of hyperthyroidism patients No the number of women without hyperthyroidism OR odds ratio CI confidence interval ER estrogen receptor 0cYang BMC Medicine Page of association however was not statistically significant inthe KARMA cohort probably due to a limited numberN The associationof breast cancer patientsbetween hyperthyroidism and breast cancer was furthersupported by an increased risk of breast cancer amongwomen with high serum thyroxine T4 [ ] Despitethis the effect of serum triiodothyronine T3 on breastcancer risk is still conflicting in different studies [ ]which might be influenced by nonthyroidalillnesssyndrome in cancer patients []Several biological pathways may exist between hyperthyroidism and breast carcinogenesis T3 could activatethe PI3K pathways mediated by integrin αvβ3 andstimulate breast cancer cell invasion [] In additionT4 induces MAPKmediated nuclear ERα phosphorylation and promotes cell proliferation to an extent comparable to the effect of estradiol [] which could beinhibited by the T4 analog tetrac []In addition to the known association between thyroidhormones and breast cancer risk it has been hypothesized that I131 radioactive iodine treatment for hyperthyroidism may also increase the risk of breast cancerHowever majority of the studies did not find a significant association between I131 treatment and breastcancer [] After surgery or radioactive iodine treatment hyperthyroidism patients may subsequently reacha hypothyroid state and thus be prescribed thyroidreplacement medication eg levothyroxine [] Despite this a recent metaanalysis did not find an increasedrisk of breast cancer after subsequent hypothyroidism[] nor following treatment with levothyroxine []Moreover considering the potential late effect of treatment on breast cancer the observed association betweenhyperthyroidism and breast cancer in our study particularly for short term risk cannot be explained by theeffect of treatmentIn this study hyperthyroidism was associated withseveral risk factors for breast cancer Both Gravesdisease and toxic nodular goiter are strongly influencedby pregnancy [ ] which could result in early cessation of breastfeeding and supports our finding of anassociation between reduced breastfeeding duration andhyperthyroidism The strong association between hyperthyroidism and breast cancer observed among womenaged below years in both the national and KARMAcohortsfurther suggests that closer surveillance forbreast cancer may be useful among women diagnosedwith hyperthyroidism during their reproductive yearsWe found a significantly higher BMI among patientswith hyperthyroidism despite previous knowledge that reduced weight is associated with hyperthyroidism [] Thisdisparity could be explained by treatments for hyperthyroidism which might eventually result in excess weightgain for patients [ ] Moreoverthe associationbetween hyperthyroidism and BMI was mainly driven bytoxic nodular goiters This finding is consistent withprevious studies indicating that obesity is associated withthyroid nodules [ ]Independent of BMI toxic nodular goiters were associated with higher mammographic density While oneprevious study did not find a significant association between thyroid disorders including hyperthyroidism andthyroid nodules and mammographic densitythis islikely due to having a limited sample size [] Thisfinding in the KARMA cohort was consistent with thepronounced association between breast cancer and toxicnodular goiter in the national cohort Unlike Gravesdisease which is an autoimmune disease toxic nodulargoiters are likely to be hormonerelated and thereforemore closely linked to breast cancer Nodular thyroiddiseases are associated with benign breast disease andbreast cancer [ ] and some shared pathways are involved in the proliferation of thyroid and breast tissues[ ] Hence the association between hyperthyroidism and breast cancer could result from both a hormonal effect and tissue proliferation and therefore possiblymediated through mammographic densityAdditionally we found a significant association between breast cancer PRS and hyperthyroidism Amongthose GWAS significant SNPs for breast cancer previous studies show that one SNP in the ABO gene is associated with thyroid function [ ] Another geneoverlapping the GWAS for thyroidstimulating hormoneand breast cancer is IGFBP5 [ ]indicating theshared growth hormoneinsulinlike growth factor GHIGF pathways between thyroid function and breast cancer Although insignificant at the GWAS level one SNPin the DIO1 gene has been associated with both free T4and breast cancer [] suggesting the role of deiodinaseactivity in the association between thyroid function andbreast cancer [] Overall given the genetic associationwas only significant for the ERpositive breast cancerPRS and not for ERnegative PRS we hypothesize thatgenetic pleiotropy between hyperthyroidism and breastcancer is involved in the shared hormonalendocrinepathways for both diseasesConsidering the phenotypic and genetic associations between hyperthyroidism and breast cancer as well as thebeneficial effect of introduced euthyroid hypothyroxinemia in the setting of breast cancer [] hyperthyroidismshould be considered in the evaluation of womens risk ofbreast cancer Despite concerns of possible overdiagnosisand anxiety caused by mammographic screening [ ]better adherence to the scheduled breast cancer screeningprogram is recommended for patients with hyperthyroidism in order to detect breast cancer at an early stageThe main strength of our study is having a large sample size and a populationbased cohort design including 0cYang BMC Medicine Page of both Swedish health registers and a mammographicscreening cohort Other strengths include the KARMAcohort containing rich lifestyle mammographic andgenetic data which allowed us to explore underlyingmechanisms for the associations investigated We alsoacknowledge severallimitations The validity of ICDcodes in the Swedish patient register is about [] which indicates potential misclassification Therefore we tried to minimize any potential misclassificationby using main diagnoses only Due to increased medicalsurveillance of patients with hyperthyroidismthesewomen may be diagnosed earlier with breast cancer thanwomen without hyperthyroidism While this possiblebias might slightly shift the temporal risk pattern amongwomen with hyperthyroidism it would not strongly influence the overall association An older age at breastcancer diagnosis among patients with hyperthyroidismsee Additional file Table S1 further argues againstthis surveillance bias In the national cohort we werenot able to adjust for breast cancer risk factors Nevertheless in the KARMA cohort which contains detailedinformation on these confounders multivariable adjustment did not change the point estimates indicating aweak confounding effect ofthese breast cancer riskfactors In the analysis of breast cancer risk using theKARMA cohort we started the followup before womenentered the KARMA study which could have introducedsurvivor bias However we believe the effect of any suchbias would be minimal given that hyperthyroidism isnot a deadly disease and the same estimates were foundusing the national cohortConclusionIn conclusion we found that women diagnosed withhyperthyroidism were associated with an increasedrisk of breast cancer and major predictors for breastcancerincluding mammographic density and polygenic risk score These findings may partly be explained byfactorsbetween these two diseases and may contribute to amorecomprehensive understanding of hormonalendocrinal factors contributing to breast cancer riskshared geneticand hormonalSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1291602001690yAdditional file Table S1 Characteristics of women in the national andKARMA cohorts by hyperthyroidism status Table S2 Single nucleotidepolymorphisms SNPs used for constructing the polygenic risk score PRS forbreast cancer Table S3 The association between hyperthyroidism and majorbreast cancer risk predictors among the KARMA cohort by type ofhyperthyroidism N Table S4 The association betweenhyperthyroidism and mammographic density among KARMA women withoutcancer by type of hyperthyroidism n Figure S1 Sample attritionfor the analyses using the KARMA cohortAbbreviationsBMI Body mass index CI Confidence interval ER Estrogen receptWAS Genomewide association studies ICD International Classification ofDiseases IRR Incidence rate ratio KARMA Karolinska Mammography Projectfor Risk Prediction of Breast Cancer OR Odds ratio PRS Polygenic risk scoreSNPs Single nucleotide polymorphisms T3 Triiodothyronine T4 ThyroxineAcknowledgementsWe thank all the participants in the KARMA studyAuthors contributionsHY had full access to all of the data in the study and takes responsibility forthe integrity of the data and the accuracy of the data analysis KC NH andHY conceived and designed the study All authors acquired analyzed orinterpreted the data HY and NH drafted the manuscript All authors criticallyrevised the manuscript for important intellectual content HY performed thestatistical analysis KC and HY obtained the funding All authors read andapproved the final manuscriptAuthors informationNot applicableFundingThis work was supported by the Swedish Research Council [grant no ] Swedish Cancer Society [grant no CAN190266] and FORTE [grant no] We would like to also acknowledge Märit and Hans Rausings Initiative against Breast Cancer Haomin Yang is supported by Startup Fund forhighlevel talents of Fujian Medical University [grant no XRCZX2020007] andStartup Fund for scientific research Fujian Medical University [grant no2019QH1002] access funding provided by Karolinska InstituteAvailability of data and materialsThe KARMA data used in the present study are available from thecorresponding author upon reasonable request The registerbasednationwide cohort datasets linked and analyzed in the current study are notpublicly available due to Swedish law but are available by applying throughthe Swedish National Board of Health and Welfare and Statistics SwedenDetailed information on data application can be found using the followinglinks httpsbestalladatasocialstyrelsensedataforforskning and httpswwwscbsevaratjansterbestallamikrodataEthics approval and consent to participateThe study was approved by the Regional Ethical Review Board in StockholmDnr and KARMA participantsconsented to participate in the study with written informed consentConsent for publicationAll authors approved the manuscript and consented to its publicationCompeting interestsThe authors declare no competing interestAuthor details1Department of Epidemiology and Health Statistics School of Public HealthFujian Medical University Xuefu North Road University Town Fuzhou China 2Department of Medical Epidemiology and BiostatisticsKarolinska Institutet SE17177 Stockholm Sweden 3Department of OncologySouth General Hospital SE11883 Stockholm SwedenReceived February Accepted June ReferencesGlobal Burden of Disease Collaboration Global regional and nationalcancer incidence mortality years of life lost years lived with disability anddisabilityadjusted lifeyears for cancer groups to a systematicanalysis for the global burden of disease study JAMA Oncology Allred DC Brown P Medina D The origins of estrogen receptor alphapositive and estrogen receptor alphanegative human breast cancer BreastCancer Res 0cYang BMC Medicine Page of Hall LC Salazar EP Kane SR Liu N Effects of thyroid hormones onhuman breast cancer cell proliferation J Steroid Biochem Mol BiolConde I Paniagua R Zamora J Blanquez MJ Fraile B Ruiz A Arenas MIInfluence of thyroid hormone receptors on breast cancer cell proliferationAnn Oncol Dinda S Sanchez A Moudgil V Estrogenlike effects of thyroid hormone onthe regulation of tumor suppressor proteins p53 and retinoblastoma inbreast cancer cells Oncogene Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast canc | Thyroid_Cancer |
pathogenesis of multiple myeloma MM is not completely known Uncovering the potential mechanism of MM initiation and progression is essential for identifying novel diagnostic and therapeutic targets Herein we explored the function and the working mechanism of circular RNA circ_0007841 in MM progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was employed to detect the expression of circ_0007841 microRNA3383p miR3383p and bromodomain containing BRD4 Cell proliferation ability was analyzed through cell counting kit8 CCK8 assay colony formation assay and flow cytometry Transwell assays were conducted to measure the migration and invasion abilities of MM cells Cell apoptosis was also assessed by flow cytometry The interaction between miR3383p and circ_0007841 or BRD4 was confirmed by dualluciferase reporter assay and RNApull down assayResults Circ_0007841 was highly expressed in bone marrow BMderived plasma cells of MM patients and MM cell lines than that in healthy volunteers and normal plasma cell line nPCs Circ_0007841 promoted the proliferation cell cycle and metastasis and impeded the apoptosis of MM cells miR3383p was a direct target of circ_0007841 in MM cells and circ_0007841 accelerated the progression of MM through targeting miR3383p BRD4 could directly bind to miR3383p in MM cells and miR3383p exerted an antitumor role through targeting BRD4 Circ_0007841 promoted the activation of PI3KAKT signaling via miR3383pBRD4 axis Exosomes generated from mesenchymal stromal cells MSCs elevated the malignant behaviors of MM cells via circ_0007841Conclusion Circ_0007841 acted as an oncogene to promote the proliferation cell cycle and motility and restrain the apoptosis of MM cells through sequestering miR3383p to upregulate the expression of BRD4Keywords Multiple myeloma circ_0007841 miR3383p BRD4 ExosomeBackgroundMultiple myeloma MM is a kind of hematologic cancer featured by malignant proliferation of plasma cells [] The therapeutic strategies for MM patients include chemotherapy radiotherapy and targeted therapy [] However MM is still incurable by current treatment Correspondence wy1782126com Department of Hematology The Fifth Affiliated Hospital of Zhengzhou University No3 Kangfuqian Street Zhengzhou Henan ChinaFull list of author information is available at the end of the methods Uncovering the molecular mechanism behind the progression of MM and intercellular interaction is important to find more effective treatment methods for MM patientsNoncoding RNAs ncRNAs are a class of RNAs that are unable to code proteins generally and they are abundant in human genome to regulate cellular processes including proliferation metastasis and apoptosis [] Circular RNAs circRNAs are a kind of ncRNAs that characterized by covalently closed loop structure [] CircRNAs are more stable than linear RNAs and they The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of are resistant to exonuclease due to their loop structure [] CircRNAs engaged in the pathogenesis of cancers through serving as microRNAs miRNAs sponges to modulate the abundance of downstream genes linked to proliferation metastasis and apoptosis [ ] The roles of circRNAs in hematological cancers have been reported before [ ] For instance circCBFB contributed to the proliferation ability while suppressed the apoptosis of chronic lymphocytic leukemia cells through targeting miR607FZD3Wntbetacatenin signaling [] However the functions of circRNAs in MM remain to be uncoveredMiRNAs belong to another class of ncRNAs that involved in the progression of cancers through inducing degradation or translational repression of target messenger RNAs mRNAs [] The dysregulation of miRNAs was involved in the pathogenesis of MM [ ] We concentrated on the role of miR3383p miR3383p suppressed the development of many cancers [] As for MM Cao et a0al reported that miR3383p suppressed the proliferation and accelerated the apoptosis of MM cells via CDK4 [] Nevertheless the function of miR3383p in MM is largely unexploredBromodomain containing BRD4 is a crucial epigenetic protein and it has been reported to elevate the levels of oncogenic proteins and accelerate the progression of cancers [] Zheng et a0al claimed that H19 accelerated the development of MM through upregulating BRD4 via sponging miR1523p [] Here the direct interaction between miR3383p and BRD4 was first found in MM and the function of BRD4 in MM was investigatedIn this study circ_0007841 was found to be abnormally upregulated in MM Lossoffunction experiments revealed that circ_0007841 silencing blocked the proliferation cell cycle progression migration and invasion while induced the apoptosis of MM cells The underlying mechanisms behind the oncogenic role of circ_0007841 in MM were further exploredMaterials and a0methodsPatientsPlasma cells from MM patients n and healthy volunteers n in The Fifth Affiliated Hospital of Zhengzhou University were collected to detect the expression of circ_0007841 miR3383p and BRD4 via qRTPCR and Western blot assayCell cultureMM cell lines H929 and OPM2 and normal plasma cell line nPCs were purchased from BeNa Culture Collection Beijing China and maintained in Roswell Park Memorial Institute1640 RPMI1640 medium Gibco Carlsbad CA USA added with fetal bovine serum FBS Gibco unitsmL penicillin and a0μgmL streptomycin Cell culture plates were placed in a CO2 incubator at a0°C and cells were collected in the log phase of growthQuantitative realtime polymerase chain reaction qRTPCRAfter measuring the concentration using NanoDrop Invitrogen Carlsbad CA USA RNA sample a0ng was used to synthesize complementary DNA cDNA with ReverTra Ace qPCR RT Kit for circ_0007841 BRD4 and U6 Takara Dalian China and AllinOne¢ miRNA glyceraldehyde3phosphate dehydrogenase GAPDH First stand cDNA Synthesis Kit for miR3383p GeneCopoeia Rockville MD USA U6 served as the internal control for miR3383p while GAPDH acted as the internal reference for circ_0007841 and BRD4 PCR amplification reaction was conducted with SYBR Green PCR Master Mix Applied Biosystems Foster City CA USA on an ABI thermocycler Applied Biosystems The quantification of circ_0007841 miR3383p and BRD4 was carried out with the ÎÎCt method The specific primers in this study were synthesized from Sangon Biotech Shanghai China and listed as below circ_0007841 ²CTA ACA TCT GTG AAA CCA TCGT3² Forward Reverse ²TCA TCA CAT ACA CGA TAG ACTGG3² miR3383p Forward ²UCC AGC AUC AGU GAU UUU GUUG3² Reverse ²CAA CAA AAU CAC UGA UGC UGGA3² BRD4 Forward ²GTG GTG CAC ATC ATC CAG TC3² Reverse ²CCG ACT CTG AGG ACG AGA AG3² U6 Forward ²CTC GCT TCG GCA GCACA² Reverse ²AAC GCT TCA CGA ATT TGC GT3² GAPDH Forward ²GCG ACA CCC ACT CCT CCA C3² Reverse ²TCC ACC ACC CTG TTG CTG TAG3²and interfering RNAs siRNAs including sicirc_00078411 Cell transfectiontargeting Three small ²UGU circ_0007841 sicirc_00078412 UAG UUG CAA UGA AGA GAG3² si²UAA UGA UCA UGC CAA AUA CUC3² circ_00078413 ²UCA CAU ACA CGA UAG ACU GGC3² its negative control siNC circ_0007841 overexpression plasmid circ_0007841 its control Vector BRD4 overexpression plasmid BRD4 its control pcDNA miR3383p mimics miR3383p its control miRNC miR3383p inhibitor inmiR3383p and its control inmiRNC were obtained from Genepharma Shanghai China MM cells were seeded into 24well plates at a density of à cellswell overnight and transfection was conducted with Lipofectamine Invitrogen 0cWang a0et a0al Cancer Cell Int Page of Cell counting kit CCK8 assayMM cells were plated in 96well plates at the density of à cellswell and cultured overnight After transfection for indicated time points a0h a0h a0h or a0h MM cells were incubated with μL CCK8 Sigma St Louis MO USA for a0h The absorbance at a0 nm was detected by a microplate reader BioTek Winooski VT USAColony formation assayA total of cells were seeded onto the 6well plates to settle down The culture medium was replenished every d After 2week incubation the colonies were immobilized using poly methanol Sangon Biotech for a0 min followed by staining using crystal violet Sangon BiotechFlow cytometry for a0cell cycle and a0apoptosis detectionFor cell cycle analysis MM cells were collected using cold phosphate buffer saline PBS and then immobilized using cold ethanol solution overnight Prior to propidium iodide PI Solarbio Beijing China staining RNase was used to remove RNA in the samples The percentage of MM cells in different phases of cell cycle was detected on the FACSCalibur BectonDickinson San Jose CA USA and analyzed using Cell Quest software BectonDickinsonFor apoptosis analysis after transfection for a0 h MM cells were collected with PBS and then these cells were suspended in binding buffer Annexin Vcombined fluorescein isothiocyanate Annexin VFITC Solarbio and PI Solarbio were added to the reaction mixture and MM cells were simultaneously incubated with Annexin VFITC and PI at a0°C for a0min in a dark room The apoptotic MM cells were identified by FACSCalibur BectonDickinson and analyzed using Cell Quest software BectonDickinsonTranswell assaysIn transwell migration assay cell suspension MM cells suspended in μL serumfree medium was added into the upper chambers Costar Corning NY USA A total of μL culture medium with FBS was added into the lower chambers FBS acted as the chemotactic factor in this study After 24h incubation MM cells remained in the upper surface were removed with the cotton swab and the migrated MM cells were fixed with paraformaldehyde Sigma for a0 min and stained with crystal violet Sigma The number of migrated MM cells in five random visual fields was counted by the microscope Olympus Tokyo JapanIn transwell invasion assay the upper chambers were precoated with μL Matrigel Sigma to mimic the extracellular matrix The detection of cell invasion was conducted through using these precoated transwell chambers following the similar procedureBioinformatic prediction and a0dualluciferase reporter assayThe targets of circ_0007841 and miR3383p were predicted by circinteractome and targetscan software respectivelyThe wildtype partial sequence in circ_0007841 that predicted to bind to miR3383p along with the mutanttype sequence with miR3383p in circ_0007841 that was synthesized through using Sitedirected gene mutagenesis kit Takara Dalian China was amplified and cloned into pGL3 luciferase reporter vector Promega Madison WI USA termed as circ_0007841 WT or circ_0007841 MUT MM cells were cotransfected with a0 nM miRNC or miR3383p and a0 ng circ_0007841 WT or circ_0007841 MUT After 48h transfection MM cells were harvested and the luciferase activity was detected with the dualluciferase reporter assay system kit Promega using the luminometer Plate Chameleon V Hidex Finland according to the manufacturers instructions Firefly luciferase activity in each group was normalized to Renilla fluorescence intensityThe wildtype fragment of BRD4 ² untranslated region ²UTR that predicted to bind to miR3383p and the mutant type fragment of BRD4 ²UTR were also amplified and inserted into pGL3 luciferase reporter vector Promega to generate BRD4 ²UTR WT and BRD4 ²UTR MUT Cotransfection of MM cells with BRD4 ²UTR WT or BRD4 ²UTR MUT and miRNC or miR3383p was conducted following the similar procedureRNApull down a0assayRNApull down assay was conducted to test the interaction between circ_0007841 and miR3383p Biotin RNA Labeling Mix Roche Shanghai China was used in this study The wildtype and mutanttype binding sites in circ_0007841 that were predicted to bind to miR3383p were biotinylated to obtain Biocirc_0007841 WT and Biocirc_0007841 MUT MM cells were disrupted and incubated with BioNC Biocirc_0007841 WT or Biocirc_0007841 MUT The abundance of miR3383p was measured by qRTPCRWestern blot assayProteins were obtained using whole cell lysis buffer Roche Basel Switzerland for a0min on the ice Protein samples were quantified using Pierce BCA Protein Assay kit Thermo Fisher Scientific Rockford IL USA Then a0 µg of proteins were run on sodium dodecyl sulfate 0cWang a0et a0al Cancer Cell Int Page of polyacrylamide gel electrophoresis SDSPAGE gel and transferred to the polyvinylidene fluoride PVDF membrane Millipore Billerica MA USA After blocking with wv nonfat dry milk for a0h primary antibodies were used to probe the indicated proteins followed by incubation with the secondary antibody ab205718 Abcam Cambridge MA USA The protein bands were measured using the enhanced chemiluminescent ECL system Beyotime Shanghai China according to the manufacturers instructions Gray analysis was conducted to quantify the expression of proteins using ImageJ software Primary antibodies including antiBRD4 ab128874 antiphosphorylatedphosphatidylinositol 3kinase antipPI3K ab70912 antiPI3K ab32089 antipAKT serinethreonine kinase pAKT ab38449 antiAKT ab64148 antiCD63 ab59479 antiCD81 ab79559 and antiβactin ab8226 were purchased from AbcamExosome isolationExosome isolation kit Qiagen Frankfurt Germany was used to extract exosomes from the culture medium of MM cells according to previous studies [ ]Statistical analysisAll statistical data in three independent experiments were shown as mean ± standard deviation SD Data were analyzed using GraphPad Prism The differences between two groups or among more than two groups were assessed through using Students t test or oneway analysis of variance ANOVA followed by Tukeys test The comparison between groups was considered significant when P value less than Linear correlation was analyzed using Spearmans correlation coefficientResultsCirc_0007841 elevates the a0malignant behaviors of a0MM cellsCirc_0007841 was abnormally upregulated in bone marrow BMderived plasma cells from MM patients compared with that in healthy individuals Fig a01a Meanwhile the level of circ_0007841 was higher in MM cell lines than that in normal plasma cell line nPCs Fig a01b The dysregulation of circ_0007841 in MM attached our attention Circ_0007841 specific small interfering RNAs were used to knockdown circ_0007841 to uncover its biological functions in MM cells As mentioned in Fig a0 1c and d the level of circ_0007841 was downregulated with the transfection of sicirc_00078411 sicirc_00078412 or sicirc_00078413 Among these three siRNAs sicirc_00078411 was chose for the following assays due to its highest knockdown efficiency Fig a01c d Cell proliferation was assessed through CCK8 assay colony formation assay and flow cytometry According to the results of CCK8 assay sicirc_00078411 transfection significantly inhibited the proliferation of MM cells Fig a0 1e f The number of colonies was markedly reduced with the knockdown of circ_0007841 compared with siNC group Fig a0 1g The cell cycle of MM cells was arrested in G1S transition in sicirc_00078411 group than that in siNC group Fig a01h These findings together demonstrated that circ_0007841 silencing hampered the proliferation ability in MM cells Whats more circ_0007841 interference notably suppressed the migration and invasion of MM cells via transwell migration and invasion assays Fig a01i j The apoptosis rate of MM cells was increased in sicirc_00078411 group compared with that in siNC group Fig a01k Overall circ_0007841 accelerated the proliferation cell cycle progression and metastasis and inhibited the apoptosis of MM cellsmiR3p could directly interact with a0circ_0007841 in a0MM cellsTo address the mechanism by which circ_0007841 functioned in MM cells circinteractome website was used to seek the targets of circ_0007841 As shown in Fig a0 2a miR3383p possessed the complementary sites with circ_0007841 The luciferase activity was dramatically reduced in circ_0007841 WT group when cotransfected with miR3383p suggesting the target relationship between circ_0007841 and miR3383p in MM cells Fig a02b c We also constructed mutant type luciferase plasmid circ_0007841 MUT to investigate if UGC UGG in circ_0007841 was the binding sequence with miR3383p The luciferase intensity remained unaffected in circ_0007841 MUT group with the cotransfection of miRNC or miR3383p Fig a02b c suggested that circ_0007841 bound to miR3383p via its UGC UGG sequence RNApull down assay revealed that miR3383p could be pulleddown when using Biocirc_0007841 WT proving the target relationship between miR3383p and circ_0007841 Fig a0 2d e An obvious decrease in the level of miR3383p was observed in BMderived plasma cells from MM patients in contrast to that in normal volunteers Fig a02f Additionally there was a prominent reduction in the expression of miR3383p in MM cell lines than that in nPCs cell line Fig a0 2g The expression of miR3383p was negatively correlated with the level of circ_0007841 in BMderived plasma cells from MM patients Fig a02h The overexpression efficiency of circ_0007841 was high in MM cells and circ_0007841 accumulation caused a notable decrease in the level of miR3383p in MM cells Fig a02i j In summary circ_0007841 could inversely regulate the expression of miR3383p through direct interaction 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 elevates the malignant behaviors of MM cells a The enrichment of circ_0007841 was examined in BMderived plasma cells of MM patients and normal volunteers by qRTPCR b The expression of circ_0007841 was measured in MM cell lines and normal plasma cell line nPCs by qRTPCR c d The level of circ_0007841 was detected in H929 and OPM2 cells transfected with siNC sicirc_00078411 sicirc_00078412 or sicirc_00078413 by qRTPCR ek MM cells were transfected with siNC or sicirc_00078411 e f CCK8 assay was employed to assess the proliferation ability of MM cells g Colony formation assay was performed for the determination of cell proliferation ability in transfected MM cells h Flow cytometry was carried out to detect the influence of circ_0007841 silencing on the cycle of MM cells i j The metastasis ability of MM cells was evaluated by transwell assays k The apoptosis of MM cells was analyzed by flow cytometry P P P P if circ_0007841 exerted Circ_0007841 plays an a0oncogenic role through a0targeting miR3p in a0MM cellsTo disclose its oncogenic role through targeting miR3383p we conducted rescue experiments through cotransfecting H929 and OPM2 cells with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p As mentioned in Fig a03a sicirc_00078411 transfection increased the level of miR3383p and the introduction of inmiR3383p reversed the influence of circ_0007841 silencing in the expression of miR3383p Sicirc_00078411mediated inhibitory effect on the proliferation of MM cells was counteracted by the interference of miR3383p via CCK8 assay Fig a03b c Circ_0007841 silencing restrained the colony formation ability while the addition of miR3383p inhibitor partly recovered the colony formation ability in MM cells Fig a0 3d Additionally cell cycle of MM cells was arrested at G1S transition in sicirc_00078411 group and this suppressive impact in the cell cycle of MM cells was attenuated by the addition of inmiR3383p Fig a03e f The migration and invasion of MM cells were suppressed by the knockdown of circ_0007841 and the metastasis ability was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a0 3g h Sicirc_00078411induced apoptosis of MM cells was 0cWang a0et a0al Cancer Cell Int Page of Fig miR3383p could directly interact with circ_0007841 in MM cells a miR3383p was predicted as a candidate target of circ_0007841 by circinteractome software b c Dualluciferase reporter assay was conducted to verify whether miR3383p could bind to circ_0007841 in MM cells d e RNApull down assay was performed to confirm the target relationship between miR3383p and circ_0007841 in MM cells f g The expression of miR3383p was detected in BMderived plasma cells of MM patients and healthy volunteers MM cells and nPCs cells by qRTPCR h The correlation between the expression of miR3383p and circ_0007841 was analyzed using Spearmans coefficient i j The abundance of circ_0007841 and miR3383p was examined in H929 and OPM2 cells transfected with Vector or circ_0007841 by qRTPCR P P P P attenuated by the addition of inmiR3383p Fig a0 3i Overall circ_0007841 could promote the malignant potential of MM cells through sponging miR3383pBRD4 is a0validated as a0a a0target of a0miR3p in a0MM cellsBRD4 was predicted as a direct target of miR3383p by targetscan database and the wild type or the mutant type binding sequence between miR3383p and BRD4 was shown in Fig a04a As exhibited in Fig a04b c the luciferase activity was markedly decreased in miR3383p and BRD4 ²untranslated region ²UTR WT cotransfected group while miR3383p transfection had no effect on the luciferase activity in BRD4 ²UTR MUT group compared with that in miRNC and BRD4 ²UTR MUT cotransfected group suggesting the interaction between BRD4 and miR3383p BRD4 was conspicuously upregulated in BMderived plasma cells of MM patients compared with that in healthy individuals Fig a0 4d Meanwhile BRD4 was also found to be upregulated in MM cell lines than that in nPCs cells Fig a0 4e The expression correlation between BRD4 and circ_0007841 or miR3383p was analyzed using Spearmans correlation coefficient As shown in Fig a0 4f g there was an inverse correlation between the levels of BRD4 and miR3383p while the expression of BRD4 was positively correlated with the level of circ_0007841 miR3383p overexpression significantly downregulated the expression of BRD4 in MM cells suggesting the negative regulatory relationship between BRD4 and miR3383p in MM cells Fig a04h Circ_0007841 and miR3383p were cotransfected into MM cells to uncover the relationship among circ_0007841 miR3383p and BRD4 As presented in Fig a0 4i circ_0007841 overexpression upregulated the level of BRD4 and the expression of BRD4 was decreased in circ_0007841 and miR3383p cotransfected group Collectively BRD4 was a target of miR3383p and circ_0007841 could elevate the expression of BRD4 through sponging miR3383pBRD4 overexpression attenuates the a0effects of a0miR3p accumulation on a0MM cellsmiR3383p and BRD4 were cotransfected into MM cells to explore whether miR3383p exerted an antitumor role in MM cells through targeting BRD4 As shown in Fig a0 5a the addition of BRD4 overexpression plasmid recovered the expression of BRD4 in MM cells that was downregulated by the accumulation of miR3383p miR3383p overexpression inhibited the proliferation cell cycle and metastasis of MM cells and these inhibitory effects were attenuated by the 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 plays an oncogenic role through targeting miR3383p in MM cells ai MM cells were transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p a The level of miR3383p was examined in MM cells by qRTPCR assay b c The proliferation of MM cells was measured through conducting CCK8 assay d The proliferation capacity in transfected MM cells was assessed by colony formation assay e f The percentage of MM cells in G0G1 S or G2M phase was analyzed using flow cytometry g h The migration and invasion abilities of MM cells were evaluated by transwell assays i The apoptosis rate of MM cells in different groups was analyzed by flow cytometry P P P addition of BRD4 overexpression plasmid Fig a0 5bh The apoptosis of MM cells was induced by the transfection of miR3383p and the introduction of BRD4 overexpression plasmid recovered the viability of MM cells Fig a0 5i In conclusion miR3383p accumulation restrained the malignant behaviors of MM cells through targeting BRD4Circ_0007841 activates PI3KAKT signal pathway through a0targeting miR3pBRD4 axisThe activation of PI3KAKT signal pathway is linked to the promotion of cell proliferation and metastasis and the inhibition of cell apoptosis Herein we examined the phosphorylation levels of PI3K and AKT to illustrate the influence of circ_0007841miR3383pBRD4 axis in the See figure on next pageFig BRD4 is validated as a target of miR3383p in MM cells a The complementary sites between miR3383p and the ²UTR of BRD4 were predicted by targetscan software b c The luciferase activity was measured in H929 and OPM2 cells transfected with miRNC or miR3383p and BRD4 ²UTR WT or BRD4 ²UTR MUT d The protein level of BRD4 in BMderived plasma cells of MM patients and healthy volunteers was detected by Western blot assay e The level of BRD4 in H929 OPM2 and nPCs cells was evaluated by Western blot assay f g The linear relationship between BRD4 and miR3383p or circ_0007841 was analyzed using Spearmans coefficient h The expression of BRD4 was detected in MM cells transfected with miRNC or miR3383p by Western blot assay i The protein level of BRD4 was detected in MM cells transfected with Vector circ_0007841 circ_0007841 miRNC or circ_0007841 miR3383p by Western blot assay P P P P 0cWang a0et a0al Cancer Cell Int Page of 0cWang a0et a0al Cancer Cell Int Page of Fig BRD4 overexpression attenuates the effects of miR3383p accumulation on MM cells ai MM cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 a qRTPCR was employed to measure the expression of BRD4 in MM cells b c CCK8 assay was applied to assess the proliferation ability of MM cells d Colony formation assay was performed to analyze the influences of miR3383p and BRD4 on the proliferation of MM cells e f Flow cytometry was conducted to detect the cell cycle of MM cells g h Transwell assays were performed to detect the metastasis of MM cells i The apoptosis rate of MM cells was examined by flow cytometry P P P activation of PI3KAKT signaling Circ_0007841 silencing downregulated the level of BRD4 and the level of BRD4 was recovered in sicirc_00078411 and inmiR3383p cotransfected group Fig a06a b The activation of PI3KAKT signaling was suppressed with the silencing of circ_0007841 and the addition of inmiR3383p recovered the phosphorylation levels of PI3K and AKT Fig a06a c Meanwhile H929 and OPM2 cells were transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 As mentioned in Fig a06d e miR3383p overexpression downregulated the level of BRD4 and the introduction of BRD4 overexpression plasmid regained the level of BRD4 in MM cells The addition of BRD4 alleviated the inhibitory influence of miR3383p overexpression on the activation of PI3KAKT signaling in MM cells Fig a0 6d f Taken together circ_0007841 accelerated the progression of MM through miR3383pBRD4PI3KAKT axisMesenchymal stromal cells MSCsgenerated exosomes accelerate the a0malignant potential of a0MM cells via a0circ_0007841MSCs exert crucial roles in the progression of MM Herein we explored whether exosomes derived from MSCs could regulate the proliferation cell cycle metastasis and apoptosis of MM cells via circ_0007841 MSCs were isolated from the adjacent tissues of MM and normal tissues The expression of circ_0007841 was higher in MSCs and MSCsderived exosomes from adjacent tissues than that in normal tissues Fig a07a b The markers of exosomes CD63 and CD81 were notably upregulated in exosomes of MSCs instead of cell lysate Fig a07c As mentioned in Fig a07d we established a working model as previously described to explore if MSCsderived exosomes could regulate the proliferation cell cycle motility and apoptosis of MM cells [] In this model only exosomes could be transmitted through the filter to the upper chambers As presented in Fig a07ek sicirc_00078411 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0007841 activates PI3KAKT signal pathway through targeting miR3383pBRD4 axis ac Western blot assay was performed to detect the levels of BRD4 and PI3KAKT signalingrelated proteins in MM cells transfected with siNC sicirc_00078411 sicirc_00078411 inmiRNC or sicirc_00078411 inmiR3383p and gray analysis was used to assess the abundance of these proteins df The expression of BRD4 and PI3KAKT signalingassociated proteins in MM cells transfected with miRNC miR3383p miR3383p pcDNA or miR3383p BRD4 was examined by Western blot assay P P P transfection inhibited the malignant behaviors of MM cells in Mock sicirc_00078411 group compared with that in Mock siNC group Besides MSCsderived exosomes MSCs siNC group promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells than that in Mock siNC group and these effects were attenuated by the silencing the circ_0007841 suggested that MSCsderived exosomes could promote the progression of MM via circ_0007841 Whats more the exosomes generated from MSCs accelerated the activation of PI3KAKT signaling while this effect was counteracted with the transfection of sicirc_00078411 Fig a0 7l Collectively MSCsderived exosomes could facilitate the progression of MM via circ_0007841DiscussionMM is an incurable cancer currently Because many MM patients were diagnosed at late stage the treatment outcomes of MM patients were unsatisfactory [] Therefore finding crucial markers in MM is urgent to improve the prognosis of MM patientsCircRNAs are featured by closely loop structure and they are widely distributed in human tissues Due to the stability and the universality of the distribution circRNAs are identified as ideal biomarkers for human cancers and other diseases [] For example the high expression of circ_0004277 was associated with the better prognosis of AML patients [] Xia et a0al claimed that circCBFB was highly expressed in chronic lymphocytic leukemia and circCBFB accelerated the proliferation and suppressed the apoptosis of chronic lymphocytic leukemia cells [] Circ_0007841 was found to be overexpressed in BMderived plasma cells of MM patients and MM cells Further studies suggested that circ_0007841 promoted the proliferation cell cycle metastasis and inhibited the apoptosis of MM cells These findings | Thyroid_Cancer |
"enzymatic cascade for posttranslational protein modiï¬cation It includes the ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 RINGtype E3 ubiquitin ligases catalyse theposttranslational proteolytic and nonproteolytic functions in various physiological and pathological processes such asammationassociated signal transduction Resulting from the diversity of substrates and functional mechanisms RINGtypeligases regulate microbe recognition and ammation by being involved in multiple ammatory signalling pathways Theseprocesses also occur in autoimmune diseases especially ammatory bowel disease IBD To understand the importance ofRINGtype ligases in ammation we have discussed their functional mechanisms in multiple ammationassociatedpathways and correlation between RINGtype ligases and IBD Owing to the limited data on the biology of RINGtype ligasesthere is an urgent need to analyse their potential as biomarkers and therapeutic targets in IBD in the future IntroductionUbiquitination is a crucial part of a diverse range of physiological and pathological processes such as protein degradationand ammationassociated signalling [ ] It is a threestep enzymatic process that consists of ubiquitinactivatingenzyme E1 ubiquitinconjugating enzyme E2 and ubiquitin ligase E3 [] E3 ligases transfer activated ubiquitin fromE2 to speciï¬c substrates thereby forming mono or polyubiquitinated proteins to activate proteasomemediated proteolysis signal transduction endocytosis etc [] E3 ligases arecrucial as they catalyse target ubiquitination and enable theformation of polyubiquitin chains that enhance the complexity of ubiquitination in physiological and pathological processes Although dysregulated ubiquitination is involved inthe development of various types of immune pathologieseg systemic lupus erythematosus rheumatoid arthritisand ammatory bowel disease [IBD] [ ] there is limitedknowledge regarding the role of RINGtype ligases inammationassociated pathways In this review we havefocused on IBD owing to its complex pathogenesis involvement in a wide range of etiological factors including dysregulated ubiquitination The term IBD is used for a group ofchronic autoimmune gastrointestinal disordersincludingmainly Crohns disease CD and ulcerative colitis UC []Chronicity of IBD often causes intestinal complications hospitalisation steroid dependency and surgery in diagnosedpatients [] Although significant progress has been made inunderstanding the nature of IBD the underlying interactingmechanism involving ubiquitination RINGtype ligases andonset of IBD remains to be fully understood Ubiquitination Mediated by RINGType LigasesHuman cells express more than E3 ubiquitin ligases thatare classiï¬ed into three types based on their catalyticdomains RING HECT homologous to the E6AP carboxylterminus and a recently identiï¬ed RBR RINGbetweenRINGRING type of E3 ligases [ ] The RING domainhas a crossbraced structure with two atoms of zinc that catalyse the direct transfer of ubiquitin from the E2Ubiquitin 0cMediators of Inï¬ammationthioester to the substrate [] Figure Apart from catalysing monoubiquitination RINGtype E3 ligases also elongatehomotypic polyubiquitin chains with varying linkage speciï¬cities such as that on Lys48 during the proteasomal targeting ofsubstrates and Lys63 in signal transduction thereby modulating proteins for proteolytic and nonproteolytic activity []However their roles in catalysing other less common typesof ubiquitination including atypical homotypic eg Lys6Lys11 Lys27 Lys29 Lys33 and Met1 [] heterotypic andbranched polyubiquitination remain ambiguous The eï¬ciency of RINGtype E3 ligases in ubiquitination depends onmultiple factors such as substrate modiï¬cation phosphorylation of E2E3 enzymes autoubiquitination by E3 ligases andpseudosubstrate competition [] The role of the RINGtype ligases and their sophisticated functional mechanism ofubiquitination will be discussed in the following sections Signalling Pathways Regulated by RINGType LigaseslectinlikereceptorsRLRs Ctype Pathogen Recognition Under physiological conditionspattern recognition receptors PRRs comprise tolllikereceptors TLRs retinoic acid inducible gene RIG Ilikereceptorsandnucleotidebinding oligomerisation domainlike receptorsPRRs recognise pathogenassociated molecular patternsPAMPs and trigger the activation of downstream eï¬ectorsin innate immune responses [] For ammatory diseasesthat are closely associated with microbiome dysbiosis such asIBD [ ] dysregulation of PRRs and relevant RINGtypeligases may be involved in pathogeninduced ammation TLR Signalling Upon recognising a wide range ofmicrobial components such as lipopolysaccharides ï¬agellinand microbial nucleic acids activated TLRs expressed onantigenpresenting cells trigger eï¬ector T cell responses inammatory diseases [] RINGtype ligases modulatethe activation of PAMPinduced TLRs By directly bindingwith TLR3 ring ï¬nger protein RNF170 catalyses theLys48linked ubiquitination and proteasomal degradationof TLR3 thereby suppressing TLR3mediated innate immunity in macrophages [] On the other hand FcÎ receptorFcÎR IIb an inhibitory FcR on antibodydependent monocyte phagocytosis is targeted by MARCH3 RNF173 forubiquitination and degradation in lipopolysaccharideinduced TLR4 activation [] RIGI Signalling RIGI is a cytoplasmic PRR that recognises viral RNA and triggers the activation of downstreamimmune responses that are associated with both viral infections and noninfectious autoimmune diseases such asenterocolitis [] The deï¬ciency of RIGI aggravates virusinduced cell death in intestinal epithelial cells and inducessusceptibility to chemically induced colitis in mice suggesting the importance of RIGI signalling in intestinal antiviralimmune response [ ] E3 ligaseslike RNF122 andRNF125 mediate Lys48linked RIGI ubiquitination andproteasomal degradation leading to the reduced expressionof infection inducedproammatory cytokines includingIL6 and type I interferons α and [ ] In contrastindependent of its E3 ligase activity RNF123 binds with theCARD domain of RIGI and melanoma diï¬erentiationassociated gene to compete with the mutual downstreamadaptor mitochondrial antiviral signalling protein MAVSand inhibit RLRmediated antiviral response [] Unlikethe aforementioned RINGtype ligases that directly targetRLRs RNF114 negatively regulates RLR signalling by polyubiquitinating and inducing the proteasomal degradation ofMAVS []is essential Proammatory Pathways Nuclear Factor Kappa B NFκB Signalling The NFκBpathway is one of the most wellstudied proammatorypathways regulated by ubiquitination [] TRAF6 RNF85ubiquitinates the evolutionarily conserved signalling intermediate in TLR activation thatfor TLR4dependent NFκB activation [] RNF183 promotes NFκBsignalling by inducing the ubiquitindependent degradationof IκBα [] TRAF2 RNF117 and TRAF3 RNF118induce Lys48linked ubiquitination and proteasomal degradation of cRel and interferon regulatory factor therebyprohibiting the synthesis of proammatory cytokines inmacrophages [] MKRN2 RNF62 mediates the polyubiquitination and degradation of the p65 subunit of NFκBthereby inhibiting NFκB signalling [] RNF114 negativelyregulates NFκB signalling and T cell activation by ubiquitinating and stabilising NFκB signalling inhibitors A20 andIκBα [ ] It has also been reported that RNF20 downregulation decreases histone H2B monoubiquitination and leadsto the NFκBdependent transcription of proammatorycytokines such as IL6 and IL8 [] Nevertheless despitethe formation of heterodimers of RNF40 with RNF20RNF40 alone activates NFκB signalling and upregulatesNFκBdependent transcription by promoting IκB kinaseIKK phosphorylation and p65 nuclear translocation indicating the involvement of NFκBdependent transcriptionin the ubiquitination of substrates other than H2B []MARCH3 RNF173 mediates Lys48linked ubiquitinationand lysosomal degradation of IL1 receptor I and therebyinhibits IL1triggered NFκB activation [] Independentof its E3 ligase activity RNF8 inhibits TNFαinduced NFκBactivation by directly binding with the kinase domain of IKKand interfering with IKKα phosphorylation [] RNF11also exerts a noncanonical role in negatively regulating NFκB signalling RNF11 has high aï¬nity for the E2 enzymeUbc13 and minimal E3 ligase activity that subsequently outcompetes E1 enzymes and other E3 enzymes such as TRAF6[] and impedes the activation of NFκB signalling [] MitogenActivated Protein Kinase MAPK SignallingMAPKs are another family of proteins closely related toammationassociated pathologies such as IBD []Upon stimulation by TNFα TRAF2 is autoubiquitinatedon the Lys63 residue that enables its translocation to thecytoskeletal fraction and activates JNK signalling [ ]In vitro experiments have shown that JNK signalling issuppressed and enhanced in cells overexpressing RNF213 0cMediators of Inï¬ammationUbUbE2LysSubstrateRING E3 ligasePolyubiquitinationchain initiationUbE2SubstrateRING E3 ligaseMonoubiquitinationE2SubstrateRING E3 ligaseMultiple monoubiquitinationUbUbE2SubstrateRING E3 ligaseHomotypic polyubiquitinationmodification sites of ubiquitin include Lys6 Lys11 Lys27 Lys29 Lys33 Lys48Lys63 and Met1 SubstrateUb Ub Ub Ub UbChain elongationSubstrateUbUb Ub Ub UbUbUbHeterotypic polyubiquitinationSubstrateUbUbUb Ub UbE2SubstrateRING E3 ligaseBranched polyubiquitinationSubstrateUb Ub Ub Ub UbUb Ub Ub UbFigure Functional mechanisms of RINGtype E3 ligase Acting as a scaï¬olding RINGtype E3 ligase recruits a E2ubiquitin thioester and asubstrate and allows the lysine of substrate to attack the thioester for ubiquitin transfer RINGtype E3 ligases catalyse monoubiquitination ormultiple monoubiquitination by transferring a single ubiquitin to one or several residues of the substrate For polyubiquitination ubiquitinsform eight diï¬erent linkage types including Met1 Lys6 Lys11 Lys27 Lys29 Lys33 Lys48 and Lys63 Apart from homotypic chains RINGtype E3 ligases also catalyse heterotypic chains and branched ubiquitin chains by adopting multiple linkage types and branched topology inthe formation of polyubiquitin chainsand RNF186 respectively [ ] however the mechanismsinvolved in this regulation remain to be understood TRAF7RNF119 upregulatesthe kinase activity of mitogenactivated protein kinase via the WD40 domain and potentiates cell apoptosis via the RING ï¬nger domain []Similarly RNF13 mediates endoplasmic reticulum ERstressinduced JNK activation and subsequent cell apoptosisby binding with and promoting the phosphorylation ofthe ER stress sensor endoplasmic reticulum to nucleussignalling [] Janus Kinase JAKSignal Transducer and Activator ofTranscription STAT3 Signalling JAKSTAT3 is one of themajor proammatory signalling pathways that orchestrate 0cRINGtype ligaseRNF11RNF183RNF2RNF135RNF125TRAF6cCblZNRF2Table RINGtype ligases targeted by noncoding RNAs and the relevant pathwaysMediators of Inï¬ammationNoncoding RNAmiR19b3pmiR7miR1395pmiR4853pmiR15bmiR124miR146miR216alncRNA TTN antisense RNA acts as a ceRNA for miR1533pRelevant pathwayNFκB signallingNFκB signallingER stressinduced apoptosisMAPK signallingMAPKERK signallingRIGI signallingTLR signallingTLR signallingPI3KAKT signallingPI3KAKT signallingReference[][][][][][][][][][]the progression of ammatory and autoimmune diseases[] A number of RINGtype ligases modulate JAKSTAT3signalling RNF6 and RNF38 function in catalysing theubiquitinationinduced proteasomal degradation of SH2containing protein tyrosine phosphatase thattargetsphosphorylated STAT3 thereby maintaining STAT3 phosphorylation and activating STAT3 signalling [ ] Incontrast TRAF6 promotes Lys63linked ubiquitination ofSTAT3 and represses STAT3mediated transcription ofdownstream ammationrelated genes such as Creactiveprotein [] Interestingly RNF41 modulates the cell surfaceexpression of JAK2associated cytokine receptors by blocking the cleavage of receptors and enhancing receptor shedding in a ubiquitinationdependent manner [] Phosphatidylinositol 3Kinase PI3K Signalling PI3K isanother classical pathway involved in ammation whereinRINGtype ligases are of crucialimportance MKRN1RNF61 functions in the positivefeedback regulation of sustained PI3KAKT activation upon stimulation by epidermalgrowth factor AKT activation phosphorylates and stabilisesE3 ligase MKRN1 that further ubiquitinates and degradesphosphatase and tensin homologue a PI3KAKT inhibitor[] MKRN2 RNF62 induces the ubiquitindependent degradation of the p85α subunit of PI3K and downregulated AKTphosphorylation suggesting a negative regulatory role ofMKRN2 in PI3KAKT signalling [] Downregulation ofUHRF1 RNF106 represses the phosphorylation of PI3Kand AKT which reveals an underlying interaction betweenUHRF1 and PI3KAKT signalling [] Transforming Growth Factor TGF Signalling TGF signalling functions in immunosuppression and inhibitingthe activity of eï¬ector T cells maintaining Treg diï¬erentiation reducing B cell responsiveness and inducing macrophage anergy [] RNF11 plays a dualrole in themodulation of TGF signalling By competing with Smad7for Smurf2 RNF11 is a positive regulator for TGF signalling and reduces the formation of Smad7Smurf2 complexesthat degrade TGF receptors [] RNF11 is also responsiblefor the ubiquitinationmediated stabilisation of Smad4 thatenhances Smad4dependent TGF signalling by directinteraction [] Notably RNF11 may negatively regulatesignallingbytheenablingformationTGFofSmurf2RNF11 complexes and inducing the ubiquitinationand degradation of the associated molecule with the SH3domain of STAM that promotes TGF signalling []PRAJA RNF70 mediates the ubiquitinationinduced proteasomal degradation of embryonic liver fodrin a Smad4adaptor protein thereby negatively regulating TGF signalling [] Autophagy Accumulating evidence reveals that autophagy contributes extensively to immune cell developmentand cell death and its dysregulation has been implicated inmany autoimmune diseases [] TRAF6 catalyses Lys63linked ubiquitination of BECN1 and stimulates TLRinduced autophagy in macrophages upon proammatorystimulation [ ] RNF166 has a novel role in antibacterialhost defence owing to its function in inducing the Lys29 andLys33linked ubiquitination of autophagy adaptor p62which mediates the recruitment of p62 to bacteria and initiates bacteria engulfment [] Noncoding RNAs Since dysregulated noncoding RNAsare involved in the progression of ammatory diseases[] the posttranscriptional regulation of RINGtype ligasesby noncoding RNAs may play critical roles in potentialammationrelevant signalling pathways Until now quitea few microRNAs have been proved to posttranscriptionallyregulate RINGtype ligases by hampering translation orinducing mRNA degradation Table [ ] Nevertheless although the other two major types of noncodingRNAs long noncoding RNAs and circular RNAs also havediverse functions in ammatory diseases eg competingendogenous RNA [ceRNA] transcription regulation andRNAbinding protein sponges [ ] to what extent theymodulate RINGtype ligases awaits further analysis RINGType Ligases in IBD Pathogenesis of IBD The pathogenesis of IBD has beenelucidated over the past years More than loci have beenimplicated in increased genetic risk for IBD that correlate withthe functioning of cellular processes such as innateadaptiveimmune responseintestinal mucosal barrier homeostasis 0cMediators of Inï¬ammationTable Roles of RINGtype ligases in IBD patients and animal models of colitisRINGtypeligaseRoleIBD patientsAnimal model of colitisReferenceRNF186ControversialIncrease risk of UC and ERstressinduced apoptosisAttenuate ER stress and maintain intestinalpermeability in DSSinduced mouse model[]of colitisRNF20RNF40AntiammatoryProammatoryDecrease in the colonic tissueProtect mice from DSSinduced colitis andfrom UC patientsmaintain intestinal barrierActivate NFκB signalling in DSSinducedmouse model of colitis[][][][][ ]RNF183ProammatoryIncrease in the colonic tissuefrom CD and UC patientscorrelate with endoscopicindex of disease severityUHRF1AntiammatoryTRAF2AntiammatoryIncrease in the colonic tissuefrom CD and UC patientsTRAF3AntiammatoryTRAF5AntiammatoryIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in the plasma andcolonic tissue from CD andUC patientsIncrease in TNBSinduced mouse modelof colitisRegulate TNFα expression in mice withDSSinduced colitis and zebraï¬shModulate the proliferation anddiï¬erentiation of Treg cellsModulate colonic microbiotacomposition proammatorycytokine expression and immunecell ltration in mice withDSSinduced colitisRegulate proammatory cytokineexpression and mitigate ammatorydamage in DSSinduced mousemodel of colitis[ ]Inhibit IL17mediated proammatorypathway in TNBSinduced mousemodel of colitisControl proammatory cytokineexpression and protect mice againstexperimental colitis[]and autophagy suggesting the involvement of multiple factorsin shaping the procolitogenic environment during the development of IBD [ ] IBD patients manifest with abnormalities in the composition of gut microbiota such as decreasedbacterial diversity increased proportion of harmful bacterialstrains and decreased proportion of protective probioticswhich trigger proammatory intestinal pathogenic immuneresponses and contribute to the pathogenesis of IBD [ ]Elevated levels of proammatory cytokines eg IL1 IL6and IL23 and activation of adaptive eg Th1 Th2 Th9and Th17 cells and innate immune cells eg neutrophilsand NK cells constitute a synergistic ammatory networkthat induces intestinal mucosal ammation and sustainedactivation of multiple proammatory signalling pathways[ ] IL1 familyinduced NFκB IL6induced STAT3MAPK and PI3K signalling pathways are pivotal in intestinalammation [ ] TGF signalling can mitigateimmune cell hyperactivation but also causes the formation ofintestinal strictures in chronic intestinal ammation []Autophagy is involved in the regulation of immune cell function thus defective autophagy also plays an important role inIBD pathogenesis [] A dysfunctional gut barrier and subsequent increased intestinal permeability are also consideredimportant etiologic factors in the development of IBD thatresult in the uncontrolled exchange of materials between theintestinal lumen and internal environment A compromisedgut barrier is often attributed to the proammatory stimulation and subsequent downregulation of sealing tight junctionproteins eg claudin5 and claudin8 and upregulation ofporeforming tight junction proteins eg claudin2 []Since multiple etiologic factors function in the pathogenesisof IBD in a synergistic manner further research is warrantedto understand the dynamics between these players Role of RINGType Ligases in IBD Despite the limitedknowledge on RINGtype ligases in IBD research suggestsa correlation between RINGtype ligases and IBD pathogenesis Table Genomewide association studies have identiï¬ed RNF186 as one of the genes associated with susceptibilityto UC the diseasecoding variant of RNF186 involves analtered RING domain [ ] The truncated RNF186 lacking the second transmembrane domain is associated withprotecting individuals against developing UC by inhibitingthe ER localisation of RNF186 and subsequent Lys29 andLys63linked polyubiquitination of proapoptotic BCL2 interacting protein under ER stress [ ] Notably RNF186functions diï¬erently in a dextran sulfate sodium DSSinduced mouse model of colitis RNF186deï¬cient mice 0cMediators of Inï¬ammationdevelop more severe colitis during DSS administration andtheir colonic epithelial cell exhibits enhanced signs of ER stressand apoptosis [] RNF186 also modulates intestinal barrierfunction by mediating the Lys48linked ubiquitination of tightjunction protein occludin and RNF186 deï¬ciency increasesintestinal permeability in RNF186 knockout mice [] ThusRNF186 targets diï¬erent substrates and has a complex association with gut ammationApart from the reduced expression of RNF20 in the colonsamples from UC patients homozygous RNF20knockoutmice die due to embryonic lethality and heterozygous miceare susceptible to DSSinduced colitis with increased intestinal permeability suggesting the antiammatory role ofRNF20 [] RNF40 knockout mice exhibit mitigated gutammation upon treatment with DSS this can be attributed to the attenuated activation of NFκB signalling []The upregulation of RNF183 in the colonic tissue from IBDpatients and 246trinitrobenzenesulfonic acid TNBSinduced mouse model of colitis indicates its proammatory function probably by promoting the ubiquitinationinduced degradation of IκBα []Because UHRF1catalysed histone H3 monoubiquitination recruits and stimulates DNA methyltransferase toDNA methylation sites and thereby maintains DNA methylation UHRF1 participates in the epigenetic control of multiple genes such as TNFα [ ] Mice with macrophagesdeï¬cient for UHRF1 manifest with TNFα overexpressionand aggravated DSSinduced colitis Also the loss of functionin UHRF1 reduces the methylation of the TNFα promoterin macrophages indicating the regulatory role of UHRF1 inthe mouse model of colitis [] Similarly an in vivo studyin zebraï¬sh has revealed that loss in function of UHRF1 leadsto defects in the epigenetic regulation of TNFα promotermethylation and elicits elevated TNFα expression in ammatory processes including intestinal epithelial cell apoptosis neutrophil recruitment and weakened intestinal barrierfunction [] However UHRF1 may eï¬ect diï¬erentlyamong subtypes of regulatory T Treg cells as UHRF1 maintains the proliferation and maturation of colonic Treg cellsbut inhibits the diï¬erentiation of peripheral induced Tregcells in the development of colitis [ ]Studies have shown the diverse roles involved with theupregulation of TRAFs including TRAF1235 in the bloodor colonic mucosa of IBD patients [ ] DSSinducedcolitis models of TRAF2 and TRAF3deï¬cient mice revealsimilar functions of TRAF2 and TRAF3 as negative regulators of experimental colitis by decreasing proammatorycytokines and reducing the ltration of immune cells inthe colon [] In another study TRAF2deï¬cient micedevelop severe spontaneous colitis and exhibit altered colonicmicrobiota composition indicating the antiammatoryrole of TRAF2 in controlling colonic microbiota [] TRAF3also acts as a colitis regulator by binding with the IL17receptor and interfering with the IL17mediated proammatory pathway in mice with TNBSinduced colitis []Although TRAF5 RNF84 promotes the ubiquitinationand stabilisation of the retinoic acidrelated orphan receptorÎt that mediates proammatory Th17 cell diï¬erentiationand IL17AIL17F expression [ ] TRAF5deï¬cientmice exhibit aggravated experimental colitis and upregulation of proammatory cytokines [] The complex function of TRAF5 needs further analysis DiscussionAs the importance of RINGtype E3 ligases is graduallyunveiled there are still problems to be solved Firstly apartfrom the canonical role of RINGtype ligases in modulatingkey signalling pathways and their downstream adaptors asE3 ubiquitin ligases some RINGtype ligases interfere withthe ubiquitination cascade by competition or direct interaction with other E3 ligases [] Owing to the variety ofRINGtype ligases and substrate speciï¬city of E3 ligases thepotential competition among RINGtype ligases in regulatingimmune response remains to be fully understood Secondlydiï¬erences in RING E3 ligasemediated target ubiquitinationcan also be attributed to the variance in length and linkagetype of ubiquitin chains Although RINGtype ligases function in proteolytic degradation and signal transduction bycatalysing Lys48linked and Lys63linked ubiquitinationrespectively their roles in catalysing less common linkagetypes of homotypic polyubiquitin chains such as Lys11linked ubiquitination [] and the outcome of such polyubiquitination are still obscureRINGtype ligases form a sophisticated but importantubiquitination network wherein the expression and functionof RINGtype ligases are also uenced reciprocally in physiological and pathological processes Understanding themechanisms employed by RINGtype ligases in modulatingammationassociated pathways by catalysing atypicallinkages and aï¬ecting signaltransduction may furtherexplain the interaction between RINGtype ligases and IBDSimilarly research on heterotypic polyubiquitin chains isalso important to unveil these underlying mechanismsIn this review we have highlighted the roles of RINGtype ligases in PAMP recognition and modulation ofammationassociated pathways that are crucial etiologicalfactors in the development of autoimmune diseases Accumulating evidence shows that many RINGtype ligases areinvolved in ammationassociated pathways such as proammatory NFκB MAPK JAKSTAT3 and PI3K signalling and antiammatory TGF signalling Subsequentlywe have discussed the role of RINGtype ligases in the pathogenesis of IBD via ammationrelated pathways Patientswith IBD exhibit the diï¬erential expression of speciï¬c RINGtype ligases such as TRAFs However there are limited studies on the potential clinical value of RINGtype ligases inpredicting or treating IBD Thus far there have been a fewattempts to use RINGtype ligases as predictive biomarkersand therapeutic targets in treating cancer RNF43 modulatesWnt signalling and has been used to target colorectal cancerand pancreatic ductal carcinoma [ ] Neverthelessthe potential of RINGtype ligases in autoimmune diseasesespecially in IBD needs to be understood in greater detailfuture research on the expression proï¬le ofThereforeRINGtype ligases in the gastrointestinaltract and thedetailed mechanisms is warranted 0cMediators of Inï¬ammationConflicts of InterestThe authors have no conï¬ict of interest to discloseAuthors ContributionsThe guarantor of the article is Shenghong Zhang ShenghongZhang designed the study Shenghong Zhang Liguo ZhuYing Li and Longyuan Zhou wrote and revised the manuscript Guang Yang Ying Wang Jing Han and Li Li revisedthe important intellectual content of the manuscript All theauthors approved the ï¬nal version Liguo Zhu Ying L andLongyuan Zhou contributed equally to the workAcknowledgmentsThis study was supported by the National Natural ScienceFoundation of China under Grants and Guangdong Science and Technology Department under Grant 2017A030306021 and FundamentalResearch Funds for the Central Universities under Grant19ykzd11References[] D Popovic D Vucic and I Dikic Ubiquitination in diseasepathogenesis and treatment Nature Medicine vol no pp [] D Aki Q Li H Li Y C Liu and J H Lee Immune regulation by protein ubiquitination roles of the E3 ligases VHLand itch Protein Cell vol no pp [] C M Pickart Mechanisms underlying ubiquitinationAnnual Review of Biochemistry vol no pp [] H Hu and S C Sun Ubiquitin signaling in immuneresponses Cell Research vol no pp [] R Hodson Inï¬ammatory bowel disease Nature vol no p S97 [] L PeyrinBiroulet E V Loftus Jr J F Colombel and W JSandborn The 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for intestinal bacteriaviruses and fungi in pathogenesis of ammatory bowel disapproaches Gastroenterologyeasesvol no pp 339e4 therapeuticand[] T Kawai and S Akira The role of patternrecognition receptors in innate immunity update on tolllike receptors NatureImmunology vol no pp [] J Q Chen P Szodoray and M Zeher Tolllike receptorpathways in autoimmune diseases Clinical Reviews inAllergy and Immunology vol no pp [] J H Park L PeyrinBiroulet M Eisenhut and J I ShinIBD immunopathogenesis a comprehensive review ofammatory molecules Autoimmunity Reviews vol no pp [] X Song S Liu W Wang Z Ma X Cao and M Jiang E3ubiquitin ligase RNF170 inhibits innate immune responsesby targeting and degrading TLR3 in murine cells Cellular Molecular Immunology vol no pp [] K Fatehchand L Ren S Elavazhagan TolllikereceptorIIbFcÎRIIb via MARCH3 proteinmediated ubiquitinationThe Journal of Biological Chemistry vol no pp ligands downregulate FcÎ receptor[] T Matsumiya and D M Staï¬orini Function and regulationof retinoic acidinducible geneI Critical Reviews in Immunology vol no pp [] Y Hirata A H Broquet L Menchén and M F Kagnoï¬Activation of innate immune defense mechanisms by signaling through RIGIIPS1 in intestinal epithelial cellsJournal of Immunology vol no pp [] W Wang M Jiang S Liu RNF122 suppresses antiviraltype I interferon production by targeting RIGI CARDs tomediate RIGI degradation Proceedings of the NationalAcademy of Sciences ofthe United States of Americavol no pp [] K Arimoto H Takahashi T Hishiki H Konishi T Fujitaand K Shimotohno Negative regulation of the RIGI signaling by the ubiquitin ligase RNF125 Proceedings of theNational Academy of Sciences of the United States of Americavol no pp [] S Wang Y K Yang T Chen RNF123 has an E3ligaseindependentreceptormediated antiviral signaling EMBO Reports vol no pp in RIGIlikefunction[] B Lin Q Ke H Li N S Pheifer D C Velliquette and D WLeaman Negative regulation of the RLH signaling by the E3ubiquitin ligase RNF114 Cytokine vol pp [] J Chen and Z J Chen Regulation of NFκB by ubiquitination Current Opinion in Immunology vol no pp [] Y Min M J Kim S Lee E Chun and K Y Lee Inhibitionof TRAF6 ubiquitinligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation Autophagy vol no pp [] Q Yu S Zhang K Chao E3 ubiquitin ligasein ammatory bowelRNF183 is a novelregulator 0cMediators of Inï¬ammationdisease Journal of Crohn's Colitis vol no pp [] J Jin Y Xiao H Hu Proammatory TLR signallingis regulated by a TRAF2dependent proteolysis mechanismin macrophages Nature Communications vol no article [] C Shin Y Ito S Ichikawa M Tokunaga K SakataSogawa and T Tanaka MKRN2 is a novel ubiquitin E3ligase for the p65 subunit of NFκB and negatively regulates ammatory responses Scientiï¬c Reports vol no article [] M S Rodriguez I Egaña F LopitzOtsoa The RINGubiquitin E3 RNF114 interacts with A20 and modulatesNFκB activity and Tcell activation Cell Death Diseasevol no article e1399 [] K Heyninck and R Beyaert A20 inhibits NFkappaB activation by dual ubiquitinediting functions Trends in Biochemical Sciences vol no pp [] O Tarcic I S Pateras T Cooks RNF20 links histoneH2B ubiquitylation with ammation and ammationassociated cancer Cell Reports vol no pp [] R L Kosinsky R L Chua M Qui Loss of RNF40decreases NFκB activity in colorectal cancer cells andreduces colitis burden in mice Journal of Crohn's Colitisvol no pp [] H Lin D Gao M M Hu MARCH3 attenuates IL1triggered ammation by mediating K48linked polyubiquitination and degradation of IL1RI Proceedings of theNational Academy of Sciences of the United States of America | Thyroid_Cancer |
"Sphingosine1phosphate receptor S1PR1 is involved in vascular development a key process intumorigenesis This study aimed to evaluate its roles in tumor development and prognosisMethods S1PR1 expression levels were analyzed using TIMER and Oncomine database and the prognosticsignificance of S1PR1 was assessed using PrognoScan and KaplanMeier plotter databases The relationship betweenS1PR1 and tumorinfiltrated immune cells was analyzed using TIMERResults S1PR1 expression was remarkably lower in breast and lung cancer tissues than in the correspondingnormal tissues Lower expression was related to poor overall survival and diseasefree survival in breast invasivecarcinoma BRCA lung adenocarcinoma LUAD and lung squamous cell carcinoma LUSC A functional networkanalysis confirmed the function of S1PR1 in regulating vasculogenesis In addition S1PR1 levels were significantlynegative with regard to the tumor purity of BRCA r P 176e66 LUAD r P 605e16 andLUSC r P 520e20 Furthermore S1PR1 levels were significantly positive with regard to infiltratingCD8 r P 591e35 and CD4 T cells r P 103e26 macrophages r P 367e12neutrophils r P 203e7 and dendritic cells DCs r P 914e11 in BRCA S1PR1 levels weresignificantly positive with regard to CD8 T cells r P 361e12 macrophages r P 101e17neutrophils r P 415e8 and DCs r P 416e6 in LUAD and positive with regard to B cells r P 157e15 CD8 r P 383e26 and CD4 T cells r P 398e14 macrophages r P 261e45 neutrophils r P 179e25 and DCs r P 212e40 in LUSCConclusions S1PR1 levels are positively correlated with multiple immune markers in breast and lung cancer Theseobserved correlations between S1PR1 and the prognosis and immune cell infiltration provide a foundation forfurther research on its immunomodulatory role in cancerKeywords S1PR1 Breast cancer Lung cancer Tumorinfiltrating Prognosis biomarker Correspondence caodl126com wenrenlyf2008163com1The First Affiliated Hospital Guangzhou University of Chinese Medicine No Airport Road Baiyun District Guangzhou China2Department of Laboratory Medicine Guangdong Second Provincial GeneralHospital No Xingang Middle Road Haizhu District Guangzhou Guangdong Province ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhong BMC Cancer Page of BackgroundSphingosine1phosphate S1P produced by sphingosine kinase Sphkis a biologically active signalinglipid [] S1P regulates vascular development andfunction including vascular maturation [ ] S1P receptor S1PR1is a biologically active sphingolipidmetabolite that mediates S1P activity and promotescell proliferation and survival [ ] S1PR1 is widelyexpressed in vascular endothelial cells and is requiredfor embryonic vascular development and maturation[] Estrogen the growthstimulating hormone inbreast cancer cells was shown to stimulate endothelial cell growth via S1PR1 [ ] In the tumor microenvironment S1P exhibits multiple functionsitincreases the survival of macrophages b it serves asthe comeandgetme signal of dead cells attractingand enhancing macrophage migration by combiningwith S1PR1the polarization ofTAMM2 macrophages by activating S1PR124 [] Accumulating evidence demonstrated that tumorprogression requires new blood vessel growth whichis achieved by producing angiogenic factors that canactivate vascular endothelial cells [] Tumor cellsreleasevascularendothelial growth factor VEGFa which leads toangiogenesis and tumor growth [] Studies haveshown that S1PR1 inhibits VEGF signaling by prointeraction between VEcadherin andmoting theVEGFR2thereby inhibiting VEGFinduced vascularsprouting [ ]angiogenicstimulatesstimulicitasuchasHowever the role of S1PR1 in tumorigenesis andits prognostic value are unclear A preclinical studyon human breast cancer cells found that S1PR1 antibody can enhance the cytotoxic and antiproliferativeeffect of carboplatin on MDAMB231 and SKBR3HER2 subtype cells respectively [] Lei et alfound that S1PR1 signaling has tumorsuppressiveeffects and survival benefits in breast cancer []Therefore it is necessary to clarify the role of S1PR1in tumor development and progression Transcriptome analysis can be used to predict important issues such as the intrinsic subtype of the primarytumor tumor grade drug reactivity and recurrencerisk []Herein we used Oncomine KaplanMeier plotterPrognoScan UALCAN and GEPIA datasetstoanalyze S1PR1 expression and its relationship withthe prognosis of cancer patients Furthermore westudied the correlation between S1PR1 and tumorinfiltrated immune cells in the tumor microenvironment using TIMER Our results shed light on theimportant role of S1PR1 in breast and lung cancerand determined that it is closely related to tumorimmunityMethodsOncomine database analysisThe Oncomine database wwwoncomineresourceloginhtml was used to evaluate the expressionlevel of S1PR1 in various types of cancers [] Thethresholds were a Pvalue of fold change of and data type was mRNAPrognoScan database analysisThe PrognoScan database wwwprognoscan wasused to test S1PR1 expression and survival in varioustypes of cancers [] The threshold was an adjustedCox Pvalue of CBioPortal database analysiscBioPortal httpcbioportal contains multidimensional cancer genomics data sets [] S1PR1 mutationsand copy number variation CNV in breast and lungcancers were analyzed using cBioPortal The OncoPrinttab was used to obtain an overview of the genetic alterations for each samplethe prognosticKaplanMeier plotterKaplanMeier Plotter kmplotcom was applied to assessvalue of S1PR1Grouped according to the median expression ofS1PR1 high vs low expression all patients were analyzed for overall survivalOS and progressionfreesurvival PFS and KaplanMeier was used to draw asurvival chart []Immune infiltrates analysis using the TIMERTIMER cistromeshinyappsiotimer wasused to analyze immune infiltrates across different typesof cancer [] Especially the expression of S1PR1 in different cancer types and the correlation between the expression of S1PR1 and the abundance ofimmuneinvasion was determined In addition the correlation between S1PR1 expression and tumor infiltrating immunecell gene markers was also explored through relatedmodulesGene correlation analysis using GEPIAGEPIA httpgepiacancerpkucnindexhtml wasused to confirm the genes with significantly correlatedexpression levelsin TIMER [] The Spearmanmethod was used to determine the correlation coefficients The tumortissue datasets were used foranalysisLinkedOmics database analysisThe LinkedOmics database httpwwwlinkedomicsloginphp was used to analyze S1PR1 coexpressioncorrelationPearsonsbasedon 0cZhong BMC Cancer Page of coefficients The results were visually evaluated usingvolcano plots and heat maps The function moduleof LinkedOmics was used to analyze gene ontologyGOKyotoEncyclopedia of Genes and Genomes KEGG pathways by a gene set enrichment analysis GSEA Therank criterion was FDR and simulationswere performed []biologicalprocessesBPandUALCAN database analysisUALCAN httpualcanpathuabeduincluded theCancer Genome Atlas TCGA level RNA sequencesClinical data from cancer types were used toanalyze the relative expression of genes in tumorand normalstagetumor grade or other clinicopathological characteristics []according to tumorsamplesS1PR1 mRNA expression level analysisGene expression data of breastinvasive carcinomaBRCA lung adenocarcinoma LUAD and lung squamous cell carcinoma LUSC in TCGA were downloadedin UCSC Xena xenabrowsernet S1PR1 mRNAexpression level was compared between cancerous andnormal tissue using MannWhitney test with P setting as a cutoffStatistical analysisGene expression data in the Oncomine database wasanalyzed using pvalue fold change and mRNA datatype The survival curves were generated via KaplanMeier plots and PrognoScan database are displayedwith HR and P or Cox Pvalues from a logrank testSpearman correlation analysis was used to evaluatethe correlation of gene expression in TIMER and LinkedOmics databases P was considered statistically significantResultsS1PR1 mRNA expression levels in different types ofhuman cancersThe Oncomine database was used to analyze S1PR1mRNA levels in tumor tissues and normal tissues ofvarious cancer types S1PR1 expression was lower inmost tumor tissues including sarcoma bladder brainleukemiacentral nervous system breast colorectallung myeloma and ovarian cancer tissuesthan innormal tissues Fig 1a The mRNAseq data fromTCGA were analyzed using TIMER to verify thesefindings Data from TCGA shown that the differentialexpression of S1PR1 between the tumor and adjacentnormal tissues is shown in Fig 1b Compared withadjacent normal tissues S1PR1 expression was significantlycarcinomaBLCA BRCA cholangiocarcinoma CHOL colonadenocarcinomacarcinomaESCA head and neck squamous cell carcinomaHNSC kidney chromophobe KICH kidney renalpapillary cell carcinoma KIRPliver hepatocellularcarcinoma LIHC LUAD LUSC prostate adenocarREADcinomaskinstomachrectum adenocarcinomacutaneous melanomain bladderesophagealurothelialSKCMreducedCOADPRADFig S1PR1 expression levels in different types of human cancers a Differences in S1PR1 between cancer tissues and normal tissues based ondata in the Oncomine database P 1E04 Fold change Data type mRNA b Human S1PR1 expression levels in different tumor types fromTCGA database were determined using TIMER P P P 0cZhong BMC Cancer Page of adenocarcinoma STAD and uterine corpus endometrial carcinoma UCEC However S1PR1 expression was significantly higher in kidney renal clear cellcarcinoma KIRC and thyroid carcinoma THCAthan in adjacent normal tissues Fig 1b These datashowed that alterations in S1PR1 expression dependon the tumor type suggesting that this gene exertsdiverse functions in various tumorscancerand GSE12276in three breastPrognostic evaluation of S1PR1 in cancersWe investigated whether S1PR1 expression is relatedto prognosis The effect of S1PR1 expression on survival wasevaluated by PrognoScan Two probes204642_at and 239401_at matching S1PR1 were detected NotablycohortsGSE1456GPL96 GSE7378lowS1PR1 expression was significantly associated with apoorer prognosis breast cancer Fig 2af We usedthe KaplanMeier plotter database to further examinethe prognostic value of S1PR1 in breast cancer Poorprognosis based on recurrencefree survival RFS inbreast cancer was significantly correlated with lowS1PR1 expression HR P 71e13 but a significant correlation was not observed for overall survival OS HR P and postprogressionsurvival PPS HR P Fig 2gi Its determined that the low expression of S1PR1 is an inderisk factorpendentfor poor prognosis of breastcancer In additionlow S1PR1 expression was alsorelated to poor prognosis in two cohorts of patientswith lung cancer GSE31210 and GSE8894 as determined using two probes 204642_at and 239401_atFig 2jl KaplanMeier plotter database also showedthat low expression of S1PR1 was an independent riskfactor for poor prognosis of lung cancer overall survival HR P 69e08 recurrencefree survivalHR P but notrelated to postin lung cancer HR P progression survival Fig 2moFurthermore we found that low S1PR1 expressionwas associated with a poor prognosis in patientswith soft tissue blood and brain cancers Fig S1ac In contrastlow S1PR1 expression was an independent risk factor for a good prognosis in gastricFig S1dg These results confirmed thecancerprognostic value of S1PR1 in specific types of cancer both high and low S1PR1 expression was associated with prognosis depending on thetype ofcancer Based on the consistent results for the associations between S1PR1 expression and survivalinbreast and lung cancers we focused on the preciseeffects of S1PR1 in these two cancer types as wellas the underlying mechanismsP Correlations between clinical characteristics and S1PR1expression in breast cancer and lung cancerWe used the KaplanMeier plotter to study the relationship between S1PR1 expression and clinical characteristics in patients with breast cancer and lungcancer Low expression of S1PR1 was associated withworse overall survival OS in male and female patients with lung adenocarcinoma P Table In particular low S1PR1 mRNA expression was correlated with worse OS in stage P 920E13 andearlystage AJCC stage Mlung cancerTable Low S1PR1 mRNA expression was relatedto poor OS in patients with P or withoutP a smoking history Table In additionlow S1PR1 mRNA expression was related to worseOS in patients who did not receive chemotherapy orradiotherapy These findings strongly suggest that lowS1PR1 mRNA expression is correlated with poor OSin lung cancer Table In BRCA low S1PR1 mRNAexpression was related to poor OS in ERpositive orHER2negative patients and in the luminal androgenreceptor subtype Table Taken together high expression of S1PR1 could be considered a good prognostic indictor for breast and lung cancers dependingon the clinical characteristicsDecreased expression of S1PR1 in breast cancer and lungcancer patientsWe further analyzed the expression of S1PR1 inbreast and lung cancers Gene expression data ofbreast invasive carcinoma BRCAlung adenocarcinoma LUAD and lung squamous cell carcinomain TCGA were downloaded and S1PR1LUSCmRNA expression level was compared between tumorand normal tissue As shown in Fig 3a the expression of S1PR1 was significantly decreased in tumortissues of BRCA LUAD and LUSC Fig 3a In comparison with normal control tissues breast cancer andlung cancertissues presented lower expression ofS1PR1 which was also observed by GEPIA analysisFig 3b Furthermore we analyzed TCGA data usingthe UALCAN database Compared to normal tissuesS1PR1 mRNA expression was significantly decreasedin primary tumors and tumor stages stage stage stage and stage of BRCA LUAD and LUSCFig 3ce Taken together these data confirmed thedownregulation of S1PR1 expression in breast cancerand lung cancer patientsRegulators of S1PR1 in breast cancer and lung cancerWe used the LinkedOmics function module to detectthe S1PR1 regulatory network to further understandthe biological role of S1PR1 in breast cancer and lung 0cZhong BMC Cancer Page of Fig See legend on next page 0cZhong BMC Cancer Page of See figure on previous pageFig Prognostic value of S1PR1 in cancers af KaplanMeier survival curves comparing high and low expression of S1PR1 in breast cancersusing PrognoScan Survival curves based on OS DSS and DFS in three breast cancer cohorts [GSE1456GPL96 n GSE7378 n andGSE12276 n ] gi Survival curves for breast cancers based on mRNAseq data from TCGA of KaplanMeier plotter databases jl KaplanMeier survival curves comparing high and low expression of S1PR1 in lung cancers using PrognoScan Survival curves based on RFS in two threelung cancer cohorts [GSE31210 n and GSE8894 n ] mo Survival curves for lung cancers based on mRNAseq data from TCGA ofKaplanMeier plotter databases OS Overall survival RFS RelapseFree Survival PPS Postprogression survival DSS Diseasespecific survivalDFS Diseasefree survivalcancer Figure 4ac shows genes with significantlypositive dark red dots and negative dark greendots correlations with S1PR1 false discovery rateFDR The top positively and negatively related genes are shown in a heat map in Fig 4df AGene Ontology GObased gene set enrichment analysis GSEA showed that genes that are coexpressedwith S1PR1 are enriched for vasculogenesis and thepurinergic receptor signaling pathway while genes related to mitochondria and RNA transcript processingwere inhibited in breast cancer Fig 4g SimilarlyGO annotation resultscoexpressed with S1PR1 are primarily associated withvasculogenesis the purinergic receptor signaling pathway and the phospholipase Cactivating G proteincoupled receptortRNAmetabolic process RNA modification and RNA transcript processing werecancerFig 4hi A KEGG pathway analysis showed enrichlineage Staphylococcusmentaureusinboth breast cancer and lung cancer SpliceosomeDNA replicationand proteasome pathways wereinhibited in both tumor types Fig 4jl These resultssuggest that S1PR1 contributes to various processesin tumor development at least partially through regulate vasculogenesisinfection and renin secretion pathwayssignaling pathway whilefor hematopoietic cellshowedthatgenesinhibited in lungamplifications and deletionsGenomic alterations in S1PR1 in breast cancer and lungcancercBioPortal database was used to determine the typesand frequencies of S1PR1 alterations in BRCA LUADand LUSC S1PR1 was altered in of patients withBRCA These alterations included mRNA missensemutationsFig 5aS1PR1 was altered in of patients with LUAD and of patients with LUSCincluding mRNA missense mutations truncating mutations amplificationsand deletions Fig 5a Moreover S1PR1 CNV wasassociated with OS in LUAD but not with OS or DFSin BRCA and LUSC Fig 5bd These results suggestthat mutations in S1PR1 are associated with prognosisin LUADRelationship between immune and S1PR1 expression inbreast cancer and lung cancerTumor infiltrating lymphocytes TIL are lymphocytes that leave the blood circulation and migrate tothe vicinity of the tumor The amount of TIL in thetumor is an important indicator to predict the prognosis of cancer patients and the response to immunotherapy [ ] Tumor purity is a key factorin analyses ofimmune infiltration by genomic approaches [] Therefore we use TIMER to investigate whetherthe expression of S1PR1 in breastcancer and lung cancer is related to immune infiltration We found a significant negative correlation between the S1PR1 expression level and tumor purityin both breast cancer and lung cancer Fig 6afLeft S1PR1 was a determinant of immune infiltration in BRCA tumor purity r P 176e including subtypes of BRCA BRCABasal r P 128e06 BRCAHer2r P 444e06 and BRCALuminal r P 915e S1PR1 was related to immune infiltration inincluding LUAD tumor purity r lung cancer P 605e16 and LUSC tumor purity r P 520e20Furthermore the relationship between S1PR1 andspecific immune infiltrates in breast cancer and lungcancer were analyzed The S1PR1 expression levelwas significantly positively correlated with levels ofr P 597e35infiltrating CD8 T cellsCD4 T cells r P 103e26 macrophagesr P 367e12 neutrophilsr P 203e07 and DCs r P 914e11 in BRCAFig 6a In BRCABasal there were slight positivecorrelations between S1PR1 expression levels andinfiltrating CD8 T cells r P levels of176e03 and CD4 T cells r P 852e03Similarly there were positive correlations with infiltrating levels of CD8 T cells r P 113e CD4 T cells r P 200e05 neutror philsP 135e04 in BRCAHer2 S1PR1 expression levelswere positively correlated with levels ofinfiltratingCD8 T cells r P 343e21 CD4 T cellsr P 626e14 macrophages r P r P 857e03 and DCs 0cZhong BMC Cancer Page of Table Correlation between S1PR1 mRNA expression andprognosis in lung cancer with respect to clinicopathologicalfactorsClinicopathologicalcharacteristicsOverall survivalNHazard ratioPvalueTable Correlations between S1PR1 mRNA expression andclinical prognosis in breast cancer with respect toclinicopathological factorsClinicopathologicalcharacteristicsOverallNHazard ratioPvalue 490E05ER statusER positiveER negativePR status 590E06PR positivePR negativeHER2 status920E13HER2 positive113E01920E01HER2 negativeIntrinsic subtypeSexFemaleMaleHistologyAdenocarcinomaSquamous cell carcinomaStageGradeIIIIIIAJCC stage TAJCC stage NAJCC stage MSmoking historyExclude those never smokedOnly those never smokedChemotherapyNoYesRadiotherapyNoYes NA NA NANA NA NABasalLuminal ALuminal BHER2Lymph node statusLymph node positiveLymph node negativeGradeTP53 statusMutatedWild typePietenpol subtypeBasallike Basallike ImmunomodulatoryMesenchymalMesenchymal stem likeLuminal androgen receptorSystemically untreated patientsIncludeExclude 71E13Bold values indicate P NA noneBold values indicate P NA none414e04 neutrophils r P 667e04 andDCs r P 644e07tumors Fig 6a We also found that S1PR1 expressionlevels were positivelycorrelated with levels ofin BRCALuminalr P 361e12infiltrating CD8 T cellsr P 101e17 neutrophilsmacrophagesr P 415e08r P 416e06 in LUAD In addition there were positiveinfiltrating B cells r correlations with levels ofand DCs 0cZhong BMC Cancer Page of Fig Decreased expression of S1PR1 in breast and lung cancer patients a Gene expression data of breast invasive carcinoma BRCA lungadenocarcinoma LUAD and lung squamous cell carcinoma LUSC in TCGA were downloaded in UCSC Xena S1PR1 mRNA expression level wascompared between cancerous and normal tissue using MannWhitney test with P setting as cutoff b The expression of S1PR1 in BRCALUAD and LUSC were analysis using GEPIA T tumor N normal tissue NUM number ce S1PR1 mRNA expression level was expressed as boxplots using the UALCAN database mRNA expression of S1PR1 in normal control and BRCA LUAD and LUSC tumors Left primary tumors Rightindividual cancer stage P P P 0cZhong BMC Cancer Page of Fig See legend on next page 0cZhong BMC Cancer Page of See figure on previous pageFig S1PR1 coexpression genes in breast and lung cancer ac The S1PR1 highly correlated genes identified by Pearson test in BRCA a LUADb and LUSC c df The heat map shows that in BRCA d LUAD e and LUSC f the first genes are positively red and negatively bluecorrelated with S1PR1 gi Significantly enriched GO annotations of S1PR1 in BRCA g LUAD h and LUSC i jl Significantly enriched KEGGpathways of S1PR1 in BRCA j LUAD k and LUSC lin LUSC These results strongly suggest P 127e15 CD8 T cells r P r P 398e14383e26 CD4 T cellsr P 261e45 neutrophilsmacrophagesr P 179e25 and DCs r P 212ethatS1PR1 plays a special role in the immune infiltrationof breast and lung cancers and has a particularlystrong effect on T cells macrophages neutrophilsand DCs These observed correlations betweenS1PR1 and various types of immune cells in breastand lung cancers indicated that S1PR1 may havehigh prognostic valuefurthercorrelationsimmune cellsevaluated theCorrelations between S1PR1 expression and immunemarkersbetweenWeS1PR1 and markers of variousinbreast cancer and lung cancer using TIMER Table and GEPIA databases Table S1 The correlationsbetween S1PR1 expression and immune markergenes for different immune cell populations including CD8 T cells T cells general B cells monocytes TAMs M1 and M2 macrophages neutrophilsNK cells DCs and various functional T cells suchas Th1 cells Th2 cells Tfh cells Th17 cells andTregs as well as exhausted T cells were analyzed byTIMER After adjusting for tumor purity S1PR1 expression levels were significantly positively correlatedwith marker sets for various immune cells exceptfor NK cells Th17 and T cell exhaustion in BRCATable and Fig However S1PR1 expressionlevels were highly positively correlated with most immune marker sets and both T cell populations andexhausted T cells in LUAD and LUSC Table andFig We further analyzed the correlation betweenS1PR1 expression and the markers using the GEPIAdatabaseincluding data for BRCA LUAD andLUSC The results for correlations between S1PR1and markers of immune infiltrating cells were similarto those of the TIMER analysis Table S1 This further confirms that S1PR1 is significantly related toimmune infiltrating cells in lung and breast cancersuggesting that high levels of S1PR1 could induceimmune activity in the lung and breast cancermicroenvironmentin normalDiscussionWe systematically analyzed the expression levels ofS1PR1 and the prognostic value in different types ofcancers Compared with levelstissuesS1PR1 expression was significantly lower in BLCABRCA CHOL COAD ESCA HNSC KICH KIRPLIHC LUAD LUSC PRAD READ SKCM STADand UCEC and was significantly higher in KIRC andTHCA Accordingly S1PR1 expression patterns depend on the type of cancer Prognostic data fromKaplanMeier plotterlow levels ofS1PR1 are significantly related to poor prognosis inbreast cancer and lung cancershowed thatsuch a complex biologicalThe downregulation of S1PR1 was associated withworse prognosis in breast cancer and lung cancer andwassignificantly related to clinical characteristicssuch as gender population smoking status and stageThese results suggested that S1PR1 is a prognosticbiomarker in breast cancer and lung cancer Howeversome literatures have reported the oncogenic role ofS1PR1 in breast cancer Lee H demonstrated thatStat3induced S1PR1 expression as well as S1PS1PR1 pathway is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression [] This maybe one of the molecular mechanisms by which S1PR1mediatesresponse Weconsidered that the main reason for this inconsistencyis that our study analyzed the expression of S1PR1 atthe overalllevel We further verified the significantreduction of S1PR1 expression in breast cancer andlung cancer patients through TCGA analysis Anotherstudy has also claimed a survival function benefit ofS1PS1PR signaling in BRCA patients which mightexplain the obstacle to relative antagonist therapy inclinics [] A recent study determined that attenuated endothelial S1PR1 function led to increasedtumor growth and metastasis whereas S1PR1 overexpression led to smaller tumors and strategies to enhance S1PR1 function in the tumor vasculature maypotentiate the efficacy of cytotoxic and targeted anticancer therapies [] These studies support our findings that high expression of S1PR1 is beneficialfortumor survivalThe tumor microenvironment refers to noncancerimmunecells in and around tumorsinfiltrated of 0cZhong BMC Cancer Page of Fig S1PR1 genomic alterations in breast and lung cancer a OncoPrint of S1PR1 alterations in BRCA LUAD and LUSC Different types of geneticalterations highlighted in different colors bd The relationship between genetic alterations and S1PR1 OSDFS in BRCA b LUAD c and LUSCd Logrank test was used in analysis of OSDFScells in the tumor microenvironment plays a vitalfunction in the occurrence and development of tumors [ ] Lymphocyte infiltration in the tumormicroenvironment is an independent predictor of cancer patient survival and lymph node metastasis [] Studies have shown that S1PR1 can affect the 0cZhong BMC Cancer Page of Fig Correlations between S1PR1 expression and immune infiltration levels in breast and lung cancer a S1PR1 expression was significantlynegatively related to tumor purity and significantly positively correlated with infiltrating levels of CD8 T cells CD4 T cells macrophagesneutrophils and dendritic cells in BRCA n b S1PR1 expression was significantly negatively related to tumor purity and was significantpositively correlated with infiltrating levels of CD8 T cells CD4 T cells and dendritic cells in BRCABasal n c S1PR1 expression wassignificantly negatively related to tumor purity and was significantly positively correlated with infiltrating levels of CD8 T cells CD4 T cellsneutrophils and dendritic cells in BRCAHer2 n d S1PR1 expression was significantly negatively related to tumor purity and wassignificantly positively correlated with infiltrating levels of CD8 T cells CD4 T cells macrophages neutrophils and dendritic cells in BRCALuminal n e S1PR1 expression was significantly negatively related to tumor purity and was significantly positively correlated withinfiltrating levels of CD8 T cells macrophages neutrophils and dendritic cells in LUAD n f S1PR1 expression was significantly negativelyrelated to tumor purity and was significant positively correlated with infiltrating levels of B cells CD8 T cells CD4 T cells macrophagesneutrophils and dendritic cells in LUSC n Spearmans correlation coefficients were used for analyses P P P proliferation and differentiation of lymphocytes in thetumor microenvironment [] The evaluation of immune cell infiltration in breast and lung cancers usingthe TIMER database revealed strong negative correlations between S1PR1 and tumor purity in BRCALUAD and LUSC Furthermore the S1PR1 expression level was positively correlated with levels ofCD8 T CD4 T neutrophils macrophages and DCsin BRCA The correlation between S1PR1 expressionand immune cell marker genes suggests that S1PR1regulates lung cancer tumor immunity through multiple immune cell populations These results indicatethat high levels of S1PR1 could increase the cytotoxicity of the immune system and immune activation in 0cZhong BMC Cancer Page of Table Correlations between S1PR1 and related genes and markers of immune cells as evaluated using TIMERLUSCPurityLUADPurityBRCAPurityDescriptionGene markersCD8 T cellT cell generalB cellMonocyteTAMvarXCD8ACD8BCD3DCD3ECD2CD19CD79ACD86CD115 CSF1RCCL2CD68IL10M1 MacrophageINOS NOS2M2 MacrophageIRF5COX2 PTGS2CD163VSIG4MS4A4ANeutrophilsCD66b CEACAM8CD11b ITGAMNatural killer cellDendritic cellTh1Th2CCR7KIR2DL1KIR2DL3KIR2DL4KIR3DL1KIR3DL2KIR3DL3KIR2DS4HLADPB1HLADQB1HLADRAHLADPA1BDCA1CD1CBDCA4NRP1CD11c ITGAXTbet TBX21STAT4STAT1IFNg IFNGTNFa TNFGATA3STAT6corp126E17242E08471E12715E19320E10738E07198E08128E01129E10468E04463E01835E02176E16618E01490E28772E02114E02196E13203E01824E01155E24727E01110E01395E01263E03319E02875E01268E01389E14211E02717E07226E11176E53158E59621E03472E13592E19261E04784E01808E 143E02669E13corp219E04166E02128E02885E07400E04537E05121E04297E07310E08386E02586E11110E09793E18355E01358E02436E14975E10539E17195E08816E06257E13130E06996E04506E01104E04879E02581E01834E03413E09479E02869E07453E07100E09107E04269E03481E05366E03310E01913E02930E02301E01220E03corp651E21122E17760E21282E26799E24378E13329E13127E45267E56589E24118E30149E36864E02431E01237E06152E57477E53928E54299E06266E30170E33136E03263E07845E04485E11168E06343E01105E06386E52884E20129E48187E52869E24669E28158E24517E20473E32103E04185E02134E01300E07625E01 0cZhong BMC Cancer Page of Table Correlations between S1PR1 and related genes and markers of immune cells as evaluated using TIMER ContinuedTfhTh17TregSTAT5AIL13BCL6IL21STAT3IL17AFOXP3CCR8STAT5BTGFb TGFB1T cell exhaustionPD1 PDCD1CTLA4LAG3TIM3 HAVCR2GZMBBRCA181E07127E01352E08977E01175E01929E02394E01671E01858E20321E25412E04575E01600E04219E01782E02LUAD227E08115E01801E03234E01265E05462E01198E01461E04467E12943E06256E01727E02439E01178E06599E01LUSC422E21120E05924E01492E06609E04409E01415E19727E27247E03164E01380E16388E20311E06844E46333E09shown in recently reports endothelialBRCA LUAD and LUSC by increasing the infiltrationof CTLs CD4 T cells and DCs On the contrarylow expression of S1PR1 could lead to reduced infiltrated effector cells in the tumor microenvironmentAsloss ofS1PR1 led to a reduction in CD45 cells macrophages and DCs which influences tumor growth andmetastasis [] In addition S1P is involved in enhancing endocytosis and migration of mature dendriticcells through S1PR3 an event that may increase theimmune response to cancer cells Our findings areconsistent with such reports and these discoveriesimply that S1PR1 plays an important role in recruiting and governing immune infiltration in BRCALUAD and LUSCthe purinergicTo further elucidate the molecular mechanismsunderlying the role of S1PR1 in b | Thyroid_Cancer |
Is preoperative heart rate variability aprognostic indicator for overall survival andcancer recurrence in patients with primarycolorectal cancerM T A StrousID1 A M Daniels1 F M Zimmermann2 F N van Erning3 Y Gidron4 FJ Vogelaar1 Department of Surgery VieCuri Medical Centre Venlo The Netherlands Department of CardiologyCatharina Hospital Eindhoven The Netherlands Department of Research Netherlands ComprehensiveCancer anisation Utrecht The Netherlands Faculty of Welfare and Health University of Haifa HaifaIsrael maudstrousviecurinlAbstracta1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Strous MTA Daniels AM ZimmermannFM van Erning FN Gidron Y Vogelaar FJ Ispreoperative heart rate variability a prognosticindicator for overall survival and cancer recurrencein patients with primary colorectal cancer PLoSONE e0237244 101371journalpone0237244Editor Louise Emilsson University of OsloNORWAYReceived February Accepted July Published August Copyright Strous This is an access distributed under the terms of theCreative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement Data cannot beshared publicly because of ethical concernsPatients were included on a no objection base toconduct retrospective data studies and publishfindings but were not asked for permission topublish full encrypted data Data are available fromthe VieCuri Institutional Data Access contact viawetenschapsbureauviecurinl for researcherswho meet the criteria for access to confidentialdataBackgroundHeart Rate Variability HRV represents efferent vagus nerve activity which is suggested tobe inversely related to fundamental mechanisms of tumorigenesis and to be a predictor ofprognosis in various types of cancer HRV is also believed to predict the occurrence andseverity of postoperative complications We aimed to determine the role of preoperativeHRV as a prognostic factor in overall and cancer free survival in patients with colorectalcancerMethodsRetrospective analysis was performed in a detailed dataset of patients diagnosed with primary colorectal cancer between January and December who underwent curative surgical treatment HRV was measured as timedomain parameters SDNN StandardDeviation of NNintervals and RMSSD Root Mean Square of Successive Differencesbased on preoperative second ECGs Groups were created by baseline HRV Low HRVSDNN 20ms or RMSSD 19ms and normal HRV SDNN �20ms or RMSSD �19msPrimary endpoints were overall and cancer free survivalResultsA total of patients were included in this study HRV was not significantly associated withoverall survival SDNN 20ms vs SDNN �20ms244 vs adjusted HR p RMSSD 19ms vs RMSSD �19ms270 vs adjustedHR p or cancer recurrence SDNN 20ms vs�20ms201 vs adjusted HR p RMSSD 19ms vs�19ms vs adjusted HR p There was noPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFunding The authors received no specific fundingfor this workCompeting interests The authors have declaredthat no competing interests existsignificant association between HRV and CEAlevel at one year followup or between HRVand occurrence of a postoperative complication or the severity of postoperativecomplicationsConclusionsHeart rate variability was not associated with overall or cancer free survival in patients withprimary colorectal cancer who underwent curative surgical treatment These results do notalign with results found in studies including only patients with advanced cancer which suggests that there is only an association in the other direction cancer causing low HRVIntroductionIn there were over million newly diagnosed colorectal cancer patients worldwide andover in the Netherlands alone It is the fourth most common cause of death worldwide[] To improve survival it is of importance to get a better insight into modifiable prognosticfactors Emerging evidence suggests that vagal nerve activity indexed by heart rate variabilityHRV could be one of these prognostic factors []HRV is the physiological phenomenon of the fluctuation in time intervals between adjacentheartbeats and represents efferent vagus nerve activity to the heart [] It has been suggestedthat efferent vagal activity is inversely related with fundamental mechanisms of tumorigenesisas inflammation oxidative stress and excessive sympathetic activity [] These mechanisms arebelieved to be controlled by the vagus nerve via a bidirectional braintoimmune pathwaysthrough the release of neurotransmitters via the cholinergic antiinflammatory pathway[] A higher vagal tone may reflect a more flexible topdown regulation of the immunesystem and physiological activity moderated by the brain [] Absence of vagus activity due tovagotomy has been shown to increase the risk of developing colorectal cancer []In addition to influencing development of cancer vagus nerve activity seems to be a predictor of prognosis in various types of cancer Recent studies show an association betweendecreased activity of the vagus nerve and worse survival in patients with cancer of the gastrointestinal tract liver pancreas lung prostate and breast among others [] Also patientswith normal HRV seem to live longer in different sorts of metastatic cancer independent ofconfounders [] In patients with colorectal cancer a low HRV at baseline has shown to beassociated with higher CEA levels at months after diagnosis which predicts a poorer prognosis []In patients undergoing curative treatment for colorectal cancer HRV does not only seemto influence cancer prognosis A recent study showed that patients with lower HRV have moreintraoperative blood loss and more and more severe postoperative complications []Identifying patients with low HRV is easy and noninvasive When its predictive value forthe prognosis of cancer patients is of satisfactory significance vagus nerve activation prior toor during cancer treatment could theoretically be beneficial in improving prognosis [] Alsoif we could predict the occurrence and severity of postoperative complications based on HRVimproving HRV before surgery could possibly accelerate postoperative recovery and indirectlyaffect patients prognosis Recent studies focussing on improving HRV by improving physicalfitness by means of physical exercise show promising results in both older men and woman[] However the only previous study on colon cancer and HRV including patients receiving curative treatment included a small sample and did not examine whether HRV predictsPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancersurvival in these patients [] To clarify the predictive value of HRV in prognosis of patientswith colorectal cancer further exploration is needed Current studies identifying HRV as aprognostic factor did not specifically focus on colorectal cancer have small study populationsdid not correct for confounders and mainly focused on metastatic disease []The aim of this study was to determine the role of preoperative HRV as a prognostic factorin overall and cancer free survival in patients with primary colorectal cancer who underwentcurative surgical treatmentMethodsData collectionData from the Netherlands Cancer Registry NCR were used The NCR collects data on allnewly diagnosed cancer patients in the Netherlands Information on patient and tumour characteristics diagnosis and treatment is routinely collected from the medical records by trainedadministrators of the cancer registry Anatomical site of the tumour is registered according tothe International Classification of Diseases Oncology The tumournodemetastasis TNMclassification is used for stage notification of the primary tumour according to the editionvalid at time of cancer diagnosis Quality of the data is high due to thorough training of theregistration team and consistency checks []For the study population additional data were collected from the medical records of thepatients This encompassed information on HRV CEAlevels ASA classification comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroid disease pulmonary disease vascular disease neurological disease and otheroccurrence and severity of postoperative complications and cancer recurrence Groups ofcomorbidities were chosen based on matching features within these groups and their potentialinfluence on HRV or the endpoint being analysed Severity of the postoperative complicationsaccording to the ClavienDindo classification was also documented Medical records wereassessed between January and July and reevaluated for revision of this between the 20th and 25th of April This study was approved by the research committee and the Board of Directors of VieCuriMedical Centre Data was obtained under the law scientific research and statistics in the interest of public health where asking for permission is not possible or appropriate for several reasons in the Netherlands unless patients objected to use of their personal medical record forscientific research Data was encrypted with an encryption key provided by the NCR Encryption was shortly lifted to access the patients number for accessing hisher medical record Following extraction data were encrypted againStudy populationThe study population included all consecutive patients diagnosed with primary colorectal adenocarcinoma between January and January at VieCuri Medical Centre who underwent curative surgical treatment Patients with metastatic disease at time of surgery orcarcinoma in situ were excluded as their treatment and prognosis differs from those receivingcurative treatment for colorectal cancer Metastasis found within months after surgery wereconsidered present at time of surgery and therefore excluded Other excluded patients werepatients with neuroendocrine tumours because of different tumour characteristics and prognosis patients with cardiac arrhythmias including atrial and ventricular extrasystole pacemakers patients taking betablockers as this enhances HRV indexes or patients withbradycardia heart rate bpm or tachycardia heart rate bpm as this precluded reliable calculation of HRV [] We did not exclude patients taking alphablockers calciumPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerinhibitors diuretics amiodarone ACEinhibitors or ARBs as these types of medicationreduce central sympathetic functioning rather than peripheral and their influence on HRV istherefore less univocal and sometimes completely absent []Heart rate variabilityHeart rate variability was analysed using a 12lead 10second ECG 150Hz used for preoperative screening In case of multiple ECGs per patient the most recent ECG before date of surgery was used for HRVanalysis In case of multiple ECGs per patient on the same date theECG with the best quality was chosen meaning an ECG without motion artefacts In case ofmotion artefacts there was always an ECG without motion artefacts available recorded on thesame date Time between two consecutive Rpeaks was measured in lead II with an accuracy of02ms using MUSEECG HRV was presented using the timedomain HRV parameters SDNNStandard Deviation of NNintervals and RMSSD Root Mean Square of Successive Differences in milliseconds calculated using the following calculations []rSDNN ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRi 00 RRmeanÃ2Pnn 00 RMSSD ¼rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii¼1 ðRRiþ1 00 RRiÃ2Pn 00 n 00 ð1Ãð2ÃSDNN and RMSSD obtained from 10s ECGs were found to correlate with results of ECGsof longer durations Power spectral analysis HRV parameters as LF and HF can only beobtained in longer recording ECGs and were therefore not implementable in this study[]SDNN and RMSSD were both analysed as continuous variables as well as binary variablesusing cutoffs of 20ms versus �20ms and 19ms versus �19ms respectively In case of anSDNN 20ms or RMSSD 19ms HRV was classified as low and in case of SDNN �20ms orRMSSD �19ms as normal These cutoff values were based on cutoff values used in otherstudies showing an association between lowHRV as SDNN 20ms and RMSSD 19ms andcolorectal cancer as there is no standardised definition of low and normal HRV []Endpoints and definitionsThe primary endpoints of this study were overall and cancer free survival Overall survival wasdefined as the time between the date of surgery to the date of death or last followup date inmonths Cancer free survival was defined as the time in months from the date of surgery untilthe date of cancer recurrence defined as the first date of either radiologic or pathologic diagnosis of metastases or tumour recurrence of colorectal cancer Patients dying without cancerrecurrence were censored on day of death Secondary endpoints were elevated CEAlevel ugl at oneyear followup occurrence of postoperative complications within daysafter surgery and severity of postoperative complications according to the ClavienDindoclassificationStatistical analysisIn this retrospective observational cohort study we utilized descriptive statistics to provide anoverview of control variables of the study population patient characteristics as age sex BMIcomorbidities and ASAclassification heart rate and tumour characteristics as TNMstagetumour localisation and tumour differentiation and their association with HRV andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerprognosis Normal distribution of the continuous variables heart rate age and BMI as well asSDNN and RMSSD were tested with a KolmogorovSmirnov test Because of normal distribution heart rate age and BMI were compared between HRVgroups using unpaired ttest Allother variables were categorical and were compared between HRVgroups using Chisquarestatistics as groups were all of sufficient powerDifferences in overall survival and cancer free survival in months according to SDNN andRMSSD were visualized by means of KaplanMeier curves and statistically tested using the logrank test Multivariate coxregression analyses were conducted to calculate the prognosticassociation between HRV and overall and cancer free survival while adjusting for other prognostic variables Multivariate logistic regression was used to assess the independent effect ofSDNN and RMSSD on CEAlevels and the occurrence and severity of postoperative complications Variables included for adjustment were chosen by means of forward stepwise selectionbased on clinical judgment differences at baseline eg differences on any predictor betweenpatients who later died or not and database availability and depended on the analysed endpoint Those included patient demographics age sex bodymassindex comorbidities identified at admission divided into groups cardiac disease hypertension diabetes mellitus thyroiddisease pulmonary disease vascular disease neurological disease other including Crohnsdisease hepatitis kidney failure disorders anaemia depression arthritis tumour characteristics localisation stage differentiation and the occurrence of postoperative complicationswhen the later was not an outcome Differences in CEAlevel at baseline and one year checkup between and within groups of low HRV and normal HRV were assessed with a repeatedmeasures linear model and tested using the tukey test To test the implication of a longer timebetween ECG and treatment all analyses were repeated after excluding patients with an ECGolder than months A twotailed pvalue � was considered significant in all analysesData were analysed using IBM SPSS Statistics version IBM Corp NY Armonk USAResultsOf colorectal cancer patients that underwent a surgical resection a total of patientswere included in this study Reasons for exclusion are presented in Fig Median SDNN andRMSSD were 204ms interquartile range IQR 115ms351ms and 175ms IQR 99ms299ms respectively Table shows descriptive data of the included patients by HRV groupsBaseline heart rate and age were negatively associated with HRV The group of patients withlow HRV contains more patients with a history of cardiac disease regardless of the HRV defining parameter When defining low HRV by RMSSD 19ms more patients in this group havehypertension as comorbidity This group also contains more patients with an ASA classification greater than oneDuring a median followup of months IQR all causedeath occurred in patients Cancer recurrence occurred in patients during a median followup of months IQR To rule out any distort in results caused by a delay between ECG and treatment all analyseswere repeated after exclusion of ECGs older than months This did not lead to any new significant results Therefore these results were not displayed in detail in this paperSurvivalIn low HRV groups slightly more patients died compared to normal HRV groups SDNN20ms versus �20ms versus respectively RMSSD 19ms versus�19ms versus respectively These observed differences between HRVgroups in overall survival were not significant Fig A SDNN p B RMSSDPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Flowchart of the study101371journalpone0237244g001p After adjustment for some potential confounders these associations remained notsignificant SDNN 20ms versus �20ms HR p and RMSSD19ms versus �19ms HR p Tables and Age cardiac disease tumour stage and postoperative complications had a significant influence on overall survival Age also differed at baseline and was identified as a confounder When defining low andnormal HRVgroups by SDNN cardiac disease was identified as confounder When conducting sensitivity analyses with SDNN and RMSS as continuous variables results remained thesame There was no association between HRV and overall survival SDNN HR p and RMSSD HR p In low HRV groups slightly more patients had recurrence of cancer compared to normalHRV groups SDNN 20ms versus �20ms versus respectivelyRMSSD 19ms versus �19ms versus respectively These observed differences between HRV groups in cancer free survival were not significant Fig A SDNNp B RMSSD p As in overall survival after adjustment for some potentialconfounders these associations remained not significant SDNN 20ms versus �20msHR p and RMSSD 19ms versus �19ms HR p Tables and In SDNNbased groups baseline heart rate was identified asa confounding variable Sensitivity analyses with SDNN and RMSSD as continuous variablesPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Descriptive data of included patients according to prespecified HRV groupsSDNN 20ms n SDNN �20ms n pRMSSD 19ms n RMSSD �19ms n pHRV and prognosis in colorectal cancerHeart Rate�Age�Age n year� yearSex nMaleFemale [] [] [] [] [] [] [] [] Mean BMI SD [] [] [] []Comorbidities nCardiac diseaseHypertensionDiabetes MellitusThyroid diseasePulmonary diseaseVascular diseaseNeurological diseaseOtherASA nASA1ASA2ASA34Localisation tumour nRight colonLeft colonRectumTumour stage nIIIIII Differentiation grade nWellmoderate Poorundifferentiated � Non normaldistributed data presented as median with interquartile range101371journalpone0237244t001did not alter these results There was no association between HRV and cancer free survivalSDNN HR p and RMSSD HR p CEAlevelCEAlevel at baseline and one year checkup was registered in patients and elevated in of these patients This was elevated at one year checkup in only patients Low HRV was notsignificantly associated with elevated CEAlevels at one year checkup as shown in Table Sensitivity analyses with SDNN and RMSSD as continuous variables did not alter these resultsSDNN HR p and RMSSD HR PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for overall survival in groups of low HRV and normal HRV presented as A SDNN andB RMSSD101371journalpone0237244g002PLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of SDNN and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerSDNN20ms�20msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI reference reference p reference reference reference reference reference reference reference reference reference Postoperative complicationNoYes reference reference 101371journalpone0237244t002p Adjusting for covariates was not possible because of the small number of patientswith an elevated CEAlevelDifferences between CEAlevel at baseline and one year checkup were compared betweenand within HRVgroups Results were displayed in Fig There were no significant differencesin CEAlevel at baseline and one year checkup between the low HRV group and normal HRVgroup defined by SDNN and RMSSD p and p respectively Also there were nosignificant differences in CEAlevel at baseline and at one year checkup within the low HRVgroup and normal HRV group defined by SDNN and RMSSD p and p respectivelyPostoperative complicationsIn patients one or more postoperative complications occurred within days after surgeryThe occurrence of a postoperative complication was not significantly associated with lowHRV defined as SDNN 20ms or RMSSD 19ms even after adjustment for some potentialconfounders Table Heart rate age cardiac disease and hypertension were identified asconfounding covariates When conducting sensitivity analyses with SDNN and RMSS as continuous variables the association between occurrence of a postoperative complication withPLOS ONE 101371journalpone0237244 August PLOS ONE 0cTable Multivariate analyses of associations of RMSSD and covariates with overall and cancer free survivalHRV and prognosis in colorectal cancerRMSSD19ms�19msHeart RateAgeBMICardiac diseaseNoYesHypertensionNoYesASAclassificationASA1ASA2ASA34LocalisationRight colonLeft colonRectumTumour stageIIIIIIOverall survivalHR CIpCancer free survivalHR CI reference reference p reference reference reference reference reference reference reference reference reference reference Postoperative complicationNoYes reference reference 101371journalpone0237244t003baseline HRV remained not significant SDNN HR p andRMSSD p The same applied when postoperative complicationswere graded according to the ClavienDindo classification and the complication that is gradedthe highest for each patient is compared to the absence of postoperative complicationsTable DiscussionIn this observational cohortstudy we determined the Heart Rate Variability in preoperativeECGs of patients with primary colorectal cancer who underwent curative surgical treatment HRV refers to physiological variations in beattobeat intervals It was presented intimedomain parameters SDNN and RMSSD HRV was not significantly associated with overall survival or cancer free survival independent of some risk factors Also this study showedno significant differences in CEAlevels at one year checkup between patients with low HRVand patients with normal HRV Patients with low HRV did not have more or more severepostoperative complications compared to patients with normal HRVTumorigenesis has three fundamental mechanisms inflammation promoting oxidativestress and stimulating tumour growth oxidative stress causing DNAdamage andPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig KaplanMeier curves for cancer free survival in groups of low HRV and normal HRV presented as A SDNNand B RMSSD101371journalpone0237244g003PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Univariate analyses of low HRV and risk of elevated CEAlevel at one year checkupCEA μglOR CIpCEA μglOR CIIndependent of baseline CEAIndependent of baseline CEASDNN 20ms n RMSSD 19ms n SDNN �20ms n Normal baseline CEA � μglSDNN 20ms n SDNN �20ms n Elevated baseline CEA μglSDNN 20ms n reference reference RMSSD �19ms n Normal baseline CEA � μglRMSSD 19ms n RMSSD �19ms n Elevated baseline CEA μglRMSSD 19ms n reference reference SDNN �20ms n referenceRMSSD �19ms n referencep101371journalpone0237244t004interfering with subsequent repair mechanisms and sympathetic neurotransmitters stimulating tumour metastasis and progression [] It has been suggested that afferent fibres of thevagus nerve inform the brain about peripheral inflammation This is followed by a braintoimmune response via the efferent route of the vagus nerve that modulates the function ofimmuneregulatory cells through the release of neurotransmitters via the cholinergic antiinflammatory pathway Malfunctioning of this route may play part in the onset of cancer []This theory has been supported by studies of patients with gastric ulcers who underwent avagotomy who then showed an increased risk of developing lung and colorectal cancer [] The vagus nerve is also believed to modulate tumour progression An active vagus nervecan inhibit inflammation oxidative stress and the release of sympathetic neurotransmittersthat stimulate tumour metastasis and progression [] Absence of this inhibitory effect inturn results in metastasis and tumour progression as shown in a study of Erin showingthat mice who underwent chemical vagotomy developed more metastasis of breastcancer cellsthan controls [] Epidemiologically low HRV has been associated with worse overall survivalover time in patients with recurrent or metastatic cancer of various types even after correctionfor confounders [] However the results of the present study do not support thesefindingsTo our knowledge this is the first study including only patients with colorectal cancer whoare eligible for curative treatment by partial bowel resection and not those receiving palliativetreatment De Couck studied the relationship between HRV and tumour burden in bothcurative and palliative patients with prostate cancer or nonsmall cell lung cancer Independent of confounders the hypothesised inverse relationship of HRV and the tumour markerPSA at and months after diagnosis was only significant in patients with stage IV prostatecancer not stage II and III [] In colorectal cancer Mouton found that low HRV definedas SDNN ms predicted significantly higher levels of the tumour marker CEA at months after diagnosis Again these results were only found in patients receiving palliativetreatment not curative [] Only one previous study showed a significant inverse relationshipbetween HRV and mortality in cancer in general independent of stage [] This study of Guo had a large study population of patients with various types of cancer Low HRV wasdefined as SDNN 70ms and was significantly associated with shorter survival times Thissuggests that HRV is a predictive indicator of survival time not only in palliative but also incurative patients However results were not controlled for cancer type which could affect bothHRV and survival and should therefore be interpreted with caution [] The fact that thepatients recruited in the present sample were only with less advanced cancer may partlyexplain the lack of prognostic role in HRV in this samplePLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerFig Bar chart for CEAlevel at baseline and one year checkup in groups of low HRV and normal HRV presented as ASDNN and B RMSSD101371journalpone0237244g004PLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and risk of occurrence of a postoperative complication within daysPostoperative complicationOR CIUnadjustedSDNN 20ms n SDNN �20ms n referenceAdjusted�SDNN 20ms n SDNN �20ms n referenceUnadjustedRMSSD 19ms n RMSSD �19ms n referenceAdjusted�RMSSD 19ms n RMSSD �19ms n reference�adjusted for heart rate age cardiac disease and hypertension101371journalpone0237244t005pSome of these previous studies suggest a bidirectional relationship between vagus nerveactivity and cancer [] However based on current evidence on this subject we cannot supportthis hypothesis The positive association between low HRV and worse prognosis found inpatients with colorectal cancer receiving palliative treatment but not in patients receivingcurative treatment suggest that this relation is not bidirectional but that advanced cancer isassociated with low HRV Midlatestage tumours are often accompanied by damage to autonomic nerves resulting in decreased HRV [] A study of De Couck showed that cancerpatients in general have a significantly lower HRV than healthy people [] The same resultswere found in studies of Bijoor where RMSSD was found to be significantly lower inpatients with early and advanced stage cancer compared to a healthy control group []When comparing patients with advanced stage cancer TNM III and IV to patients with anearly stage of cancer TNM I and II RMSSD was found to be significantly lower in patientswith advanced stages of cancer [] Thus though experimental studies in animals showthat vagal nerve functioning can causally slow tumorigenesis the human data suggests that themalignant tumour causes vagal nerve dysfunction and therewith decreased HRV []Besides the proposed influence of low HRV on survival of colorectal cancer patientsthrough development and increased progression of cancer Reimer suggested that lowHRV could influence survival of those undergoing surgical treatment due to more and moresevere postoperative complications [] However the results found in this study were notconcurrent with those of Reimer who included patients with ASA undergoingelective surgery Their analysis of HRV was through powerspectrum parameters based on longer ECGrecordings instead of the timedomain parameters based on 10s ECGs used in thisstudy But the difference in used parameters used in both studies is probably not the explanation for the differences in results between both studies since it has been demonstrated thatRMSSD and SDNN based on ECG recordings of 10s are in substantial agreement with those of45min and a 10s ECG therefore suffices to determine time domain HRV parameters HoweverReimer did not correct for possible confounders In their study patients with low HRVwere more likely to have diabetes a known risk factor for postoperative ileus and wound infections both found to be common postoperative complications in their low HRV group Correcting for this comorbidity may change the significance of their findings [] A study ofPLOS ONE 101371journalpone0237244 August PLOS ONE 0cHRV and prognosis in colorectal cancerTable Low HRV and severity of postoperative complications according to ClavienDindo classificationUnadjustedMinor grade I and IIOR CIpMajor grade III IV VOR CISDNN 20msn n SDNN �20msn referencen referenceAdjusted�SDNN 20msn SDNN �20msn referenceUnadjustedRMSSD 19msn RMSSD �19msn referenceAdjusted�n n referencen n referenceRMSSD 19msn n RMSSD �19msn referencen referencep�adjusted for heart rate age categories vs � and hypertension101371journalpone0237244t006Scheffler did not show any significant preoperative differences in HRV between patientswith or without postoperative complications or between patients with postoperative complications of different severity levels [] Thus also in relation to predicting postoperative complications results are not uniform | Thyroid_Cancer |
"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ ] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen BMC Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ¥ mmolL onehour bloodglucose postoral sugar ¥ mmolL or twohour bloodglucose postoral sugar ¥ mmolL Preterm birth wasdefined as delivery at ¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Students ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolL nmolL¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ¥ y y ¥ y y ¥ y y ¥ y Low birth weight y ¥ y Macrosomia y ¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child NutrYun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency 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DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet GynecolJolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril 1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132 Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J MedLiu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
performance demonstration of a hybrid compton camera with an active pinhole for wideband Xray and gammaray imagingAkihisa omata1 Jun Kataoka1 Kazuya fujieda1 Shogo Sato1 eri Kuriyama1 Hiroki Kato2 Atsushi toyoshima3 takahiro teramoto3 Kazuhiro ooe2 Yuwei Liu2 Keiko Matsunaga2 takashi Kamiya2 tadashi Watabe2 eku Shimosegawa2 Jun Hatazawa2Xray and gammaray imaging are technologies with several applications in nuclear medicine homeland security and highenergy astrophysics However it is generally difficult to realize simultaneous wideband imaging ranging from a few tens of keV to MeV because different interactions between photons and the detector material occur depending on the photon energies for instance photoabsorption occurs below keV whereas Compton scattering dominates above a few hundreds of keV Moreover radioactive sources generally emit both Xray and gammaray photons in this study we develop a hybrid compton camera that can simultaneously achieve Xray and gammaray imaging by combining features of compton and pinhole cameras in a single detector system Similar to conventional compton cameras the detector consists of two layers of scintillator arrays with the forward layer acting as a scatterer for highenergy photons keV and an active pinhole for lowenergy photons keV The experimental results on the performance of the hybrid camera were consistent with those from the Geant4 simulation We simultaneously imaged Am keV and Cs keV in the same field of view achieving an angular resolution of ¦ FWHM for both sources in addition imaging of At was conducted for the application in future nuclear medicine particularly radionuclide therapy the initial demonstrative images of the At phantom were reconstructed using the pinhole mode using keV and Compton mode using keV exhibiting significant similarities in sourceposition localization We also verified that a mouse injected with MBq of At can be imaged via pinholemode measurement in an hourIn the field of nuclear medicine it is essential to visualize the distribution of radioisotopes in a patients body Particularly a radiological diagnosis that enables noninvasive visualization of the pathology from outside the body is important The general approach is to visualize nuclear gamma rays emitted from radioactive tracers Two common techniquessingle photon emission computed tomography SPECT and positron emission tomography PETplay important roles in the diagnosis However they image a specific energy range of either Xrays or gamma rays SPECT can image gamma rays with energies less than a0keV with the use of the collimator whereas PET can image positron emitters that emit 511keV gamma rays These lead to a limited number of radioactive tracers that can be imaged only with current SPECT and PET scanners In this context a Compton camera12 that can conduct imaging in a wide energy band is reckoned to cause a breakthrough in nuclear medicine34 Attempts are being made to develop Compton cameras aiming the image of prompt gamma rays emitted from the patients body during proton therapy5 In addition several works have aimed to realize tracer visualization using the Compton camera For example Fontana et a0al describe the Compton camera as an 1Graduate School of Advanced Science and Engineering Waseda University Tokyo Japan 2Graduate School of Medicine Osaka University Osaka Japan 3Institute for Radiation Science Osaka University Osaka Japan email omt22fujiwasedajpScientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Comparison of the simulated energy response of the intrinsic efficiency left and angular resolution right with the results of actual measurementselectronically collimated SPECT device which is however optimized for higher energies above a few hundreds of keV9 Yoshida et a0al proposed whole gamma imaging WGI wherein a conventional PET scanner is converted by inserting an additional scatterer to create a Compton camera10 They succeeded in realizing the triple gamma ie PETCompton imaging of 44Sc which emits keV and a0keV gamma rays however the application to imaging SPECT tracer remains to be researched in the future In this context the simultaneous capture of SPECT and PET images has also been reported using a Compton camera consisting of SiCdTe semiconductors which is however limited both in terms of detection efficiency and angular resolution11 We argue that such difficulties can be easily overcome with the hybrid camera proposed in this paper This increases the variety of radioactive tracers available for nuclear medicine14 The emergence of new tracers may solve current problems such as manufacturing costs of tracers in addition to achieving improvements in the diagnosis quality A Compton camera also enables the simultaneous imaging of multiple tracers this significantly increases the information obtained from living anisms in a single diagnosisMoreover nuclear medicines involving radioactive sources are applied not only in the diagnosis but also in the treatment of diseases such as cancer Particularly radionuclide therapy RNT1920 which uses the targeted radionuclide by administering radioisotopes to patients is widely used because it damages cancer cells while limiting the exposure of healthy tissues to radiation For instance RNT with α ps is receiving significant attention because of its high therapeutic potential owing to the higher ionization power for damaging cancer cells2122 Nevertheless once administered into the body it is difficult to determine the distribution and pharmacokinetics of a radionuclide For the safety and effectiveness of RNT it is conceivable to visualize the characteristic Xrays and nuclear gammarays emitted simultaneously with the alpha decay Some of these characteristic X rays and nuclear gamma rays can be visualized by SPECT Furthermore the use of a Compton camera for the in a0vivo visualization of 223Ra an αp emitter used for RNT was suggested in our previous study23 However these radionuclides emit several Xrays and gamma rays with different branching ratios Some emit strong characteristic Xrays that can be imaged with a SPECT whereas others emit highenergy gamma rays that can be imaged only with a Compton camera see Table a0 Imperatively a simple and costeffective imaging system that is sensitive to both Xrays and gamma rays is highly desiredThis paper proposes a hybrid Compton camera that realizes simultaneous wideband imaging from a few tens of keV to approximately an MeV combining some features of Compton cameras and pinhole cameras in a single detector system Although the hybrid camera consists of two layers of positionsensitive detectors similar to a Compton camera1323 the front detector has a hole in the center Compton and pinhole imaging are enabled using the front detector as a scatterer for highenergy photons a0 keV and an active pinhole for lowenergy photons a0 a0keV We developed a prototype of the hybrid camera Simulation and experimental results depicted resolutions better than ¦ full width at half maximum FWHM in the range of a0keV In addition the imaging of At is receiving attention as a source applicable to RNT with αps24 We initially investigated the capability of our hybrid camera system with a simple phantom of At and thus conducted mouse imagingResultsperformance evaluation The imaging performance of the hybrid camera was initially evaluated by simulations using Geant428 The simulation configuration includes the scintillator arrays the MPPC arrays and the metal case which are detailed in the Methods section A monochromatic point source placed a0cm from the camera and at the center of the field of view FOV was imaged The detector performance was investigated between and a0keV for both the pinhole and Compton modes Considering the energy and spatial resolution of the actual detectors we utilized a reconstruction flow as described in the Methods section The simulation showed that pinhole imaging is difficult over a0keV whereas Compton imaging is active at approximately a0keV or higher Figure a0 left details the intrinsic detection efficiency that indicates the proportion of events detected either as the pinhole or the Compton mode to all radiation emitted towards the detector The intrinsic detection efficiency Ç«int is expressed by the following equation using the absolute detection efficiency Ç«absScientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Upper The experimental configuration of the simultaneous measurement of Am and 137Cs Lower The MLEM reconstructed images of the Am a0keV source analyzed by the pinhole mode left and the Cs a0keV source analyzed by the Compton mode rightÇ«int 4Ï�ǫabswhere 01 denotes the solid angle of the detector viewed from the source Figure a0 right shows the angular resolution of the pinhole and Compton images as a function of energy For Compton imaging we use the angular resolution measure ARM that is commonly used to measure the angular resolution of Compton cameras For measuring the angular resolution of pinhole imaging the �θ value is obtained by geometrically converting the position resolution of the image to angular resolution Note that for the source placed at the center of the FOV the angular resolution �θ can be calculated as follows�θ arctan�x2lwhere 01x and l denote the position resolution FWHM of the pinhole image and the distance between the camera and the source respectively The angular resolution was better than ¦ in the range of keV exhibiting the higher efficiency of the proposed hybrid camera than those of the conventional camerasSubsequently the fundamental imaging performance of the hybrid camera was evaluated by performing experiments under the same geometry as in the simulation The measurements were recorded at energies of a0keV and a0keV for pinhole imaging and a0keV and a0keV for Compton imaging The obtained angular resolutions were consistent with the values predicted by the simulation as represented by the red plots in Fig a0 rightExperimental demonstration of wideband imaging To examine the performance of the hybrid camera we conducted simultaneous imaging of Am and Cs sources As shown in Fig a0 upper these sources were placed cm away from the camera and at ¦ and ¦ respectively from the center of the FOV The Am source was reconstructed in the pinhole mode a0keV whereas the Cs source was reconstructed in the Compton mode a0keV From Fig a0 lower each convergence indicated the correct positions depicting the potential of broadband imaging using the hybrid cameraAs a next step extended sources were measured to examine the validity of the camera system including the image reconstruction technique The extended a0mm à a0mm Lshaped sources were reproduced by moving on a stage that automatically moved at a constant speed a0mmmin The image of an Lshaped Am source Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cFigure a0 The MLEM reconstructed images of Lshaped sources Pinhole reconstruction of the Am source left and Compton reconstruction of the Cs source right that were measured separatelyFigure a0 Energy spectrum of At obtained by a LaBr3 scintillator coupled to a PMTwas reconstructed by the pinhole mode using events with energies around a0keV Subsequently the image of an Lshaped Cs source a0keV was also reconstructed by the Compton mode MLEM reconstruction images of each measurement are presented in Fig a0 At imaging of a small bottle Furthermore we investigated the performance of the hybrid camera with the imaging of a At source which is an αps source for RNT Initially the energy spectrum of At was obtained by a LaBr3 scintillator coupled to a photomultiplier tube PMT Figure a0 shows peaks of characteristic Xrays mainly a0keV and gamma rays with energies of and a0keV Furthermore the intensity of Xrays is approximately three orders of magnitude higher than that of the gamma raysNext placing a0cm away from the hybrid camera at ¦ the center of the FOV and ¦ to the right a small bottle was imaged with μL of At a0MBq The measurement times were and a0min at ¦ and ¦ respectively The images were reconstructed using a0keV Xrays in the pinhole mode and a0keV gamma rays in the Compton mode The measurements resulted in pinhole events and Compton events at ¦ and pinhole events and Compton events at ¦ The pinhole images of the a0keV Xrays and the Compton images of the a0keV gamma rays are presented in Fig a0 At imaging of a mouse To investigate the capability of the hybrid camera for animal imaging imaging of a mouse [8weekold male ICR mouse SLC Japan Hamamatsu Japan] with a At tracer was conducted The mouse length a0mm weight a0g was injected with At a0kBq a0h before the measurement The mouse was euthanized by an overdose of isoflurane a0h after the injection The hybrid camera was positioned on the right side of the mouse a0mm from the body axis as shown in Fig a0 left The measurement time was a0h resulting in pinhole events and Compton events From Fig a0 center the pinhole image depicted that the distribution of the At converged on the thyroid stomach and bladder from events obtained after a0h of measurement Moreover although the Compton image confirms the concentration near the Scientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The pinhole MLEM reconstruction image left and the Compton MLEM reconstruction image right of a bottle with At at the center of the FOV upper and ¦ to the right lowerFigure a0 Left Experimental configuration of the measurement of the mouse administered with 211At Center The pinhole MLEM reconstructed image obtained by a0h of measurement Right The Compton MLEM reconstructed image obtained by a0h of measurementcenter accumulation is splitting possibly owing to the lack of event statistics as shown in Fig a0 right All the animal experiments were approved by the animal ethics committees of Osaka University and were performed according to the institutional guidelinesScientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cRadionuclide211At213Bi225Ac223Ra212BiX a0ray keV Gamma ray keV absolute intensity Pinhole Comptoncid31cid31cid31cid31cid31cid31Table Examples of αp emitters for RNT and their main Xrays and gamma rays and their absolute intensities in percentage accompanied by the adaptability of the pinhole mode andor the Compton modeFigure a0 The configuration of the hybrid camera left Schematic view of the pinhole event center for the lower energy range and the Compton event right for the higher energy range that are used for the pinholeCompton reconstruction in the hybrid cameraDiscussionIn the imaging experiment with the At inside a small bottle the source position was obtained by pinhole reconstruction using the events accumulated in the first s The time taken to localize the convergence through the pinhole mode was of that thorough the Compton mode This is due to the intense Xray emission from the 211At as shown in Fig a0 a0keV Xrays are statistically advantageous over a0keV gamma rays for imaging 211At In this regard it is comprehensible that Compton events are not enough to reconstruct the distribution of the At tracer although it can be imaged through the pinhole mode There are several αemitting nuclides that can be used for RNT in the future2122 The distribution of these nuclides should be comprehensible and controlled by monitoring characteristic Xrays and nuclear gamma rays from outside the body Table a0 summarizes several properties of αemitting nuclides that are planned for clinical use Moreover Xray or gammaray energy suitable for a specific imaging application may use different nuclides and consequently different energies as the a0keV for At investigated here Although some nuclides are easy to image with lowenergy Xrays others are suitable for gammaray imaging for example Ac and 212Bi The wide scope of the hybrid cameras allows us to select the appropriate energy from lowenergy Xrays to highenergy gamma rays and thus potentially cover a wide energy range including the conventional SPECT a0keV PET a0keV and Compton camera subMeV to MultiMeV for various applications This leads to a reduction in the measuring time thus reducing the burden on the patient In addition wideband imaging can image multiple tracers simultaneously which significantly increases a patients complete medical information obtained from a single diagnosisTo improve the performance for further applications the hole size in the front detector should be adjusted as a tradeoff between the efficiency and the resolution of pinhole imaging Additionally the application of the depthofinteraction technique29 to the backward detector may improve the resolution Therefore the energy range of the pinhole and Compton modes can be adjusted based on the density andor thickness of the detectors By revising the structure of the detector imaging in a wider band can be realized Particularly Compton cameras developed for gammaray astronomy have realized imaging in high energy bands such as a0MeV32 By applying the configuration of the hybrid camera as proposed in this paper these Compton cameras can perform pinhole imaging without compromising on the performance of the original Compton camera Currently we are developing a new hybrid camera that covers a0keV to a0MeV by adopting the configuration of the scintillator as described in Kishimoto et a0al and Hosokoshi et a0al MethodsConfiguration of the hybrid camera The configuration of the hybrid camera is detailed in Fig a0 left The hybrid camera consists of a pair of positionsensitive detectors capable of receiving the reaction position and the energy deposit of each event The detectors are composed of Cedoped Gd3Al2Ga3O12 GAGG scintilScientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Left Energy spectrum of all the events detected in either detector black the events accumulated by with the front detector green the events obtained only with the rear detector blue and the events reacted with both detectors red accumulated from placing Am and Cs sources simultaneously in front of the detector Right 2D energy spectrum of coincidence events from the front detector scatterer and the rear detector absorber The area painted red corresponds to the energy cut range for a0keV Compton events The brightest area Efront ¼ a0 a0keV corresponds to backscattering eventsModePinholeComptonCoincidence selectionAnticoincidence rearCoincidence front and rearEnergy cutRearSum and frontTable Factors of event selection for each reconstruction modelator arrays3334 coupled with multipixel photon counter MPPC arrays29 A à array of à à a0mm3 GAGG pixels is used as the front detector and a à array of à à a0mm3 GAGG pixels is used as the rear detector The front detector has an active à mm2 pinhole in its center The distance between the detectors is a0cm The energy resolutions of the front and rear detectors are and FWHM at a0keV respectively The position resolution for gammaray interaction is equal to the pixel size both for the pinhole and Compton mode The time range of the coincidence is set to be ± a0 a0μs and the count rate capability is ¤ a0 a0kHz The camera is enclosed in a mmthick metal mainly tungsten density a0gcm3 case except the front surfaceThe proposed hybrid camera enables imaging similar to that of a pinhole camera in addition to the conventional Compton camera Commonly the Compton camera which consists of a scatterer and an absorber uses events that undergo Compton scattering in the scatterer and photoabsorption in the absorber as shown in Fig a0 right For each event the scattering angle of Compton scattering is calculated using Compton kinematics restricting the arrival direction in the conical area called the Compton cone The position of the radiation source is identified by superimposing the Compton cones However photons with energy lower than several hundreds of keV cannot be imaged by the Compton camera owing to the increased probability of photoabsorption in the front scatterer The hybrid camera can also make use of such photoabsorption events We have devised a method to operate the camera as a Compton and a pinhole camera by utilizing the front scatterer with a hole in the center As shown in Fig a0 center the arrival directions of lowenergy photons are limited by analyzing the scatterer as an active pinhole Among lowenergy events detected in the rear detector the events that are not detected in the front detector can be considered to have passed through the hole in the front detector Note that Compton andor pinhole reconstruction can be selected analytically after the measurementReconstruction flow for each mode The selection of valid events for Compton and pinhole modes are parameterized with two factors coincidence selection and energy cut Coincidence is an event pattern hitting one or both of the detectors Specifically only anticoincidence events wherein only the rear detector is triggered are used for pinhole mode imaging whereas coincidences between the front and rear detectors are used for Compton mode reconstruction An energy cut is used to restrict the energy range of photons deposited in each detector The event selection criteria for each mode are summarized in Table a0 From Fig a0 left blue the spectrum from the rear detector in the anticoincidence mode simultaneously irradiated with Am and Cs sources shows that a0keV Xray is accurately masked in the front detector Therefore the pinhole events Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0cRadionuclide241Am137Cs211At211AtTarget keV ModePinholeComptonPinholeComptonEnergy cut keV Erear Efront Efront Erear Erear Efront Efront Erear Table The numbers for applied energy cutsare selected by the energy cuts with the corresponding energy range On the other hand Compton events are chosen from candidates of coincident events Figure a0 left red shows the spectrum from coincidence events obtained with mixed Am and Cs measurement Therefore Compton events are chosen from the region of interest reddiamond restricted from the total energy deposit a0keV a0 Efront Erear a0keV and energy deposit of front detector to reject the backscattering events Efront a0keV Furthermore the events whose scattering angles cannot be determined geometrically are deleted A red diamond in the 2D spectrum of the coincidence events shown in Fig a0 right corresponds to valid events for Compton image reconstruction The quantitative numbers applied for energy cut are summarized in Table a0 In image reconstruction maximum likelihoodexpectation maximization MLEM a statistical approximation method153536 is used to improve the image quality This method uses statistical iterations to locate sources with greater accuracy and better signaltonoise ratio alternate to the simple back projection method The number of iterations is for each mode except for the Lshaped Compton image which is In the pinhole mode the signaltonoise ratio is also improved by subtracting the background image that was reconstructed from the events next to the source energy rangeReceived May Accepted August References Schönfelder V et al The imaging Compton telescope COMPTEL on the gamma ray observatory IEEE Trans Nucl Sci 101109TNS198443333 Schöenfelder V et al Instrument description and performance of the imaging gammaray telescope COMPTEL aboard the Compton gammaray observatory Astrophys J Suppl Ser 10108619179 Todd R Nightingale J Everett D A proposed γ camera Nature 10103825113 2a0 Kataoka J et al Ultracompact Compton camera for innovative gammaray imaging Nucl Instrum Methods Phys Res Sec A Acceler Spectrom Detect Assoc Equip 101016jnima201709048 Koide A et al Precision imaging of MeV gamma rays using a 3D position sensitive Compton camera Sci Rep 101038s4159 Mochizuki S et al Highprecision Compton imaging of MeV prompt gammaray toward an online monitor for proton therapy Nucl Instrum Methods Phys Res Sect A Detect Assoc Equip 101016jnima201811032 Golnik C et al Tests of a Compton imaging prototype in a monoenergetic MeV photon fielda benchmark setup for prompt gammaray imaging devices J Instrum P06009 101088174802211106P0600 Hilaire E Sarrut D Peyrin F Maxim V Proton therapy monitoring by Compton imaging influence of the large energy spectrum of the promptγ radiation Phys Med Biol 101088003191556183127 Fontana M Dauvergne D Létang J M Ley JL Testa E Compton camera study for high efficiency SPECT and benchmark with Anger system Phys Med Biol 10108813616560aa926 a Yoshida E et al Whole gamma imaging a new concept of PET combined with Compton imaging Phys Med Biol Nakano T et al Imaging of 99mTcDMSA and 18FFDG in humans using a SiCdTe Compton camera Phys Med Biol 05LT01 10108813616560ab8e8 10108813616560ab33d 10108813616560aae1d Sakai M et al In a0vivo simultaneous imaging with 99m Tc and F using a Compton camera Phys Med Biol Sakai M et al Compton imaging with 99m Tc for human imaging Sci Rep 101038s4159 z Kishimoto A et al Development of a compact scintillatorbased highresolution Compton camera for molecular imaging Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016jnima201606056 Kishimoto A et al First demonstration of multicolor 3D in a0vivo imaging using ultracompact Compton camera Sci Rep 101038s4159 w Motomura S et al Gammaray Compton imaging of multitracer in biological samples using strip germanium telescope IEEE Trans Nucl Sci 101109TNS200789420 Motomura S Kanayama Y Haba H Watanabe Y Enomoto S Multiple molecular simultaneous imaging in a live mouse using semiconductor Compton camera J Anal At Spectrom 101039B8029 64D Motomura S et al Improved imaging performance of a semiconductor Compton camera GREI makes for a new methodology to integrate biometal analysis and molecular imaging technology in living anisms J Anal At Spectrom 101039C3JA3 0185K Hamilton J G Soley M H Studies in iodine metabolism by the use of a new radioactive isotope of iodine Am J Physiol Legacy Content 101152ajple gacy19391273557 Odonoghue J Bardies M Wheldon T Relationships between tumor size and curability for uniformly targeted therapy with betaemitting radionuclides J Nucl Med Poty S Francesconi L C McDevitt M R Morris M J Lewis J S Alpha emitters for radiotherapy from basic radiochemistry to clinical studiespart J Nucl Med 102967jnume d11720465 Sgouros G et al MIRD Pamphlet No Abridged radiobiology and dosimetry of αp emitters for targeted radionuclide therapy J Nucl Med 102967jnume d10805865 Scientific RepoRtS 101038s41598020710195Vol1234567890wwwnaturecomscientificreports 0c Fujieda K et al First demonstration of portable Compton camera to visualize 223Ra concentration for radionuclide therapy Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016jnima201916280 Brown I Astatine211 its possible applications in cancer therapy Int J Radiat Appl Instrum Part A Appl Radiat Isot 101016088328898690273 X Willhauck M J et al The potential of 211Astatine for NISmediated radionuclide therapy in prostate cancer Eur J Nucl Med Mol Imaging 101007s0025 Watanabe S et al Difference in thyroid uptake between Astatine211 and Iodine123 in normal rats a comparative study between oral and intravenous administration J Nucl Med Watabe T et al Enhancement of At uptake via the sodium iodide symporter by the addition of ascorbic acid in targeted αtherapy of thyroid cancer J Nucl Med 102967jnume d11822263 Agostinelli S et al GEANT4a simulation toolkit Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016S0168 Kataoka J et al Recent progress of MPPCbased scintillation detectors in high precision Xray and gammaray imaging Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016jnima201411004 Kishimoto A et al Development of a dualsided readout PET module using largearea monolithic MPPCarrays IEEE Trans Nucl Sci 101109TNS201222332 Kataoka J et al Handy Compton camera using 3D positionsensitive scintillators coupled with largearea monolithic MPPC arrays Nucl Instrum Methods Phys Res Sect A Acceler Spectrom Detect Assoc Equip 101016jnima201307018 Hosokoshi H et al Development and performance verification of a 3D positionsensitive Compton camera for imaging MeV gamma rays Sci Rep 101038s4159 z Kamada K et al Composition engineering in ceriumdoped Lu Gd3Ga Al5O12 singlecrystal scintillators Cryst Growth Des Kamada K et al inch diameter single crystal growth and scintillation properties of CeGd3Al2Ga3O12 J Cryst Growth 101021cg200 694a 101016jjcrys gro201111085 Dimmock M R Nikulin D A Gillam J E Nguyen C V An CL implementation of pinhole image reconstruction IEEE Trans Nucl Sci 101109TNS201221977 Wilderman S a0J Clinthorne N a0H Fessler J a0A Rogers W a0L Listmode maximum likelihood reconstruction of Compton scatter camera images in nuclear medicine In IEEE Nuclear Science Symposium Conference Record IEEE Nuclear Science Symposium and Medical Imaging Conference Cat No 98CH36255 vol a0 101109NSSMI C199877387 IEEE AcknowledgementsThis research was supported by JSPS KAKENHI Grant Number JP15H05720 20H00669 and JPMJER1905 ERATOFS The At was supplied through the Supply Platform of Shortlived Radioisotopes supported by JSPS GrantinAid for Scientific Research on Innovative Areas Grant Number 16H06278Author contributionsJK conceived the concept of this research AO developed the hybrid camera AO JK KF SS and EK conducted the experiments HK AT TT KO YL KM TK TW ES and JH conducted the experiments using 211At AO wrote the manuscriptcompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to AOReprints and permissions information is available at wwwnaturecomreprintsPublishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020710195Vol0123456789wwwnaturecomscientificreports 0c' | Thyroid_Cancer |
"Older patients with cancer require specific and individualized management The 3groupMultidimensional Prognostic Index MPI based on the Comprehensive Geriatric Assessment CGA has shown apredictive interest in terms of mortality The objective of our study was to assess the prognostic value of MPI for year mortality in an external prospective French cohort of elderly patients with cancerMethods From March to March a prospective singlecenter cohort study enrolled all patients withcancer aged years and older referred to the geriatric oncology clinic We used a proportional hazard model for1year mortality adjusted for age sex tumor sites and metastatic status Cstatistics were used to assess theincremental predictive value of MPI index to these risk factorsResults overall patients underwent CGA with MPI women mean age ± years The most commontumor sites were prostate skin colorectum and breast of patients had a metastaticdisease patients belonged to the MPI1 group to the MPI2 group and patients wereclassified in the MPI3 group Oneyear mortality rate was in MPI1 in MPI2 and in MPI3 p All domains of MPI except cognition and living status were significantly associated with mortality at oneyear aswell as tumor sites and metastatic status Higher MPI was associated with a higher mortality risk adjusted HR [95CI ] and [] for MPI groups and compared to p Conclusions In addition to established risk factors MPI improves risk prediction of 1year mortality This practicalprognostic tool may help to optimize management of these vulnerable patientsKeywords Aged Neoplasms Mortality Comprehensive geriatric assessment Correspondence Evelyneliuuchupoitiersfr1Department of Geriatrics Poitiers University Hospital Poitiers France2Clinical Investigation Centre CIC1402 CHU Poitiers University of PoitiersINSERM Poitiers FranceFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiuu BMC Geriatrics Page of BackgroundIndividuals over years old are the fastest growing segment of the population and by will represent about of Americans and of Europeans [] The incidence of cancer continues to increase worldwide it is estimated at millionyear by representing anincrease of in cases compared with [] Theincidence of cancer is times higher in people over years old and people aged and older have a higherrisk of developing invasive cancer []The older population is characterized by a very heterogeneous profile especially in terms of frailty geriatriccharacteristics and comorbidities which explains theneed for specific and adapted care [ ] Neverthelessscientific data are scarce because older subjects are oftenunderrepresented in oncological clinical trials that setthe standards of antineoplastic treatment [ ]Over the last three decades the fiveyear survival ratefor all types of cancer has increased particularly in individuals aged to [ ] Still older patients are atmore risk of toxicity in anticancer therapies such aschemotherapy and require a benefitrisk assessmentprior to treatment [] A comprehensive geriatric assessment CGA is consequently recommended in these patients to diagnose comorbidities and optimize geriatricinterventions and to improve the functional state andpossibly the survival rate by ensuring better tolerance totreatment [ ] CGA has also shown predictive valuein identifying elderly patients with cancer who are exposed to a poor prognosisincluding a higher risk ofdeath during hospitalization [] Among the CGAbased assessment tools the Multidimensional PrognosticIndex MPI has shown a predictive interest in mortalityat months and months in Italian patients aged years and older with advanced cancers []The main objective of our study was to validate theprognostic value of the MPI for 1year mortality in an external French cohort of older patients with cancer Thesecondary objective was to assess the major risk factors associated with 12month mortality in these patientsMethodsStudy population and data collectionThis prospective singlecenter cohort study enrolledfrom March to March all patients with cancer aged years and older who were referred to thegeriatric oncology clinic of Poitiers University HospitalpriorSociodemographic data and cancerrelated information werecollected during the consultationincluding age sexmarital status social environment type of cancer metastasis status and cancerspecific treatment Tumor siteswere classified as follows colorectal breast prostateupper gastrointestinaltract stomach and esophagusanticancertreatmenttoplannedand liver urinary system bladder upper urinary tractand kidney hematologic malignancies and other tumors including ovary uterus lung head and neck skinthyroid and unknown primary The CGA was performed by a senior geriatrician specialized in oncologyand provided data necessary to calculate MPI All eligible patients who had signed the consent form were included in the study The study protocol was validated bythe Poitiers University Hospital ethics committee Poitiers France All the clinical and biological data werecollected and recorded in a cohort databaseliving with familyMultidimensional prognostic indexThe MPI based on a CGA was calculated after administration of standardized and validated tests exploringeight domains Table [] Living status was categoinstitutionalized orrized asalone and functional status was evaluated by Activitiesof Daily Living ADL ranging from total dependenceto independence and Instrumental ADL IADL [] Nutrition was assessed by the Mini NutritionalAssessmentShort Form MNASF questionnaire cognitive status was evaluated by the Short Portable MentalStatus Questionnaire SPMSQ[ ] The ExtonSmith Scale ESS estimated the risk of pressure ulcer[] Comorbidities were evaluated by the CumulativeIllness Rating Scale CIRS which scores the severity of anic systems ranging from absent to mostsevere [] Based on this scale a comorbidity indexCIRSCI records the number of moderate to severean pathologies CIRS scores from to [] Thenumber of medications is classified in three groups drugs a day to drugs or ¥ drugsThe MPI was scored by matching the results of thesetests A value of or was assigned accordingto the conventional cutoff points considering as noproblem minor problem and major problemTable The sum was then divided by to obtain thefinal MPI score which was categorized into groupsthe MPI1 group final score defining patientswith low mortality risk at year the MPI2 group moderate risk and the MPI3 groupgroup higher riskDefinition of outcomesThe primary outcome in the longitudinal analyses was1year mortality Systematic followup was performedafter discharge through clinical visits every months bythe same clinical research assistant When patients werenot present at visit phone calls were made to the general practitioners to assess vital status and to obtain thedate of death if applicable 0cLiuu BMC Geriatrics Page of No problem value ¥¥Table Multidimensional Prognostic Index score assigned to each domain according to the severity of problemAssessment tests rangeADL IADL SPMSQ 10aCIRSCI 14bMNASF ESS Number of medicationsMinor problem value Institutionalized¥ ¥Living with familySevere problem value ¥¥ ¥ Living statusAbbreviations ADL Activities of Daily Living IADL Instrumental Activities of Daily Living SPMSQ Short Portable Mental Status Questionnaire CIRSCI CumulativeIllness Rating Scale Comorbidity Index MNASF Mini Nutritional Assessment Short Form ESS Exton Smith Scalea Number of errorsb Number of pathologiesLiving aloneStatistical analysisDescriptive statistics were reported as mean ± standarddeviation SD or median 25th75th percentiles forcontinuous variables or absolute number and percentagefor categorical variables The time to event was plottedas KaplanMeiersurvival curves according to MPIgroups and comparison was made using the logranktest The hazard ratio HR of 1year mortality for eachparameter was determined by Cox proportional hazardsregression Two models were used univariate modeland models adjusted for age sex metastatic statustumor sites Interactions between sex tumor site andmetastatic status for the association between MPI and year mortality were evaluated by the addition of interaction terms into the corresponding regression modelThe Akaikes information criterion AIC was used tocompare globalfit among models with and withoutMPI and the model with the smallest AIC was considered as the best modelGeneralized cstatistics were calculated to assess improvement in 1year mortality risk prediction of MPI inaddition to traditional risk factors age sex metastaticstatus tumor sites [] The CIs for the changes inthe cstatistic were computed based on bootstrapsamples P values were considered statistically significant Statistical analyses were performed with SASversion SAS Institute Cary NCResultsBaseline characteristics of study populationDuring the recruitment period eligible patients aged years and older were included mostly males n with a mean age of ± years Table Themost common tumor sites were prostate skin and breast of patients had a metastaticdisease Anticancer treatment included chemotherapyin patients surgery in and radiotherapy in Patients had comorbid conditionsregarding the CIRSscale and medication and were frequently malnourished Table In this cohort patients were classified in the MPI1 group patients in MPI2 and patients in MPI Except for metastatic status and antineoplastic treatments all variables of interest differed between the threeMPI groups P MPI and 1year mortalityAmong the patients were lost to followup Mean followup was ± months Overall mortalityat months was in MPI1 in MPI2 and in MPI3 P Fig Since we observed significant statistical interaction between sex and tumor site P we presented resultsfor the multivariate model with the inclusion in themodel of an interaction term We found no significantinteraction between tumor site and metastatic statusP The risk of 1year mortality across MPI groups isshown in Fig All functional scoring but SPMSQ and living statusnumber of daily drugs metastatic status and tumor sitewere significantly associated with mortality Table Compared to colorectal cancer reference categorybreast cancer was associated with significantly lower year mortality and upper gastrointestinal tractliver cancer and other malignancies with significantly higher year mortalityMPI groups were associated with 1year mortality inthe univariate model and remained significantly associated even after adjustment for age sex metastatic statusand tumor site Compared to the MPI1 group patientsof the MPI2 and MPI3 groups had gradual increasedrisk of 1year mortality adjusted hazard ratio [95CI] [] and [] respectively P Table 0cLiuu BMC Geriatrics Page of Table Patients baseline characteristics and evaluation by multidimensional prognostic index MPI n Sociodemographic characteristicsAgeFemale n Oncological characteristicsMost frequent tumor sitesProstateSkinColorectumBreastHematological malignanciesBladderMetastatic status n Type of antineoplastic treatment aChemotherapySurgeryRadiotherapyTotal cohortN ± MPI1N ± Comprehensive geriatric assessment and multidimensional prognostic index bHormone therapy ADL scoreADL categoryIADL scoreIADL categoryESS scoreESS categoryMNASF scoreMNASF categorySPMSQ scoreSPMSQ categoryCIRS scoreCIRSCI scoreCIRSCI categoryNumber of medicationsNumber of medications categoryLiving status familyinstitutionalone ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± MPI2N ± ± ± ± ± ± ± ± ± MPI3N ± ± ± ± ± ± ± ± ± MPI score ± ± ± ± P Numbers are mean ± SD or n Abbreviations MPI multidimensional prognostic index SD standard deviation ADL activities of daily livings IADL instrumental activities of daily livings ESS ExtonSmith Scale MNASF mini nutritional assessment short form SPSMQ Short Portable Mental Status Questionnaire CIRS Cumulative Illness Rating Scale CIcomorbidity indexa Antineoplastic treatment may combine one or several types of treatmentb Categories are reported as number of patients with nominorsevere problem to calculate MPI scoreDiscriminationWe assessed improvement in risk discrimination for theMPI group compared with the model with traditionalrisk factors age sex metastatic status and tumor siteWe observed a small but significant improvement in year mortality risk prediction difference in Cstatistic P when including the MPI group in themodel Table 0cLiuu BMC Geriatrics Page of Fig KaplanMeier curves of overall mortality in patients according to MPI groups Dotted line MPI dashed line MPI and solid line MPI Logrank test P Discussion and implicationsOur study confirmed the predictive value of the multidimensional prognostic index for 1year mortality in olderpatients with cancer MPI group had a significantlytwo to fivefold higher rate of 1year mortality We alsoshowed that the MPI improved prediction of 1yearmortality going beyond the traditional risk factors reported in the literature []Estimation of patient survival at time of the therapeutic decision is required to assess the balance of benefits and risks of performing or not performing specificoncologic interventions taking cancerspecific mortalityinto consideration Clinicians may need to know if thepatient will die of cancer or with cancer in cases wherecomorbidities or geriatric syndromes are challengingSeveral scales have been created and validated in largeepidemiologic cohorts to estimate overall survival notably at months with the Carey and Walter indexes [ ] These two scores consider dependency comorbidities with cancer and malnutrition Walter and collaborators reported independent associations betweenoneyear mortality in multivariable analysis and risk factors including male gender two medical diagnoses congestive heart failure aOR 95CI andcancer aOR for localized cancer and aOR for metastatic cancerfunctional dependency in any ADL at discharge aOR for dependencies from to ADLs and aOR for dependencies in all ADLs and laboratoryvalues creatinine level mgdL [ μmolL] aOR and albumin level gdL aOR from to gdL and aOR forvalues below gdL [] Carey confirmed thesefindings and furthered the elaboration of a prognosticindex for mortality in communityliving frail older individuals considering eight independent risk factors ofmortality weighted using Cox regression male sex dependence in toileting malignant neoplasm and renal insufficiency [] None of these tests were specificallydeveloped in cohorts with individuals with cancer andthey may consequently not be informative enough to reflect clinical and functional variability in daily care andto provide personalized corrective interventions Recentevidence reported a positive impact of geriatric interventions and monitoring in survival increase improvementof quality of life and completion of chemotherapy [] The MPI differs from other mortality indexes because it is based on a CGA with each of the eight tests 0cLiuu BMC Geriatrics Table Univariate and multivariate analyses for oneyear mortality model for MPIgroups n VariableADL score pointPvalueAdjusted HR CIIADL score pointSPMSQ score pointCIRS score pointMNA score pointESS score pointNumber of drugs drugLiving statusliving with familyliving aloneInstitutionalizedAge yearSex male vs femaleMetastatic statusTumor sitesColorectalbreastprostateUpper gastrointestinal tractliverurinary systemhematologic malignanciesother tumorsMultidimensional Prognostic Indexgroup group group HR CI reference reference reference reference reference Page of P Abbreviations HR hazard ratio CI confidence interval MPI Multidimensional Prognostic IndexMultivariate model adjusted for age sex tumor site metastatic status and MPI groupsassessing one geriatric domain Giantin and collaboratorsconfirmed the good discriminatory power for 12monthmortality in a cohort of cancer patients older than and validated higher mortality prediction compared to astandard CGA [] Use of the MPI in clinical practicemay provide rapid and comprehensive evaluation of patients and help to adapt decisionmaking in oncologyThe MPI has been developed and validated in large cohorts of in and outpatients for many causes to predict notonly mortality but also length of hospital stay P care intensity institutionalization rehospitalization andaccess to homecare services [ ] In an internationalmulticenter cohort of hospitalized older patients patients in group MPI2 OR P and the MPI3 group OR P were at higher risk of overall mortality compared to thoseof the lower risk group at admission [] This index maybe used as a decisionmaking tree for cancer managementso as to select older patients with lower mortality risk forthe same standard treatment as younger counterpartsthose who could benefit from adapted care or an exclusively supportive strategy in patients with limited life expectancy This classification in three groups is comparableto the geriatric oncology algorithm of Balducci [] ThisTable Predictive performance of MPI during 12month followupBiomarkerclinical modelAkaike criterioncindexclinical model MPIClinical model age sex metastatic status tumor site CIdifference in CstatisticsP value 0cLiuu BMC Geriatrics Page of algorithm defines three groups of patients robust vulnerable and frail according to seven criteria age dependencemeasured by ADL and IADL comorbidities with CIRSCIcognition evaluated with MMSE minimental state examination or delirium depressive mood urinary and fecalincontinence and falls in the last months Risk of deathincreased steadily from the lowest to the highest categorycompared to the fit group the patients with a vulnerableprofile had a twofold mortality risk HR and a threefold risk in the frail group HR P [] More recent classifications were suggested to improve global management for such individualsincluding nutrition data and cognitive assessment[ ]Indeed malnutrition is highly prevalent in geriatriconcology settings [] This geriatric syndrome is a wellknown risk factor for early mortality Our findings confirmed that oneyear mortality is strongly associated withnutritional status and altered MNA in its short formSome questions in this test were selected for the elaboration of the Geriatric8 G8 index to screen for vulnerability in older patients with cancer as recommended bythe International Society for Geriatric Oncology SIOG[ ]The findings ofthis study should be interpretedwith caution Firstits design as an observationalsinglecenter study may limit the extrapolation of ourresults to a more general older population with cancer Recruited patients in this cohort may not be representative as cancer specialists may not refer alltheir patients to the geriatric oncology clinic notablythose screened as notvulnerable in geriatric termsas recommended by the SIOG and National Instituteof Cancer in a twostep approach [] Cancer management of these patients may follow standard strategy without geriatric expertise After accounting fortraditional risk factors the magnitude of the improvement in risk prediction by the addition is small butsignificant Moreover our results are consistent withexisting findings in geriatric oncology settingsthisstrategyOur research on the predictive value of MPI foroneyear mortality of older patients with cancershould serve as a foundation for future studies aiming to improve therapeutic strategies for these patients A major part ofinvolvespersonalized geriatric interventions such as specificcare monitoring by nurse and physical rehabilitationIt has shown benefits for elderly cancer patients butso far no study has demonstrated any impact onsurvival [] MPI appears to be a rapid assessmenttool helping to optimize cancer care guidepatienttailored interventions and predict early mortality These findings should pave the way for prospective interventionaltaking account ofstudiesMPI groups for decisionmaking about cancer treatments and followupConclusionsIn addition to established risk factors MPI improves riskprediction of 1year mortality in older cancer patientsThis practical prognostic tool may help to optimizemanagement of these vulnerable individualsAbbreviationsADL Activities of Daily Living AIC Akaikes information criterionCGA Comprehensive Geriatric Assessment CI confidence intervalCIRS Cumulative Illness Rating Scale CIRSCI Cumulative Illness Rating Scale comorbidity index ESS ExtonSmith Scale G8 Geriatric8 HR Hazard ratioIADL Instrumental Activities of Daily Living MNASF Mini NutritionalAssessmentShort Form MPI Multidimensional Prognostic IndexSD Standard deviation SIOG International society of geriatric oncologySPMSQ Short Portable Mental Status QuestionnaireAcknowledgmentsAuthors thank Emilie Favard for her assistance in the collection of followupdata and Jeffrey Arsham who edited the English of the manuscriptAuthors contributionsEL CH and MP designed the study EL SV and AJ were responsible for theacquisition of data EL and PJS performed the statistical analysis andinterpretation EL PJS and MP wrote the manuscript EL PJS MP TB MLBand AP substantively revised the work All authors EL CH SV TB AJ MLBAP PJS and MP read and approved the final manuscriptFundingThe authors declare no fundingAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateAll eligible patients who had signed the consent form were included in thestudy The study protocol was validated by the Poitiers University Hospitalethics committee Poitiers FranceConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Geriatrics Poitiers University Hospital Poitiers France2Clinical Investigation Centre CIC1402 CHU Poitiers University of PoitiersINSERM Poitiers France 3Department Geriatric Care Orthogeriatrics andRehabilitation Frailty Area EO Galliera Hospital Genova Italy 4Departmentof interdisciplinary Medicine Aldo Moro University of Bari Bari ItalyReceived December Accepted August ReferencesExtermann M Aapro M Bernabei R Cohen HJ Droz JP Lichtman S et alUse of comprehensive geriatric assessment in older cancer patientsrecommendations from the task force on CGA of the International Societyof Geriatric Oncology SIOG Crit Rev Oncol Hematol Cancer Research UK Worldwide cancer incidence statistics Availablefrom httpwwwcancerresearchukhealthprofessionalcancerstatisticsworld widecancer incidence Accessed Apr Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin 0cLiuu BMC Geriatrics Page of Pamoukdjian F Liuu E Caillet P Herbaud S Gisselbrecht M Poisson J Howto optimize Cancer treatment in older patients an overview of availablegeriatric tools Am J Clin Oncol Kalsi T BabicIllman G Ross PJ Maisey NR Hughes S Fields P The impact ofcomprehensive geriatric assessment interventions on tolerance tochemotherapy in older people Br J Cancer Rao AV Hsieh F Feussner JR Cohen HJ Geriatric evaluation andmanagement units in the care of the frail elderly cancer patient J GerontolA Biol Sci Med Sci Meyer AM Becker I Siri G Brinkkötter PT Benzing T Pilotto A Polidori MCNew associations of the Multidimensional Prognostic Index Z GerontolGeriatr Pilotto A Veronese N Daragjati J CruzJentoft AJ Polidori MC MattaceRasoF Using the multidimensional prognostic index to predict clinicaloutcomes of hospitalized older persons a prospective multicentreinternational study J Gerontol A Biol Sci Med Sci Ferrat E Paillaud E Caillet P Laurent M Tournigand C Lagrange JL et alPerformance of four frailty classifications in older patients with cancerprospective elderly cancer patients cohort study J Clin Oncol Caillet P Liuu E Raynaud Simon A Bonnefoy M Guerin O Berrut GAssociation between cachexia chemotherapy and outcomes in oldercancer patients a systematic review Clin Nutr Decoster L Van Puyvelde K Mohile S Wedding U Basso U Colloca G et alScreening tools for multidimensional health problems warranting a geriatricassessment in older cancer patients an update on SIOG recommendationsAnn Oncol Soubeyran P Bellera C Goyard J Heitz D Curé H Rousselot H et alScreening for vulnerability in older cancer patients the ONCODAGEprospective multicenter cohort study PLoS One 20149e115060 Caillet P CanouiPoitrine F Vouriot J Berle M Reinald N Krypciak S et alComprehensive geriatric assessment in the decisionmaking process inelderly patients with cancer ELCAPA study J Clin Oncol Galvão DA Taaffe DR Spry N Joseph D Newton RU Combined resistanceand aerobic exercise program reverses muscle loss in men undergoingandrogen suppression therapy for prostate cancer without bonemetastases a randomized controlled trial J Clin Oncol Goodwin JS Satish S Anderson ET Nattinger AB Freeman JL Effect ofnurse case management on the treatment of older women with breastcancer J Am Geriatr Soc Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsBalducci L Extermann M Management of Cancer in the older person apractical approach Oncologist Walter LC Brand RJ Counsell SR Palmer RM Landefeld CS Fortinsky RH Development and validation of a prognostic index for 1year mortalityin older adults after hospitalization JAMA Gouverneur A Salvo F Berdaï D Moore N FourrierRéglat A Noize PInclusion of elderly or frail patients in randomized controlled trials oftargeted therapies for the treatment of metastatic colorectal cancer asystematic review J Geriatr Oncol Talarico L Chen G Pazdur R Enrollment of elderly patients in clinical trialsfor cancer drug registration a 7year experience by the US Food and DrugAdministration J Clin Oncol Zeng C Wen W Mans AK Pao W Shu XO Zheng W Disparities by raceage and sex in the improvement of survival for major cancers results from theNational Cancer Institute surveillance epidemiology and end results SEERprogram in the United States to JAMA Oncol Ellis G Gardner M Tsiachristas A Langhorne P Burke O Harwood RH et alComprehensive geriatric assessment for older adults admitted to hospitalCochrane Database Syst Rev 20179CD006211 Wildiers H Heeren P Puts M Topinkova E JanssenHeijnen ML ExtermannM International Society of Geriatric Oncology consensus on geriatricassessment in older patients with cancer J Clin Oncol AvelinoSilva TJ Farfel JM Curiati JA Amaral JR Campora F JacobFilho WComprehensive geriatric assessment predicts mortality and adverseoutcomes in hospitalized older adults BMC Geriatr Angleman SB Santoni G Pilotto A Fratiglioni L Welmer AK MPI_AGEProject Investigators Multidimensional Prognostic Index in Association withFuture Mortality and Number of Hospital Days in a PopulationBasedSample of Older Adults Results of the EU Funded MPI_AGE Project PLoSOne 201510e0133789 Giantin V Falci C De Luca E Valentini E Iasevoli M Siviero P Maggi S et alPerformance of the multidimensional geriatric assessment andmultidimensional prognostic index in predicting negative outcomes inolder adults with cancer Eur J Cancer Care httpsdoi101111ecc12585 Pilotto A Ferrucci L Franceschi M D'Ambrosio LP Scarcelli C Cascavilla L Development and validation of a multidimensional prognostic indexfor oneyear mortality from comprehensive geriatric assessment inhospitalized older patients Rejuvenation Res Pilotto A Rengo F Marchionni N Sancarlo D Fontana A Panza F et alComparing the prognostic accuracy for allcause mortality of frailtyinstruments a multicentre 1year followup in hospitalized older patientsPLoS One 20127e29090Katz S Downs TD Cash HR Grotz RC Progress in development of the indexof ADL Gerontologist Lawton MP Brody EM Assessment of older people selfmaintaining andinstrumental activities of daily living Gerontologist Pfeiffer E A short portable mental status questionnaire for the assessmentof anic brain deficit in elderly patients J Am Geriatr Soc Rubenstein LZ Harker JO Salva A Guigoz Y Vellas B Screening forundernutrition in geriatric practice developing the shortform mininutritional assessment MNASF J Gerontol A Biol Sci Med Sci M366 Bliss MR McLaren R ExtonSmith AN Mattresses for preventing pressuresores in geriatric patients Mon Bull Minist Health Public Health Lab ServLinn BS Linn MW Gurel L Cumulative illness rating scale J Am Geriatr Soc Conwell Y Forbes NT Cox C Caine ED Validation of a measure of physicalillness burden at autopsy the cumulative illness rating scale J Am GeriatrSoc Pencina MJ D'Agostino RB Overall C as a measure of discrimination insurvival analysis model specific population value and confidence intervalestimation Stat Med Giantin V Valentini E Iasevoli M Falci C Siviero P De Luca E Does themultidimensional prognostic index MPI based on a comprehensivegeriatric assessment CGA predict mortality in cancer patients Results of aprospective observational trial J Geriatr Oncol Carey EC Covinsky KE Lui LY Eng C Sands LP Walter LC Prediction ofmortality in communityliving frail elderly people with longterm careneeds J Am Geriatr Soc Epub Nov 0c" | Thyroid_Cancer |
"rebound effect after stopping treatment with denosumab may be associated with rapid loss ofthe gains in bone mineral density achieved with treatment high levels of bone remodeling markers the occurrenceof vertebral fractures and even hypercalcemiaCase presentation A 64yearold osteoporotic Caucasian woman suffered from a fracture of her second lumbarvertebra in From January she was treated with denosumab for years with good densitometry resultsfor her hip and lumbar areas and no fractures over the last years of treatment Ten months after the treatmentwith denosumab was stopped a cascade of vertebral fractures including some in unusual locations third thoracicvertebra and multiple rib fractures in a context of hypercalcemia suggested possible malignancy A completeevaluation including systemic biological and biopsy analyses ruled out this hypothesis The hypercalcemia wasassociated with normal plasma phosphate and vitamin D concentrations and a high parathyroid hormone levelwith an abnormal fixation of the lower lobe of the thyroid on sestamethoxyisobutylisonitrile scintigraphyHistological analysis of the excised parathyroid tissue revealed hyperplasia The associated thyroidectomy goiterled to the discovery of a thyroid papillary microcarcinomaConclusions We consider the consequences of this rebound effect not only in terms of the major loss of bonedensity return to basal values within years and the multiple disabling fracture episodes but also in terms of thehypercalcemia observed in association with apparently autonomous tertiary hyperparathyroidism Several cases ofspontaneous reversion have been reported in children but the intervention in our patient precluded anyassessment of the possible natural course The discovery of an associated thyroid neoplasm appears to befortuitous Better understanding of the various presentations of the rebound effect after stopping treatment withdenosumab would improve diagnostic management of misleading forms as in this case Bisphosphonates couldpartially prevent this rebound effectKeywords Osteoporosis Denosumab rebound Fracture Hypercalcemia Hyperparathyroidism Correspondence yvesmaugarschunantesfrRheumatology Department Nantes University Hospital place AlexisRicordeau Nantes Cedex France The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMaugars Journal of Medical Case Reports Page of IntroductionSome warning signs need to be explored and are wellknown aftertreatment with denosumab A majorrebound effect after stopping denosumab can be responsible for rapid bone loss with vertebral crushes []Some other manifestations have been described such ashypercalcemia in both children [] and adults []hyperparathyroidism [] and vertebral osteonecrosis[] A suspected increase in the number of cases ofprimary neoplasia has been reported in a recent metaanalysis [] We discuss a new observation with allthese manifestations together which poses diagnosisproblems and several explorations to eliminate a neoplasia This could have been avoided with better knowledge of these rebound manifestationsCase presentationA 64yearold Caucasian woman suffered from a firstvertebral fracture in the second lumbar vertebra L2 in following a fall from her bicycle She did not obtainany treatment Dual Xray absorptiometry DXA relumbar Tscore of standardvealed osteoporosisdeviation SD Our patients characteristics during follow up are summarized in Table Her phosphorus andcalcium levels were normal plasma calcium concentration mmoll normal range to mmollparathyroid hormone PTH concentration was normal ngl normal range to ngl and vitamin D levelwas low ngml normal range to ngl Shewas included in the FREEDOM protocol comparingdenosumab mg subcutaneously every monthsplus mg of calcium and IU of vitamin D dailywith placebo for the treatment of postmenopausal osteoporosis in January The unblinding of the trial years later showed that she had been randomized to thedenosumab group Several vertebral fractures occurredTable Patients characteristics during follow upduring this 3year period fifth thoracic vertebra T5eighth thoracic vertebra T8 and an aggravation of theL2 fracture She continued to participate in the extension protocol in mode for years and then withdrew of her own volition with a finalinjection ofdenosumab in July there were no new vertebralfractures during this entire period DXA in September demonstrated increased bone mineral densityBMD of in her lumbar region Tscore SD and of in her total left hip Tscore SD She was a former tobacco smoker and her medicalhistory included osteoarthritis of the knee a hiatus hernia hypertensionand colonicpolyps Calcaemia monitoring revealed a return to normal values until January nmoll normal range to mmoll Checkups while our patient wasstill on denosumab yielded values of nmoll in January and in September Fig amlodipineallergyIn May our patient complained of acute intensespinal pain that resisted standard painkillers and required treatment with opiates An evaluation was carriedout in hospital in June Spinal Xrays revealed fractures of the fourth thoracic vertebra T4 wedge grade T5 biconcave grade T8 wedge grade ninththoracic vertebra T9 crush grade tenth thoracic vertebra T10 wedge grade 11th thoracic vertebra T11crush grade first lumbar vertebra L1 biconcave grade L2 wedge grade and third lumbar vertebra L3 biconcave grade Fig Bone scintigraphy revealedhypersignals in all these vertebrae except L2 and T5 andin several ribs Magnetic resonance imaging MRI identified vertebra T4 in hypersignal on a T2weighted sequence and hyposignal on a T1weighted sequence withno signs of infiltration or suspected lysis Fig T9 T10T11 L1 and L3 also showed hypersignal and T5 T8 andL2 were older vertebral fractures with no bone marrowFracturesL2Tscore lumbartotalhip BMD SDCalcaemiammollClinicalpresentationBicycle fallNo painFollow up of thepatient January FREEDOM 3yearevaluationJanuary FREEDOM extensionSeptember T5 T8 and aggravationof L2No painNo new fractureTreatmentsInitiation denosumab mg semiannuallycalcium g daily vitamin D IU dailyPursuit denosumab mg semiannuallycalcium g daily vitamin D IU dailyAll treatments stoppedNoneZoledronate two infusions of mg annuallyvitamin D IU quarterlyHospitalizationJune Acute intensedorsal painNew evaluationSeptember Chronic dorsaland lumbar painT4 T9 T10 T11 L1 L3No new fractureNew evaluationAugust BMD bone mineral density L1 first lumbar vertebra L2 second lumbar vertebra L3 third lumbar vertebra SD standard deviation T4 fourth thoracic vertebra T5fifth thoracic vertebra T8 eighth thoracic vertebra T9 ninth thoracic vertebra T10 tenth thoracic vertebra T11 11th thoracic vertebraChronic lumbarpainTreatments stoppedNo new fracture 0cMaugars Journal of Medical Case Reports Page of Fig Calcemia bone mineral density and fracture events for a patient treated with denosumab for years who experienced a rebound effectwhen treatment was stopped with a combination of hypercalcemia related to hyperparathyroidism multiple fractures and rapid bone lossedema Lumbar and dorsal pain remained severe throughout this period of exploration justifying bed rest Biologicaltests revealed hypercalcemia with plasma concentrations of mmoll for calcium normal range to mmoll Fig and mmoll for phosphate normal range to mmoll hypercalciuria mmol24 hoursnormal range to mmol24 hours a 25OH vitaminD3 concentration of ngml normal range to ngla PTH concentration of pgml normal range to ngl a Creactive protein CRP concentration of mglnormal values mgl normal protein electrophoresiswith no Bence Jones proteinuria and a plasma creatinineconcentration of μmollrange to μmoll Blood formula and plasma concentrationsthyroidstimulating hormone TSH parathyroidofhormonerelated peptideand 25OH2D were normal Carboxyterminal collagen crosslinklevels were very high μgl normal valuesCTX μgl but were difficult to interpret in the context ofvertebral fracture DXA performed year after the last injection of denosumab revealed BMD losses of in our patients lumbar region and in her total hipPTHrpnormalcortisolThe association of fractures that are unusual for osteoporosis T4 acute and persistent back pain other rib fractures and hypercalcemia were suggestive of a potentialneoplasia which led to systemic explorations vertebralbiopsy and hyperparathyroidectomy and thyroidectomyknown goiter at the ultrasound exploration A thoracicabdominalpelvic computed tomography CT scan showedonly a heterogeneous multinodular goiter Sestamethoxyisobutylisonitrile MIBI scintigraphy revealed a small areaof fixation of the posterior lower right thyroid lobe and alower lobe nodule displaying clear uptake Fineneedleaspiration results were negative A biopsy of the T4 wascarried out under CT control and produced normal resultsWith hindsight a gassy image of the upper facet of the T4was suggestive of necrosis A parathyroid neoplasia couldhave been evoked too Surgery was performed at the end ofJuly to remove the right upper parathyroid gland à à mm weight g and histological analysissuggested nodular hyperplasia Associated total thyroidectomy led to the detection of a dystrophic goiter withmacrovesicular nodules and a mm isthmic papillarymicrocarcinoma with no associated adenopathy 0cMaugars Journal of Medical Case Reports Page of Fig Lumbar and thoracic Xray in January months before denosumab was stopped and in June months after denosumabwas stopped three old vertebral fractures L2 in and T5 T8 L2 aggravation during the period 20052008stars and new fractures T4 T9T10 T11 L1 L3arrows with denosumab rebound Stars are the old fractures and arrows the new oneHer pain was initially acute but of the mechanical typewith a generally favorable outcome Her calcaemia normalized the day after surgery mmoll with a plasma PTHconcentration of ngl normal range to ngl Anew bone densitometry evaluation was carried out in October at which time bone losses of for the lumbarregion Tscore SD and for the total left hipTscore SD were recorded Fig She continued tocomplain of disabling spinal pain Her phosphorus andcalcium evaluation results remained normal as did her vitamin D levels with the continuation of substitution treatment Given the considerable decrease in BMD she wasplaced on risedronate in September but this wasbadly tolerated She was then placed on zoledronate mgHer calcaemia remained stable at mmoll normalrange to mmoll After two infusions October and October a new DXA in August showed stabilization of the lumbar BMD and a significant loss in the total hip BMD Her plasma calcium levels remained normal mmoll and she did nothave any new vertebral or peripheral fracturesDiscussion and conclusionsThis patient who was included in the initial FREEDOMprotocol [] had benefited from denosumab treatmentwith no new vertebral fractures in the last years oftreatment and an increase in BMD to values exceeding SD with no secondary effects However monthsafter stopping the treatment a cascade of vertebral fractures some in unusual locations T4 although this canbe possible when there are multiple lower vertebral fractures and multiple rib fracturesin a context of 0cMaugars Journal of Medical Case Reports Page of Fig Bone scintigraphy computed tomography scan and magnetic resonance imaging in June Magnetic resonance imaging and bonescintigraphy confirmed that there were six recent vertebral fractures fourth thoracic vertebra ninth thoracic vertebra tenth thoracic vertebra11th thoracic vertebra first lumbar vertebra and third lumbar vertebra small white arrows There were some costal fractures on bonescintigraphy sixth posterior on the right side and laterally tenth 11th and 12th on the left side black arrows A computed tomography scan alsoshowed old vertebral fractures fifth thoracic vertebra eighth thoracic vertebra and second lumbar vertebra white starssuggestedpossible malignancy Ahypercalcemiacomplete evaluation with systemic biological and biopsy T4 analyses ruled out this hypothesis Histological analysis ofthe tissue removed during theparathyroid intervention revealed hyperplasia but no adenoma The hypothesis of coincidental hyperparathyroidism had to be considered Before the treatment withdenosumab our patients calcaemia and PTH levels werenormal Based on retrospective analyses of calcaemia results we concluded that the increase in calcaemia became abnormal after years of denosumab treatmentHyperparathyroidism could have appeared during thedenosumab treatment phase with no obvious link between the two occurrences however there were no clearincreases in calcaemia during the first years and hypercalcemia was markedly aggravated by stopping denosumab The link between hyperparathyroidism anddenosumab was the subject of a recent publication []However the hyperparathyroidism described occurredrapidly after a single injection of denosumab with a fold increase in PTH levels at week normalization at months and normal calcaemia throughout [] This caseresolved within a year In our case calcaemia was highafter years of treatment No PTH determinations werecarried out in parallel during this period It is therefore difficult to determine the date of onset of the hyperparathyroidism as the denosumab treatment may have masked thehypercalcemia Similarly as our patient underwent surgery it is impossible to know what spontaneous course itwould have takenTwo similar cases of hypercalcemia during the rebound effect after stopping treatment with denosumabhave been reported one with low PTH levels and aspontaneously favorable outcome over several months[] and the other after a high dosage of denosumab mg quarterly [] The excessive bone remodelingobserved in the absence of associated fractures may havebeen due to major bone reabsorption potentially accounting for the hypercalcemia as in immobilizationrelated osteoporosis The high hypercalcemia observed isconsistent with this hypothesis Alendronate was administered and the patients plasma calcium concentrationsreturned to normal values within months In contrastour patient had a PTH concentration that was well 0cMaugars Journal of Medical Case Reports Page of nothighandanddisplayedcontrolledrapidnormalization within hours of calcaemia after theintervention Normal phosphate midupper calcaemiaand normal creatinine were not in favor of tertiaryhyperparathyroidism However the hyperparathyroidismdescribed occurred rapidly after a single injection ofdenosumab with a 22fold increase in intact PTHlevels at weeks and normal calcaemiaiPTHthroughout in a case report [] This dramatic increasein iPTH resolved spontaneously within a year Given thesurgical intervention carried out on our patient we cannot determine what the natural course of this hypercalcemia might have been in the same way that half thecases of hyperparathyroidism in kidney transplant recipients resolve within year for example [] The surgerywas carried out because we were concerned that our patient might be suffering from a parathyroid carcinomaThis rebound effect has been reported after stoppingdenosumab administered at an oncological dosage mg monthly for months [] Seven nonmalignantvertebral crushes were observed months after the lastinjection of denosumab At lower dosages denosumab mg halfyearly for years patients with breastcancer receiving aromatase inhibitors developed vertebral crushes after stopping denosumab [] The riskof vertebral fracture was higher if the treatments werelonger and if the patients had preexisting osteoporosistumorssometimesEight other cases of hypercalcemia have been reportedin children Denosumab was administered in these casesfor giantcellfibrous dysplasia brittle bonedisease and juvenile Pagets disease [] The hypercalcemia occurred early to months after the denosumab injection and wassevere plasmacalcium concentrations of up to mmoll but it occurred in a context of high doses and bone remodelingnot comparable with the contextin adults and therebound effect The hypercalcemia regressed over a fewmonths either spontaneously or on zoledronate Reactional bone hyperreabsorption was again suggestedThis hyperreabsorption seems to be related to therelease of receptor activator of nuclear factor kappaBligand RANKL with high crosslinked carboxyterminaltelopeptide of type collagen CTX1 and low Dickkopf1DKK1 and sclerostin [] Osteocytes are known to be theprincipal source of RANKL [] We have suggested thatosteocytes may undergo apoptosis during this reboundeffect when the treatment with denosumab is stoppedpotentially accounting for the necrosis and strong hyperreabsorption reported in certain patients presenting thisrebound effect []The discovery of a thyroid papillary microcarcinomaappears to have been fortuitous in this case Other studies have shown that denosumab may not only decreasethe frequency of bone tumor events but even have adirect or indirect antitumoral effect [] However ametaanalysis comparing denosumab and zoledronateand including four randomized trials patientsfound a significantly higher risk of primary neoplasiawith a cumulative annual incidence of on denosumab versus on zoledronate [] No particular cancer type profile was identified but the number of caseswas small n The cumulative doses in our patientamounted to approximately g of denosumab corresponding to months at the dose of mgmonth prescribed to prevent secondary bone tumor complicationsIt is not possible to draw any firm conclusions concerningour case as goiter is itself a risk factor for thyroid cancerwith a poorly defined incidence of between and forcancer in patients undergoing surgery for goiter []In conclusionit is important to be aware of thisrebound effect with strong bone hyperreabsorption incertain patients after stopping treatment with denosumab Several explorations to eliminate a neoplasm couldbe avoided with knowledge of these cases It can takeseveral different forms a simple loss of the BMD gainedon the treatment vertebral fractures or transient hypercalcemia which in this context may raise concerns of orsimulate malignant bone diseases or hyperparathyroidism as in our case The rebound after stopping treatment with denosumab makes it necessary to checkcalcaemia and CTX early before the risk of further vertebral fractures DXA will be carried out at the end ofthe denosumab sequence but an early new comparativeDXA would be less sensitive than CTX dosage We canpropose CTX and calcaemia months after the last injection of denosumab as a reference and then or months later A marked increase will require preventionThe most appropriate therapeutic approach remains unclear The effects of gradually decreasing the dose ofdenosumab have not yet been reported Bisphosphonatesseem to be only partially effective according to publishedpreliminary results []AbbreviationsBMD Bone mineral density CRP Creactive protein CT Computedtomography CTX Carboxyterminal collagen crosslink CTX1 Crosslinkedcarboxyterminal telopeptide of type collagen DKK1 Dickkopf1 DXA DualXray absorptiometry iPTH Intact parathyroid hormone L1 First lumbarvertebra L2 Second lumbar vertebra L3 Third lumbar vertebraMRI Magnetic resonance imaging PTH Parathyroid hormonePTHrp Parathyroid hormonerelated peptide RANKL Receptor activator ofnuclear factor kappaB ligand SD Standard deviation T4 Fourth thoracicvertebra T5 Fifth thoracic vertebra T8 Eighth thoracic vertebra T9 Ninththoracic vertebra T10 Tenth thoracic vertebra T11 11th thoracic vertebraTSH Thyroidstimulating hormone MIBI MethoxyisobutylisonitrileAcknowledgementsNAAuthors contributionsAll authors contributed to the work contribution to the observation YMCDL the discussion JMB BLG PG JG and writing the manuscript YMCDL All authors read and approved the final manuscript 0cMaugars Journal of Medical Case Reports Page of function Curr Drug Saf httpsdoi102174 Maugars Y Bart G Guillot P Multiple vertebral osteonecrosesKümmell's Disease after years on denosumab is osteocyte apoptosis toblame Calcif Tissue Int Chen F Pu F Safety of denosumab versus zoledronic acid in patients withbone metastases a metaanalysis of randomized controlled trials Oncol ResTreat Papapoulos S Lippuner K Roux C The effect of or years ofdenosumab treatment in postmenopausal women with osteoporosisresults from the FREEDOM Extension study Osteoporos Int Nakamura Y Kamimura M Ikegami S Changes in serum vitamin D andPTH values using denosumab with or without bisphosphonate pretreatment in osteoporotic patients a shortterm study BMC Endocr DisordTorres A Lorenzo V Salido E Calcium metabolism and skeletal problemsafter transplantation J Am Soc Nephrol GonzalezRodriguez E AubryRozier B Stoll D Zaman K Lamy O Sixtyspontaneous vertebral fractures after denosumab discontinuation in women with earlystage breast cancer under aromatase inhibitorsBreast Cancer Res Treat httpsdoi101007s10549019054588Tyan A Patel SP Block S Hughes T McCowen KC Rebound VertebralFractures in a Patient with Lung Cancer After OncologyDose DenosumabDiscontinuation A Cautionary Tale Mayo Clin Proc Innov Qual OutcomesFassio A Adami G Benini C Vantaggiato E Saag KG Giollo A Lippolis IViapiana O Idolazzi L Orsolini G Rossini M Gatti D Changes in Dkk1sclerostin and RANKL serum levels following discontinuation of longtermdenosumab treatment in postmenopausal women Bone Bonewald LF The amazing osteocyte J Bone Miner Res de Groot AF AppelmanDijkstra NM van der Burg SH Kroep JR The antitumor effect of RANKL inhibition in malignant solid tumors A systematicreview Cancer Treat Rev Sahbaz NA Tutal F Aksakal N Cancer frequency in retrosternal goiterAm Surg Lamy O Stoll D AubryRozier B Rodriguez EG Stopping Denosumab CurrOsteoporos Rep Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsFundingNone for this workAvailability of data and materialsAll original data are available corresponding authorEthics approval and consent to participateInclusion in the FREEDOM protocol signedConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsWe do not have any competing interestsReceived July Accepted May ReferencesAnastasilakis AD Polyzos SA Makras P AubryRozier B Kaouri S Lamy OClinical features of patients with reboundassociated vertebral fracturesafter denosumab discontinuation systematic review and additional cases JBone Miner Res Dupont J Laurent MR Dedeyne L Luyten FP Gielen E Dejaeger MReboundassociated vertebral fractures after stopping denosumab Reportof four cases Joint Bone Spine httpsdoi101016jjbspin201907010Fernández Fernández E Benavent Núñez D Bonilla Hernán G Monjo HenryI GarcÃa Carazo S Bernad Pineda M Balsa Criado A Aguado AP MultipleVertebral Fractures Following Discontinuation of Denosumab TreatmentTen Clinical Cases Report Reumatol Clin httpsdoi101016jreuma201811002 [Epub ahead of print]Florez H RamÃrez J Monegal A Guañabens N Peris P Spontaneousvertebral fractures after denosumab discontinuation A case collection andreview of the literature Semin Arthritis Rheum Popp AW Varathan N Buffat H Senn C Perrelet R Lippuner K Bone mineraldensity changes after year of denosumab discontinuation inpostmenopausal women with longterm denosumab treatment forosteoporosis Calcif Tissue Int TriptoShkolnik L Rouach V Marcus Y RotmanPikielny P Benbassat CVered I Vertebral fractures following denosumab discontinuation inpatients with prolonged exposure to bisphosphonates Calcif Tissue IntKoldkjær Sølling AS Harsløf T Kaal A Rejnmark L Langdahl BHypercalcemia after discontinuation of longterm denosumab treatmentOsteoporos Int Kurucu N Akyuz C Ergen FB Denosumab treatment in aneurysmalbone cyst Evaluation of nine cases Pediatr Blood Cancer httpsdoi101002pbc26926 Epub Dec PubMed PMID Uday S Gaston CL Rogers L Osteonecrosis of the jaw and reboundhypercalcemia in young people treated with denosumab for giant celltumor of bone J Clin Endocrinol Metab Setsu N Kobayashi E Asano N Severe hypercalcemia followingdenosumab treatment in a juvenile patient J Bone Miner Metab Gossai N Hilgers MV Polgreen LE Greengard EG Critical hypercalcemiafollowing discontinuation of denosumab therapy for metastatic giant celltumor of bone Pediatr Blood Cancer Boyce AM Chong WH Yao J Denosumab treatment for fibrousdysplasia J Bone Miner Res Trejo P Rauch F Ward L Hypercalcemia and hypercalciuria duringdenosumab treatment in children with osteogenesis imperfecta type VI JMusculoskelet Neuronal Interact Roux S Massicotte MH Huot Daneault A BrazeauLamontagne L DufresneJ Acute hypercalcemia and excessive bone resorption following antiRANKLwithdrawal Case report and brief literature review Bone Mazokopakis EE Denosumabinduced normocalcemic hyperparathyroidismin in a woman with postmenopausal osteoporosis and normal renal 0c" | Thyroid_Cancer |
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