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"Lactation has a negative effect on female sexual function Hormonal changes during lactation causechanges which might lead to dyspareunia lack of libido and anasmia There are various pharmacological andnonpharmacological approaches to treat sexual dysfunction While pharmacological treatment has multipleunwanted side effects nonpharmacological therapies such as complementary medicine are a potential saferalternative The aim of this study is to evaluate the effect of ear acupressure on sexual function of lactating womenMethodsdesign This is a randomized clinical trial with a parallel sham control group In this study lactatingwomen between months and year after childbirth were referred to health care centers in Qazvin City andwould be invited to participate Participants will be divided into intervention n and control n groupsusing simple block randomization Both intervention and sham control groups will be visited over sessionswithin a 4day interval At each visit the adhesives containing Vaccaria seed will be adhered for the interventiongroup while nonlatexbased adhesives with no Vaccaria seeds will be placed on the same ear acupoints for thesham control group Selected ear acupoints include genitalia two ear points pelvic point master shoulder andposterior pituitary gland The women will be asked to hold the seeds on their ears for days and press each earpoint three times a day for s After days they will be asked to remove the seeds from their ears and rest for day Sexual function as primary outcome in both groups will be assessed using the Female Sexual Function Indexbefore and immediately after and months after the intervention Also Sexual Quality of Life as secondaryoutcome will be assessed using Sexual Quality of LifeFemale SQOLF before and months after intervention Datawill be analyzed using repeated measure ANOVA at the significant level of Discussion This study is expected to support the impact of ear channel ear acupressure on sexual function inlactating womenTrial registration Iranian Clinical Trial Registration Center IRCT20190626044028N1 Registered on August Keywords Ear acupressure Sexual function Lactation Correspondence nbahramiqumsacir2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin IranFull 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 0cBarghamadi Trials Page of BackgroundThe postchildbearing period is a crucial stage in thewomans life that involves physical hormonal mentalsocial and cultural changes [] Fatigue insomnia somestressors such as child care and changes in the bodyimage along with hormonal changes can reduce interestin sexual life and decrease the number of sexual intercourses during this period [] Endocrine function of lactation has a negative effect on sexual function becauseprolactin decreases sexual hormones such as androgenand estrogen This mechanism reduces sexual functionbecause of vaginal dryness vaginal epithelium atrophyand dyspareunia [] About two thirds of women duringlactating period experience at least one sexual problemsuch as decreased libido lack of sexual pleasure dyspareunia and vaginal dryness which usually are resolvedwithin year after childbirth [ ] The prevalence ofsexual dysfunction increases from to in the prepartum period [] to to in the postpartum period[] The results of various Iranian studies confirmed thehigh prevalence to of sexual dysfunctionduring lactation [ ] Therefore sexual dysfunction is acommon problem during lactation that needs moreattention from health care providersIn the postchildbearing period most women try to return to their sexual life within to weeks after childbirth Howeverit may take up to year to resumesexual relationships with the same quality of beforepregnancy [] Psychotherapy and pharmacotherapy aretwo main methods for treating female sexual dysfunctionFSD [] However the US Food and Drug Administration FDA does not recommend any foods or medicines for the treatment of FSD [] Therefore nonpharmacological therapies such as complementary medicine are a potentially safer alternative to pharmacologicaltherapy Acupressure is one of the noninvasive complementary methods oftraditional Chinese medicineTCM based on the principles of acupuncture []Acupuncture stimulates medical points and meridiansthat are distributed throughout the body to regulatephysiologic reactions [] Acupressurelike acupuncture modulates a persons vital energy by stimulatingacupoints and meridians that are distributed throughoutthe body []The external ear is one of the several somatotopicmicrosystems that can be used in acupressure Ear acupressure also known as auriculotherapy or auricularacupressure [ ] was first presented in ancient Chinese medicine to years BC but Dr Paul Nogierwas the first Western physician who put forward auriculotherapy in a scientific way [ ] Accordingto this theory each part of the body is associated with aspecific part of the ear that reflects the physiological orpathological state of the body [] In this method theouter surface of the ear auricle can be stimulated to reduce pathological conditions in other parts of the body[] Ear stimulation can be performed in various wayssuch as manual finger pressure electrical stimulation lasers differenttypes of needles magnetic seeds andseeds to strengthen neural connections [] The WorldHealth anization WHO has recognized ear acupuncture as a promising therapeutic approach becauseof its efficacy in managing health disorders [] Ear acupuncture has been recognized as a form of acupuncturethat can affect all body ans [] Ear acupressure isan easy noninvasive and safe therapy [ ] It is easyto use for mothers who need to take care of their childin the postchildbearing period []There are several studies supporting the effects of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] However the effect of ear acupressure on sexualfunction has not been studiedResearch aimThis study is designed to investigate the effect of earacupressure on the sexual function of lactating womenMethodsDesign and settingThis is a randomized clinical trial with a parallel shamcontrolled group The study is designed in accordancewith the CONSORT standards Lactating women referred to health care centers in Qazvin City Iran will beinvited to participate Figure shows the CONSORTflow diagram The current protocol is anized basedon the SPIRITTCM extension []ParticipantsIn this study breastfeeding women between monthsand year after childbirth will be referred to health carecenters in Qazvin City Iran and they will be invited toparticipate in the study Inclusion criteria will be willingness to participate in the study being literate beingprimiparous breastfeeding fullterm singleton deliverylesionfree auricle no ulcer and pain in the ear and ability to present for all intervention sessions Exclusion criteria will be being away from their spouse for more than month having complications during pregnancy orhaving postpartum depression in the postchildbearingperiod These criteria were diagnosed using the Edinburgh Postpartum Depression Scale substance abuse inthe individual or the spouse and history of illnessesaffecting sexual function in a woman or her spouse egpremature ejaculation cardiovascular mental health disorders thyroid diseases any form of cancer or injuries 0cBarghamadi Trials Page of Fig The CONSORT flow diagramof the genital area The use of drugs affecting sexualfunction such as psychotropic cardiovascular neurological and hormonal drugs will also be excluded Afterthe initial screening the Demographic and ObstetricQuestionnaire and Female Sexual Function Index FSFIwill be completed by the participants before anyinterventionRole and qualification of practitionersThe research team consists of one specialist in auriculotherapy TO two specialists in reproductive health ZAand NB and one midwifery postgraduate student Theprotocol of intervention was designed based on the literature review and expert opinion of TO who is an expert inauriculotherapy and is a journal editor for several international journals Other team members have been qualified in this technique before designing present researchParticipant screening and care providing will be done bySB and ZA and NB will monitor her during the first sessions to ensure the fidelity of providing interventionSample size calculationSample size is estimated according to the study ofBokaie with the mean and standard deviation of 0cBarghamadi Trials Page of total sexual function score has been reported as ± and ± in the intervention telephonecounseling group and control groups respectively []type error α Therefore considering the first confidence the second type error β power and error d and the possibility of loss tofollowup individuals for each group are requiredSo the total sample size will be peopleInterventionEar acupressure groupThe researcher will clean the auricle using alcoholand then place Vaccaria seeds using nonlatexbased adhesives on ear acupoints for genitalia pelvis shoulderand the posterior pituitary gland Figure illustrates theintended ear acupoints Intervention is designed for sessions within a 4day interval During these sessionsthe seeds are placed on the designated ear acupressurepoints The women will be instructed to keep the seedson their ears for days during which each point shouldbe compressed three times a day for s The compression should be performed with moderate stimulationthrough pressing steadily and slightly tighter until feelinga slight tingling and discomfort After days they willbe asked to remove the seeds from their ears and restfor a day [] The women will be reminded daily bysending the text and will be reminded by phone for thenext interventional session This procedure will be performed for ten sessions for each participant It should benoted that if a participant has problems during the useof the method for any reason displacement of seedsdiscomfort etc she can remove the previous seed andask the researcher to reattach new seedsSham control groupThe control group will have sessions that will be thesame as the intervention group except that no Vaccariaseeds will be placed on the ear acupointsMeasuresIn this studycollectionfour measures will be used for dataDemographic and Obstetric QuestionnaireThe demographic section includes questions such asage education level occupation place of residence economic status age at marriage and length of marriageThe obstetric section includes questions regarding thenumber of pregnancies type of contraceptive methoddelivery date type of delivery perinealinstrumental delivery history of painful intercourse in prenatalperiod infant gender birth weight first lactation number of intercourse per month before pregnancy onset offirst intercourse after delivery and average number ofintercourses per week during lactation Validity of thisquestionnaire will be confirmed by some of the facultymembers ofthe midwifery department of QazvinUniversity of Medical Sciences IraninjuryFig The selected ear acupoints 0cBarghamadi Trials Page of Female Sexual Function Index FSFIIt has been designed by Rosen to evaluate sexualfunction in women over the past weeks [] It consistsof items subdivided into six subcategories of sex desire items arousal items lubrication items asm items satisfaction items and pain itemsThese subcategories have response ranges from or to with higher scores indicating better sexual performance [] The questionnaire has been used in manystudies abroad and has shown to have a high degree ofinternal consistency reliability and validity [] Thefindings of Fakhri and Mohammadis study showed thatthe Farsi version of FSFI FSFIIV is a reliable and validinstrument with appropriate psychometric properties toevaluate womens sexual function [ ] Reliability ofthe scale has been calculated through stability analysisor calculation of the internal consistency coefficient TheCronbachs alpha coefficient was and was higher forall domains and for the whole scale [] In addition reliability was confirmed by the testretest method and thecorrelation coefficient was reported high indicating thatFSFIIV is reusable within a 4week interval [ ]The maximum score for each domain is and for thewhole scale is Also any score less than will beused to indicate sexual dysfunction []The Edinburgh Postpartum Depression Rating ScaleThis 10item scale has been designed to detect depression from weeks after delivery The Edinburgh Scalescores vary between and with cutoff point andabove to detect postpartum depression [] Psychometric evaluation of the Farsi version of this scale is carriedout in [] The Cronbachs alpha for the Edinburgh Scale has been reported as Validity of the Edinburgh Beck Scale has been reported as Thefindings of the study confirmed the high validity of theFarsi version of the Edinburgh Scale for the diagnosis ofpostpartum depression []The Sexual Quality of LifeFemale SQOLFIt is a short tool that specifically assesses the relationshipbetween sexual function and womens quality of life Ithas been developed by Symonds [] This 18itemquestionnaire focuses on sexual selfesteem emotionalaffairs and relationships Each item is responded with aLikert scale of strongly agree score to strongly disagree score Higher scores indicate better quality ofsexual life for women [] Validity and reliability of theFarsi version ofthis scale have been confirmed byMaasoumi []Primary outcomePrimary outcome of this study is to investigate changesof sexual function using the FSFISecondary outcomeThe secondary endpoint is investigating the quality ofwomens sexuallife which will be assessed using theSexual Quality of Life Questionnaire Any harm or sideeffects will be also reportedStudy procedureAfter obtaining the necessary permissions from the Ethics Committee of Qazvin University of Medical Sciencesregistration at the Iranian Clinical Trial RegistrationCenter IRCT and obtaining the permission from Qazvin Nursing and Midwifery School the researcher willattend for recruitment in Qazvin Health Centers Thelactating women of each center will be invited for participation The process and purpose of the research willbe explained to those who meet the inclusion criteriaAlso they will be ensured that their information will bekept confidential and that the questionnaires will be anonymous and coded in accordance with the researchethics They will enter into the study after signing thewritten consent form The researcher will ensure thatthey can leave the study at any time After enrolling eligible individuals a simple block randomization will beused Participants will be randomly assigned to the intervention and control groupsThe questionnaires will be filled out by the participants before the intervention Then the interventionacupressure will be performed as mentioned aboveEvaluation of sexual function in both the interventionand control groups will be performed using the FSFIquestionnaire immediately and months after theintervention Figure provides the timeline of recruitment allocation and assessmentStatistical analysisData will be analyzed using the chisquare test Fisherexact test and independent t test via the SPSS v24 software The KolmogorovSmirnov and Shapiro tests willalso be used to check for the normal distribution of sexual function scores Descriptive statistics number meanand standard deviation will be used to describe thedemographic characteristics of the participants The repeated measure ANOVA will be used to compare themean sexual function of women before and after theintervention The significance level of all tests is set asp Methods to protect against biasRandomization and allocationSampling will be made using a twostep samplingmethod In the first step Qazvin City will be consideredas five geographical districts Next two health centerswill be randomly selected in each of these five districtsof Qazvin City Secondly women referred to selected 0cBarghamadi Trials Page of STUDY PERIODEnrolment AllocationPostallocationCloseoutTIMEPOINT1t1t2t3t4t5t6t7t8t9t10t11t12t13tXENROLLMENTEligibility screenInformed consentRandomized by independent personAllocationINTERVENTIONSGroup A Auricular acupressureGroup B ControlASSESSMENTSFSFISQOLXXXXXXX X XXFig Schedule of enrolment interventions and assessmentshealth centers will be assessed for eligibility and in caseof willingness to participate in the study will be recruited They will be randomly assigned to two groupsas follows One letter is assigned to each group A intervention group B control group and all possible conditions for the 4block will be written and numbered asfollows 1AABB 2ABAB 3BBAA 4BABA 5ABBA6BAAB In a simple block randomization method using thetable of random numbers numbers of blocks will be selected until the specified sample size is achieved Therandom allocation sequence is specified For example ifthe numbers given are and respectively theallocation sequence will be as follows AABB ABABABAB BBAA As a result each participant will have aunique code n Allocation concealmentFor this purpose after preparing the allocation sequencethe sequence is annotated on paper and placed inopaque sealed envelopes which will be numbered consecutively The questionnaires will be coded in the samemanner A questionnaire with the same code will becompleted by the person who receives code intervention The assignment sequence and its concealment willbe performed by someone outside the research teamBlindingParticipants outcome assessor and statistical analyzerwill be blind to which group a participant was placedThe individual entering the data from the participantquestionnaires on demographic information postpartumdepression female sexual functioning and female qualityof life will be blind to which participant was assigned tothe auricular acupressure seed group or the sham control groupTreatment fidelityOne of the researchers SB is responsible to performintervention She has participated specific course on auriculotherapy and is supervised by acupuncturist MHAabout how to perform the intervention Approximately of intervention sessions will be run under supervision of the acupuncturist to ensure that all acupoints arechosen correctlyData managementOne of the researchers SB will be responsible for collecting data at each study stage and will assign anotherindividual for the task of data entry into the SPSS software This process will be performed under supervisionof NB and ZA 0cBarghamadi Trials Page of Ethical and safety issuesThe research protocol will be reviewed and approved bythe Ethics and Human Research Committee of QazvinUniversity of Medical Sciences decree code IRQUMSREC1398056 It is registered on the Iranian ClinicalTrial Registration Center underofIRCT20190626044028N1 In addition the following ethical considerations will be considered throughout this research obtaining written informed consent the volunteernature of participation in the study ability to withdrawfrom the study at any time and confidentiality of information even during the publication of findings All ethical issues related to clinical trials will be considered Informedconsent during recruitment phase will be obtained by SBdecreecodeDiscussionTo the best of our knowledge this study is the first randomized clinical trial investigating the effect of ear acupressure on the sexual function of lactating women Thestrength of this study is the randomized design with parallel treatment procedures and a large sample size butdue to the importance of the treatment practitionerknowing whether they are placing an adhesive patchwith a Vaccaria seed or without one a true double blindstudy is not possible Acupressure has no known side effects is noninvasive and is easy to use [ ] Therefore the therapist and clients can develop a sense ofclosenesstrust and confidence [] Several studieshave shown promising results about the effect of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] In the study of Oakley acupuncture wasaffective in premenopausal women with sexual function[] In line with previous literature this study wouldprovide insights about the impact of ear acupressure onsexual function in lactating womenAbbreviationsCONSORT Consolidated Standards of Reporting Trials SQOLF Sexual Qualityof LifeFemaleAcknowledgementsNot any in this stageTrial statusThe recruitment has not yet begun and necessary permissions are acquiredthe estimated date of recruitment is November The expected timefor completing the recruitment is March Protocol version registered on August Authors contributionsAll authors SB ZA NB and TO contributed to the design of the study SBNB and ZA drafted the preliminary manuscript TO revised the manuscriptand prepared the final version of the manuscript All authors revised themanuscript agreed to be fully accountable for ensuring the integrity andaccuracy of the study and read and approved the final version of themanuscript to be published All the authors met the criteria for authorshipand listed as coauthors on the title pageFundingThe research deputy of Qazvin University of Medical Sciences will providefinancial support Study funders have no role in the study design thecollection management analysis and interpretation of data the writing ofthe report and the decision to submit the report for publicationAvailability of data and materialsAnalyzed data and materials will be deidentified and publishedEthics approval and consent to participateResearch protocol is approved by the Ethics and Human ResearchCommittee of Qazvin University of Medical Sciences decree codeIRQUMSREC1398056 Permissions from responsible authority will beobtained before recruitment Informed consent will be acquired beforeparticipationConsent for publicationNot applicableCompeting interestsNone to declareAuthor details1Student Research Committee Qazvin University of Medical Sciences QazvinIran 2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin Iran 3Emperors College of TraditionalOriental Medicine Santa Monica CA USAReceived November Accepted August ReferencesAbdelhakm EM Said AR Elsayed DMS Effect of PLISSIT model sexualcounseling program on sexual quality of life for postpartum women Am JNurs Sci Avery MD Duckett L Frantzich CR The experience of sexuality duringbreastfeeding among primiparous women J Midwifery Womens HealthAcele Eà Karaçam Z Sexual problems in women during the firstpostpartum year and related conditions J Clin Nurs Mohammed YF Hassan HM AlDinary AM Rashed NS Sexual function afterchild birth according to the mode of delivery AAMJ Si H Kumamoto Y Sato Y Masumori N Horita H Kato R Prevalenceof female sexual dysfunction symptoms and its relationship to quality of lifea Japanese female cohort study Urology Williams A HerronMarx S Carolyn H The prevalence of enduring postnatalperineal morbidity and its relationship to perineal trauma Midwifery Ahmad Shirvani M Bagheri NM Sexual dysfunction and related factorsamong breast feeding women Iran J Obstet Gynecol Infertility Tork Zahrani S Banaei M Ozgoli G Azad M Investigation of the postpartumfemale sexual dysfunction in breastfeeding women referring to healthcarecenters of Bandar Abbas Iran J Obstet Gynecol Infertility Yörük F Karaçam Z The effectiveness of the PLISSIT model in solvingpostpartum sexual problems experienced by women Athens J Health Davis SR Van Der Mooren M van Lunsen RH Lopes P Ribot J Rees M et alEfficacy and safety of a testosterone patch for the treatment of hypoactivesexual desire disorder in surgically menopausal women a randomizedplacebocontrolled trial Menopause Belkin ZR Krapf JM Goldstein AT Drugs in early clinical development forthe treatment of female sexual dysfunction Expert Opin Investig Drugs Ozgoli G Mobarakabadi SS Heshmat R Majd HA Sheikhan Z Effect of LI4and BL32 acupressure on labor pain and delivery outcome in the first stage 0cBarghamadi Trials Page of of labor in primiparous women a randomized controlled trial ComplementTher Med Oleson T Auriculotherapy manual Chinese and Western systems of ear Mohammadi KH Heydari M Faghihzadeh S The Female Sexual FunctionIndex FSFI validation of the Iranian version Health Monit J Iran Inst HealthSci Res Burri A Cherkas L Spector T Replication of psychometric properties of theFSFI and validation of a modified version FSFILL assessing lifelong sexualfunction in an unselected sample of females J Sex Med Sidi H Abdullah N Puteh SEW Midin M The female sexual function indexFSFI validation of the Malay version J Sex Med Sun X Li C Jin L Fan Y Wang D Development and validation of Chineseversion of female sexual function index in a Chinese populationa pilotstudy J Sex Med Ter Kuile MM Brauer M Laan E The female sexual function index FSFI andthe female sexual distress scale FSDS psychometric properties within aDutch population J Sex Marital Ther Cox JL Holden JM Sagovsky R Detection of postnatal depressiondevelopment of the 10item Edinburgh Postnatal Depression Scale Br JPsychiatry Ahmadi kani Golzar A GoliZadeh Z Validation of Edinburgh PostpartumDepression Scale EPDS for screening postpartum depression in Iran J NursEduc Symonds T Boolell M Quirk F Development of a questionnaire on sexualquality of life in women J Sex Marital Ther Maasoumi R Lamyian M Montazeri A Azin SA AguilarVafaie ME HajizadehE The sexual quality of lifefemale SQOLF questionnaire translation andpsychometric properties of the Iranian version Reprod Health Oakley SH WaltherLiu J Crisp C Pauls R Acupuncture in premenopausalwomen with hypoactive sexual desire disorder a prospective cohort pilotstudy Sexual medicine 201643e176e81Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsacupuncture Yeh ML Chang YC Huang YY Lee TY A randomized controlled trial ofauricular acupressure in heart rate variability and quality of life forhypertension Complement Ther Med Tan JY Molassiotis A Wang T Suen LK Current evidence on auriculartherapy for chemotherapyinduced nausea and vomiting in cancer patientsa systematic review of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chien LC Chiang YC Lin SW Huang CK Ren D Reduction innausea and vomiting in children undergoing cancer chemotherapy byeither appropriate or sham auricular acupuncture points with standard careJ Altern Complement Med Abbate S Chinese auricular acupuncture Florida Routledge Yeh TL Chen HH Pai TP Liu SJ Wu SL Sun FJ The effect ofauricular acupoint stimulation in overweight and obese adults a systematicreview and metaanalysis of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chiang YC Hoffman SL Liang Z Klem ML Tam WW Efficacyof auricular therapy for pain management a systematic review and metaanalysis Evid Based Complement Alternat Med Kim JY Ryu HS Nam SH Park KS Effects of auricular acupressure therapy onnocturia and insomnia in the elderly Korean J Rehabil Nurs Wang YJ Hsu CC Yeh ML Lin JG Auricular acupressure to improvemenstrual pain and menstrual distress and heart rate variability for primarydysmenorrhea in youth with stress Evid Based Complement Alternat MedShergis JL Ni X Jackson ML Zhang AL Guo X Li Y A systematicreview of acupuncture for sleep quality in people with insomniaComplement Ther Med Dantas KKdL Auriculoterapia chinesa com o uso de sementes de colza nadismenorreia primaria relato de caso Universidade Federal do Rio Grandedo Norte Portman DJ Bachmann GA Simon JA Group OS Ospemifene a novelselective estrogen receptor modulator for treating dyspareunia associatedwith postmenopausal vulvar and vaginal atrophy Menopause Oleson T Flocco W Randomized controlled study of premenstrualsymptoms treated with ear hand and foot reflexology Obstet GynecolKwon SJ Park JS Effects of auricular acupressure on chemotherapyinducednausea vomiting and serum serotonin level Korean J Adult Nurs Di YM May BH Zhang AL Zhou IW Worsnop C Xue CC A metaanalysis ofearacupuncture earacupressure and auriculotherapy for cigarette smokingcessation Drug Alcohol Depend Huang ETY Di PhD YM Acupuncture therapies for chronic obstructivepulmonary disease a systematic review of randomized controlled trialsAltern Ther Health Med Dai L Cheng CW Tian R Zhong LL Li YP Lyu AP Standard ProtocolItems for Clinical Trials with Traditional Chinese Medicine recommendations explanation and elaboration SPIRITTCM extension Chin J Integr Med Bokaie M Hajimaghsoudi S Dehghani A Hosseini F The effect of sexualhealth counselling on the sexual function and satisfaction of breastfeedingwomen in the form of group consultation and telephone consultancyJ Adv Pharm Educ Res 20199S2191 Rosen CB Heiman J Leiblum S Meston C Shabsigh R Ferguson DD'Agostino R The Female Sexual Function Index FSFI a multidimensionalselfreport instrument for the assessment of female sexual function J SexMarital Ther Fakhri A Pakpour AH Burri A Morshedi H Zeidi IM The Female SexualFunction Index translation and validation of an Iranian version J Sex Med Wiegel M Meston C Rosen R The female sexual function index FSFI crossvalidation and development of clinical cutoff scores J Sex Marital Ther 0c" | 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 |
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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doi101007s0040 McInnes MDF Moher D Thombs BD et a0al Preferred reporting items for a systematic review and metaanalysis of diagnostic test accuracy studies the PRISMADTA statement JAMA https doi101001jama201719163 Thompson M Tiwari A Fu R Moe E Buckley DI A Framework To Facilitate the Use of Systematic Reviews and MetaAnalyses in the Design of Primary Research Studies Rockville Agency for Healthcare Research and Quality US Vaarala MH Porvari KS Kellokumpu S Kyll¶nen AP Vihko PT Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues J Pathol https doi10100210969896200099999999 Hamming I Timens W Bulthuis ML Lely AT Navis G van Goor H Tissue distribution of ACE2 protein the functional receptor for SARS coronavirus A first step in understanding SARS pathogenesis J Pathol https doi101002path1570 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 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Chen Q High expression of ACE2 receptor of 2019nCoV on the epithelial cells of oral mucosa Int J Oral Sci https doi101038s4136 80200074x Xu J Li Y Gan F Du Y Yao Y Salivary glands potential reservoirs for COVID19 asymptomatic infection J Dent Res https doi10117700220 Wu C Zheng S Chen Y Zheng M Singlecell RNA expression profiling of ACE2 the putative receptor of Wuhan 2019nCoV in the nasal tissue https doi1011012020021120022 0c Head and Neck Pathology Ziegler CGK Allon SJ Nyquist SK et a0al SARSCoV2 receptor ACE2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues Cell 2020S0092 https doi101016jcell202004035 Kaye R Chang CWD Kazahaya K Brereton J Denneny JC III COVID19 anosmia reporting tool initial findings Otolaryngol Head Neck Surg https doi10117701945 Lechien JR ChiesaEstomba CM Hans S Barillari MR Jouffe L Saussez S Loss of smell and taste in European patients with mild to moderate COVID19 Ann Intern Med https 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"radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Nathalie Chaput Gras2 Emilie Lanoy3 Alicia Larive3 Celine Boutros Christine Mateus4 Emilie Routier4 Roger Sun5 Yun Gan Tao5 Christophe Massard6 Rastilav Bahleda6 Dominique Schwob3 Nathalie Ibrahim7 Rita Maria Khoury Abboud7 Caroline Caramella8 Andrea Lancia Lydie Cassard2 Severine Roy4 J C Soria10 Caroline Robert11 Eric Deutsch12 To cite Boutros a0C Chaput Gras a0N Lanoy a0E et a0al Dose escalation phase study of radiotherapy in combination with anti cytotoxic T lymphocyte associated antigen monoclonal antibody ipilimumab in patients with metastatic melanoma Journal for ImmunoTherapy of Cancer 20208e000627 101136jitc2020000627 º Additional material is published online only To view please visit the journal online http dx jitc CR and ED contributed equallyAccepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJFor numbered affiliations see end of Correspondence toDr Eric Deutsch eric deutsch gustaveroussy frBackground A synergy between radiotherapy and anti cytotoxic T lymphocyte associated antigen anti CTLA4 monoclonal antibody has been demonstrated preclinically The Mel Ipi Rx phase study aimed to determine the maximum tolerated dose MTD and safety profile of radiotherapy combined with ipilimumab in patients with metastatic melanomaPatients and methods A dose escalation design was used with and Gy dose of radiotherapy at week combined with mgkg ipilimumab every weeks for four doses Patients with evidence of clinical benefit at week were eligible for maintenance with ipilimumab mgkg every weeks starting at week until severe toxicity or disease progression The database lock occurred on April Tumor growth rate of irradiated lesions and non irradiated lesions were analyzed to assess the systemic immunologic antitumor response Blood immune monitoring was performed before and during treatment to determine if radiotherapy could modify ipilimumab pharmacodynamicsResults patients received ipilimumab between August and July Nine patients received the four doses of ipilimumab All patients received the combined radiotherapy Grade adverse events occurred in nine patients the most common being colitis and hepatitis No drug related death occurred Dose limiting toxicity occurred in two of six patients in the cohort receiving Gy The MTD was Gy Two patients had complete response three had partial response response and seven had stable disease giving an objective response rate of and a clinical benefit rate of at week The median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years The median progression free survival PFS CI was Radiotherapy combined with ipilimumab was associated with increased CD4 and CD8ICOS T cells Increased CD8 was significantly associated with PFSConclusion When combined with ipilimumab at mgkg the MTD of radiotherapy was Gy This combination of ipilimumab and radiotherapy appears to be associated with antitumor activity Increased CD8 was significantly associated with PFS Thus immune biomarkers may be useful for early response evaluationTrial registration number NCT01557114INTRODUCTIONImmunotherapy using immune checkpoint inhibition has revolutionized the management of patients with advanced stage melanoma and is an emerging approach for many other cancers1 The first immune checkpoint inhibitor that was developed was ipilimumab2 Ipilimumab targets the cytotoxic T lymphocyte associated antigen CTLA4 and significantly improves the overall survival in patients with metastatic melanoma2 However the objective response rate ORR and the disease control rates proportion of patients with a partial response PR or complete response CR or stable disease SD are relatively low and respectively3 As such increased interest has been drawn to enhance the induction of systemic immune responses of ipilimumab by combining it with radiotherapy4 One of the rationale put forward to combine these therapies is that ipilimumab is able to deplete T regulatory cells Treg cells through antibody dependent cell cytotoxicity and consequently increases the CD8 T cell to Treg ratio whereas radiotherapy promotes the diversity of the T cell receptor TCR Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access repertoire of intratumoral T cells7 When combined ipilimumab promotes the expansion of T cells while radiation enhances the TCR repertoire of the expanded peripheral clones4 Ipilimumab at a dose of mgkg is approved in several countries for the treatment of unresectable or metastatic melanoma In a recent prospective phase study patients were treated with ipilimumab mgkg combined with concurrent or sequential stereotactic ablative radiotherapy11 Dose limiting toxicities DLTs and grade toxicities were reported in and of patients respectively PR and clinical benefit were reported in and of patients respectively However few clinical studies were conducted to assess the efficacy of ipilimumab mgkg combined with radiotherapy11 although overall survival was significantly improved with the ipilimumab mgkg monotherapy compared with the mgkg dose2 Here we present the phase Mel Ipi Rx study investigating the safety and efficacy of ipilimumab mgkg combined with radiotherapy in patients with metastatic melanoma Although the treatment landscape of patients with advanced melanoma has changed since this study was initiated the increased survival benefit of ipilimumab mgkg compared with mgkg suggests that the clinical utility of ipilimumab in refractory patients with high unmet medical need could warrant further assessment The primary objective of this study was to determine the maximum tolerated dose MTD DLT and the recommended phase dose RPTD of radiotherapy administered in combination with ipilimumab at a dose of mgkg in patients with metastatic melanoma The secondary objectives were to determine the adverse event AE profiles to describe the preliminary antitumor activity following escalating doses of radiation combined to ipilimumab using the immune related response criteria irRC and to evaluate the overall survival in patients treated with this combination The exploratory objectives were to evaluate the systemic immunologic antitumor response and factors influencing this responseMETHODSPatientsEligible patients had unresectable locally advanced or metastatic melanoma with at least one measurable metastasis accessible to radiotherapy Subcutaneous nodules and lymph nodes were considered as targets for irradiation Tumor lesions located within vital ans or gastrointestinal tract were not considered as target for radiotherapy All patients had measurable and non measurable disease evaluable according to irRC Patients were ¥ years of age were able to give written informed consent had Eastern Cooperative Oncology Group ECOG performance status of to and had normal renal and liver functions on blood tests Patients were excluded if they had one of the following criteria suspected or known central nervous system tumors including brain metastasis any other malignancy with a disease free for less than years an autoimmune disease a history of prior treatment with ipilimumab prior radiotherapy within the same body area or radiotherapy in fields containing flat bones volume If chemotherapy or immunotherapy were previously used a wash out period of weeks at least was required before the first administration of ipilimumabStudy designThis phase I dose escalation study evaluated the MTD of radiotherapy in combination with ipilimumab in patients with unresectable locally advanced or metastatic malignant melanoma Eligible patients received ipilimumab at mgkg at weeks and for a total of four doses at week intervals online supplementary figure S1 Patients without progressive disease PD who tolerated the treatment continued ipilimumab dosing in week intervals until progression or withdrawal of consent Radiotherapy was delivered on week on Monday Tuesday and Friday at the time of the second cycle of ipilimumab on measurable superficial lesions including subcutaneous nodules and lymph nodes Radiotherapy was delivered using MV photons or electrons with standard field encompassing Maximal field had to be at least à cm but not be more than à cm maximal dimensions on a lesion Millimetric margins were used to take into account microscopic spreading and patient movements clinical and planning target volumes of mm Dose escalation of ionizing radiation was planned in cohorts of three to six patients depending on the occurrence of DLTs during the first combination treatment cycle A hypofractionated radiation regimen higher doses per fraction was used Dose escalation was used with total doses of and Grays Gy administered in three fractions every Monday Wednesday and Friday and planned in cohorts of three to six patients depending on the occurrence of DLTs from week to week A minimum deadline of hours between two radiotherapy sessions had to be respected If necessary the radiotherapy could be put back to Tuesday Thursday Saturday Given the small size of the radiation field and the fact that irradiated lesions were superficial vital ans and gastrointestinal tract were not involved in the irradiated fieldToxicities were evaluated according to the Common Terminology Criteria for Adverse Events CTCAE version DLT observation period ranged from week to Any toxicity before the start of radiotherapy was not considered as a DLT but as a toxicity from ipilimumab alone The DLT was defined by the appearance of at least one of the following study combination related event within weeks grade vomiting diarrhea or gastrointestinal bleeding grade or non hematologic toxicity excluding grade nausea vomiting diarrhea and transient fever or grade or radiation dermatitis except if return to grade ¤ within weeks If none of the first three subjects in a given dosing cohort had experienced DLT dose escalation was realized If Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cone of the first three subjects in a given dosing cohort had experienced DLT an additional three subjects were enrolled to that dose level before further escalation was considered A dose escalation was realized if none of these additional three subjects had experienced DLT If two of the first three subjects in a given dosing cohort had experienced DLT dose escalation was stopped Dose escalation was continued until one third or more of the subjects at a particular dose level experienced DLT or up to the fourth cohort This was considered the maximum administered dose MAD The MTD was defined as the highest dose at which less than one third of the subjects experienced DLT The RPTD was defined as the MTD or the dose that was considered to give the optimal clinical andor immunological results Tumor assessments consisting of CT scans and assessment of skin lesions were performed at weeks and every weeks thereafter until the patient withdrew from the study or entered the follow up phase MRI or CT scan of the brain had to be realized when clinically indicated Evidence of PD was confirmed by a second assessment performed weeks later Definitions of lesions were based on irRC Measurable lesions were defined as lesions that could be accurately measured in two perpendicular diameters with at least one diameter ¥ mm and the other dimension ¥ mm It was possible to consider skin lesions if they were measurable All measurable lesions up to a maximum of lesions per an and lesions in total were identified as index lesions measured and recorded at screening and follow up The index lesions were representative of all involved ans Non index lesions corresponded to measurable lesions that were recorded and evaluated at the same assessment time points as the index lesions and that were irradiated according to the trial irradiation designBlood immune monitoringBlood samples were collected at baseline T0 at week W4 before second injection of ipilimumab and at week W6 after radiotherapy and before third injection of ipilimumab online supplementary figure S2 Phenotyping was performed on fresh whole blood and peripheral blood mononuclear cells PBMCs isolated by Ficoll density gradient were frozen for later analyses Data analysis from standard blood tests was realized to estimate the derived neutrophil to lymphocyte ratio dNLR at baseline W4 and W6 to see whether or not granulocytes could impact the prognosis of patients Whole blood or PBMCs were incubated with fluorochrome conjugated antibodies for min at room temperature or at °C respectively followed by min of lysis Versalyse Beckman Coulter Mervue Galway Ireland The following fluorochrome conjugated antibodies were used fluorescein isothiocyanate anti ICOS CD278 clone DX29 phycoerythrin PE conjugated anti CD25 clone B1499 allophycocyanin cyanine APC Cy7 conjugated CD45RA clone HI100 phycoerythrin cyanine PE Cy7 conjugated anti CD45RA clone access2H4 allophycocyanin Alexa Fluor AA700 conjugated anti CD3 clone UCHT1 Pacific Blue PB conjugated anti CD4 clone 13B82 and Krome orange conjugated anti CD8 clone B911 were obtained from Beckman Coulter Stained cells were acquired using a FACS Canto II cytometer BD Bioscience or a Gallios Cytometer Beckman Coulter and analyzed using Kaluza software Beckman Coulter Conventional T cells CD3CD4 and CD3CD8 were respectively defined by CCR7CD45RA for naïve T cells CCR7CD45RA for central memory T cells TCM CCR7CD45RA for effector T cells TEM and CCR7CD45RA for terminally differentiated T cells TEMRA Treg cells were defined as CD3CD127lowCD25Tumor growth rate analysisTo assess the systemic immunologic antitumor response tumor growth rate TGR of irradiated lesions and non irradiated lesions were analyzed Briefly TGR estimates the variation in tumor volume over time using each patient as his own control and has shown to be interesting to identify the specific therapeutic effect of a treatment regardless of the disease course of each patient16 TGR was expressed as a percentage increase in tumor volume during month in accordance with Hiniker et al11 using the following formula TGR100 [exp[3LogDtDt0t] ] with D0tumor size defined as the sum of the longest diameters of the lesions at baseline Dttumor size defined as the sum of the longest diameters of the lesions at the time t of evolution evaluation in monthsTGR100 [exp3LogDtDt0t]The TGR was computed for irradiated lesions TGRirr and non irradiated target lesions defined by the radiologist for the irRC evaluation TGRnon irr for two treatment periods when data were available the Reference TGR REF TGR assessed before the onset of the treatment using the baseline CT and a CT realized before the baseline prebaseline CT the Experimental TGR EXP TGR assessed between the onset of the treatment and the first evaluation The TGR was computed using the same target lesions at each evaluation time A positive value of the EXP TGR reflected a bigger lesion at the first evaluation than at baseline Therefore new lesions related to PD were not included in the TGR computation Difference between EXP TGR and REF TGR was used to assess the effect of the treatment ÎTGREXP TGRREF TGR a negative value reflecting a slowdown of the natural course of the disease ie a slower tumor growth or a tumor response between the two periodsStatistical analysisDemographic and baseline characteristics were summarized for all registered subjects using descriptive statistics Toxicity grades per subject were tabulated for AEs and on study laboratory measurements by using the NCI CTCAE version Analyses of efficacy endpoints were based on all subjects evaluable for efficacyBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access The Kaplan Meier method was used to estimate overall survival and immune related progression free survival PFS Overall survival was defined as the time from treatment initiation to the date of death or date of last follow up in persons alive at last follow up Immune related progression was defined according to irRC An event was defined as progression or death whichever comes first PFS was defined as the time from treatment initiation to the date of occurrence of the event Follow up of patients who did not experience was censored at the date of last evaluation Duration of follow up was estimated using the Schemper and Smith method18 All statistical analyses were done using SAS software V94Evolution of innate immune cells was analyzed using GraphPAD PRISM software values at W4 and at W6 were described and compared with baseline by using Friedman test followed by Dunn's multiple comparisons testStudy oversightThe study was conducted in accordance with the protocol Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki All patients provided written informed consentRESULTSPatient populationNineteen patients with advanced melanoma were treated at Gustave Roussy in the Mel Ipi Rx phase trial between August and July online supplementary table S1 The database lock occurred on April Nine patients received the four doses of ipilimumab and two patients received maintenance ipilimumab one and two cycles respectively All patients received the combined radiotherapy at week in three fractions Thirteen patients were enrolled at dose level Gy and six patients at dose level Gy Demographic data of patients are summarized in table Overall patients presented visceral metastases M1c had elevated level of lactate dehydrogenase LDH tumors were BRAF mutated and none had a history of brain metastases All patients had received systemic therapy previously Prior therapy included chemotherapy in patients BRAF inhibitors in patients radiotherapy in patient and surgery in patients respectively and all of them were immune checkpoint inhibitor naïve Irradiation was delivered on subcutaneous lesions in patients and patients in the cohorts treated with and Gy respectively and on lymph nodes in patients and patients in the cohorts treated with and Gy respectively online supplementary table S2 The average irradiated tumor volumes were relatively homogeneous with a median of mm IQR SafetyAll patients presented at least one AE of any grade These AEs were felt to be probably related to ipilimumab The role of concurrent radiotherapy on these AEs was difficult Table Patient baseline characteristicsCharacteristics SexMaleFemaleAge yearsMedianRangeMutation BRAF statusNon mutantMutantUnknownLactate dehydrogenaseNormalElevatedM staging extent of metastasesM0M1aM1bM1cBrain metastasesNoYesSite of irradiated lesionsLymph nodesSubcutaneous nodulesVisceral ansPrevious treatments No prior systemic treatmentIpilimumabChemotherapyAnti BRAFRadiotherapySurgeryOverall populationN19 No The normal range for lactate dehydrogenase is UIL All patients were naïve to immune checkpoint inhibitorsto assess However no radiation induced necrosis or local symptoms were observed inside the radiation field AEs of all grades are summarized in table regardless of their attributionAsthenia was the most commonly reported AE of any grade It occurred in patients Among them nine patients were treated in the Gy cohort and five patients in the Gy cohort The median time to onset of asthenia was Q1 Q34 weeks and the median duration was Q1 Q34 weeks The other most common AEs of Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Adverse events of all grade safety populationNo of patients with at least one adverse eventsFatigueDiarrheaDisease related painFeverPainNauseavomitingAnorexiaConstipationColitisPruritusWeight lossAnemiaEdema of limbsDyspneaSkin eruptionVitiligoHypereosinophiliaAlanineaspartate aminotransferase increasedCoughLymphedemaLevel dose N13 Level dose N6 TotalN19 any grade included diarrhea disease related pain fever nausea and vomitingThirteen patients discontinued the study owing to treatment related AEs nine patients in the cohort receiving Gy and four patients in the cohort receiving Gy Nine of these patients had AEs of grade Multiple AEs of grade occurred in some patients AEs of grade included colitis n2 hepatitis n2 asthenia n2 thyroid disorders n1 DRESS drug rash with eosinophilia and systemic symptoms syndrome n1 and nauseavomiting n1 Online supplementary table S3 presents the grade and grade events for each dose level for all events There were no treatment related deathsFour of the patients who discontinued the study did not have grade or AEs Indeed three patients had grade colitis associated with either contraindication to corticosteroids because of uncontrolled diabetes mellitus or complete remission after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab One patient had a grade asthenia after four cycles of ipilimumab leading to regular follow up without maintenance ipilimumab accessDose escalation and DLTsEighteen patients were evaluable for DLTs in this study patients in the cohort treated with Gy and patients in the cohort treated with Gy Among them four patients experienced DLTs All the DLTs occurred outside the radiation fieldIn the cohort treated with Gy of evaluable patients experienced DLTs after two cycles of ipilimumab combined with radiotherapy Indeed one of these patients presented grade hepatitis and the other one presented grade colitis with grade hypokalemia grade anorexia and grade thyroid disorders In both patients the three fractions of radiotherapy on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsIn the cohort treated with Gy two of the six evaluable patients experienced a DLT after two and three cycles of ipilimumab respectively combined with radiotherapy One of these patients presented grade hepatitis and the other one presented grade colitis with unusually normal macroscopic colonoscopy but a total villous atrophy mimicking celiac disease on biopsies In both patients the radiotherapy fractions on Monday Wednesday and Friday were delivered on week DLTs led to permanent ipilimumab discontinuation in both patientsThe MTD of radiotherapy was thus Gy when combined with ipilimumab mgkg in the present design Consequently the RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was GyClinical outcomesAt the time of analysis the median duration of follow up was years Q145 Q368 The median overall survival CI was estimated at years The median PFS CI was Figure shows Kaplan Meier median overall survival and PFS curves with CI According to irRC the best response within the trial was CR for two patients both in the cohort receiving Gy PR for three patients two patients in the cohort receiving Gy and one patient in the cohort receiving Gy and SD for seven patients six patients in the cohort receiving Gy and one patient in the cohort receiving Gy giving an ORR of and a clinical benefit rate of at week online supplementary table S2 The initial melanoma staging of the patients who had CR was M1a for one patient multiple subcutaneous nodules and M1c multiple subcutaneous nodules and lymph nodes associated with an elevated LDH for the other patient The initial melanoma staging of the patients who had PR was M1a for one patient multiple subcutaneous nodules and M1c for two patients one of these patients had a subcutaneous nodule a lymph node and elevated LDH The other patient had multiple lymph nodes and bone metastasisBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access Figure A Waterfall plot of patients according to the variation of tumor growth rate ÎTGR between reference before treatment and experimental period on treatment For each patient specific ÎTGR of irradiated and non irradiated lesions are represented ÎTGR on treatment lesions at the first evaluation are bigger than at baseline ÎTGR of non irradiated lesions was superior to the irradiated lesion B Changes of the sum of diameters of the target lesions irradiated and non irradiated respectively at months in compared with the baselineVariation of tumor growth rate across lesionsTGR variation for irradiated and non irradiated lesions before and after the treatment was evaluable for patients figure A total of non irradiated lesions with up to lesions for one patient were evaluated for the REF and EXP TGRnon irr median IQR non irradiated lesions by patient and irradiated lesions for the REF and EXP TGRirr one patient had two irradiated lesions Median reference and experimental period were IQR and IQR months respectively The sum of diameters of lesions at the three evaluation times prebaseline baseline first evaluation and corresponding TGR are presented in table The EXP TGR on treatment was not correlated with the REF TGR before treatment Spearmans rho011 p073 Decrease of the TGR was of the tumor sizemonths IQR to for irradiated lesions and month IQR to for non irradiated lesions although the difference was not significantly different p082 Response Figure Kaplan Meier median overall survival A and progression free survival B curves with CINo pseudoprogression was observed among the patients treated in this study nor radiation induced necrosis or edema Of note three patients were not evaluable for response because they progressed and died before the radiologic assessment scheduled in this studyTreatments administered after the Mel Ipi Rx study in patients with disease progression are summarized in online supplementary figure S3Boutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0cTable Characteristics of lesions evaluated for tumor growth rate TGRPatients nLesions n sumLesions n median IQRSum of diameters prebaseline median IQRSum of diameters baseline median IQRSum of diameters at first evaluation median IQRREFperiod median IQREXPperiod median IQRREF TGR median IQREXP TGR median IQRTGRdiff median IQR accessWilcoxon p valueOverallIrradiated lesionsNon irradiated lesions to to to to to to to to to to to to to to to to to to to to to REF TGR corresponds to TGR before the start of the treatment EXP TGR corresponds to TGR between the start of the treatment and the first evaluation at monthsof non irradiated lesions seemed more representative of patient outcomes vs irradiated lesion EXP TGRnon irr was significantly higher in patients with PD Although the number of patients was too low for statistical test p values are shown for information in table figure and online supplementary figure S4Immune analysesWe analyzed immune parameters in the blood that have been previously described to be changed during ipilimumab treatment to determine if radiotherapy could modify ipilimumab pharmacodynamics19 For CD4 T cell counts and as expected ipilimumab alone W4 could favor accumulation of TEM Treg and ICOSCD4 T cells Interestingly at W6 after ipilimumabradiotherapy only TEM and ICOSCD4 T cells remained significantly increased suggesting that combination favored accumulation of activated memory CD4 T cells rather than Treg cells For CD8 T cell counts no accumulation could be observed at W4 while augmentation of TCM and TEMRA could be depicted between W4 and W6 suggesting that adjunction of radiotherapy to ipilimumab was more prone to boost these CD8 T populations online supplementary figure S2 High fold change in CD8 from baseline to week was significantly correlated with PFS p00223 but not significantly correlated with overall survival p02355 online supplementary figure S5Innate immune cells and NLR absolute neutrophils count ANC divided by the number of lymphocytes or dNLR ANC divided by the number of white blood cellsANC have been shown to have a prognostic role in patients treated with immunotherapy and even might represent a predictive biomarker of response We took advantage of standard blood tests to determine if Table Univariate analysis of tumor growth rate TGRAll lesions n37Progressive disease WilcoxonNop valueYesIrradiated lesion n13Progressive diseaseNoYesWilcoxon p valueNon irradiated lesions n24Progressive diseaseNoWilcoxon p valueYesSum of diameters prebaseline meanSum of diameters baseline meanSum of diameters at first evaluation meanREF TGR meanExp TGR meanÎTGR meanTGR is evaluated in percentage per months ÎTGR EXP TGR REF TGR A negative value corresponds to a slowdown of the tumor growthBoutros a0C et a0al J Immunother Cancer 20208e000627 101136jitc2020000627 0c access neutrophils monocytes NLR or dNLR at baseline W4 and W6 could correlate with the prognosis in our study ANC or monocytes did not correlate with the prognosis of patients while absolute count of lymphocytes significantly increased at W6 compared with baseline in only patients with a clinical benefit CRPRSD Both NLR and dNLR were significantly lower at W6 only in patients with clinical benefit online supplementary figure S6 Note that we did not found an association with the dose of radiotherapy data not shownDISCUSSIONIn this dose escalation phase study four patients experienced DLTs All the DLTs occurred outside the radiation field Therefore it was difficult to assess the role of concurrent radiotherapy on the DLTs The MTD of radiotherapy combined with ipilimumab at mgkg was Gy The RPTD of radiotherapy administered in combination with ipilimumab at mgkg in patients with metastatic melanoma was Gy A hypofractionated radiation regimen higher doses per fraction was used in this study It is usually preferred for melanoma which displays low alphabeta ratio Our three fractions hypofractionated regimen is in line with the radiation standard in the metastatic setting Moreover it has been shown recently that radiation doses per session inferior to Gy combine more favorably with immunotherapy through interferon type induction21 Of note our study is the only one that combines radiotherapy and high dose of ipilimumab mgkgThe incidence of treatment related AEs was high with this combination in our study but numerically similar to the incidence reported previously with ipilimumab monotherapy at mgkg Grade or AEs occurred in of patients in our study whereas they occurred in of patients treated with ipilimumab monotherapy at mgkg2 The overall AE spectrum of the combination in this study was consistent with previous findings with the drugs immune based mechanism of action The high rate of AEs might be partially attributed to the high dose of ipilimumab The dose of mgkg was chosen based on data from the randomized phase trial that compared various | Thyroid_Cancer |
The role of serum inflammatory cytokines andberberine in the insulin signaling pathwayamong women with polycystic ovarysyndromeHongying Kuang12¯ Yuwei Duan34¯ Dan LiID5a Yanwen Xu34b Wenxia Ai2 Wei Li1Ying Wang1 Sha Liu2 Mushan Li2 Xiaoqiu Liu2 Manqi Shao2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine Harbin China HeilongjiangUniversity of Chinese Medicine Harbin China The First Affiliated Hospital of Sun Yatsen UniversityGuangzhou China Guangdong Provincial Key Laboratory of Reproductive Medicine Guangzhou China Department of Acupuncture the Third Affiliated Hospital Beijing University Of Chinese Medicine BeijingChina¯ These authors contributed equally to this worka Current address The First Affiliated Hospital of Sun Yatsen University Guangzhou Chinab Current address Reproductive Medicine Center The First Affiliated Hospital Sun Yatsen UniversityGuangzhou Guangdong China 3509437qqcom DL Xuyanwenlivecn YXAbstractObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome PCOSMethodsSelected serum inflammatory cytokines were analyzed in the p cells which were interfered by berberine from infertile women who were to be treated with In Vitro FertilizationIVF Intracytoplasmic Sperm InjectionEmbryo Transfer icsiet Among them patientshad PCOS infertility and were nonPCOS patients whose infertility resulted from fallopian tube and male factors The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells The correlations between the seruminflammatory cytokine expression levels and the corresponding clinical hormones were analyzed The changes in the expression mRNA and protein levels of the serum inflammatorycytokines were studied by realtime quantitative PCR and protein printing Fluorescencemicroscope and flow cytometry were used to detect the glucose uptake capacity of ovariangranulosa cells in PCOS patients under the action of insulin after berberineResultsIn the PCOS group IL17a P IL1Ra P00001 and IL6 P were significantly higher than those in the nonPCOS group In the nonPCOS group AMH level wasnegatively correlated with inflammatory cytokines IL17a r 0819P IL1a r a1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Kuang H Duan Y Li D Xu Y Ai W Li W The role of serum inflammatorycytokines and berberine in the insulin signalingpathway among women with polycystic ovarysyndrome e0235404 101371journalpone0235404Editor Meijia Zhang China Agricultural UniversityCHINAReceived November Accepted June Published August Copyright Kuang 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 relevant data arewithin the manuscript and its SupportingInformation filesFunding This work was supported by theOutstanding talents funding project of HeilongjiangUniversity of Chinese Medicine China [grantnumber 2018JC05] the Foundation for youngacademic leaders of Heilongjiang University ofChinese Medicine China [grant number2018RCD08] and the Foundation for HeilongjiangProvince Postdoctoral Research Startup ChinaPLOS ONE 101371journalpone0235404 August PLOS ONE 0c[grant number LBHQ18119] the Project ofUndergraduate Colleges and Universities YouthInnovation Talents by Education Department ofHeilongjiang Province China [grant numberUNPYST2018227] the Natural ScienceFoundation of Heilongjiang Province China [grantnumber H2016083]Competing interests The authors have declaredthat no competing interests existThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway0716P IL1b r 0678P IL2 r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines Berberine significantlyreduced the expression level of mTOR mRNA P and increased the expressionlevel of IRS1 mRNA P in the PCOS granule cellsConclusionIn this study we find that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 cause women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors alter their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroyingthe normal ovulation and fertilization system of women leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation Berberine can improvethe sensitivity of insulin by regulating the signal pathway of insulin receptor substrate1IRS1 and mammalian target of rapamycin mTOR in PCOS patients and achieve a therapeutic effect of treating PCOS IntroductionPolycystic ovary syndrome PCOS is the most common endocrinopathy affecting reproductive aged women It affects reproduction infertility irregular menstruation hirsutism etcmetabolism insulin resistance impaired glucose tolerance etc and psychological characteristics anxiety depression and deterioration in quality of life [] Berberine BBR as a quaternary ammonium salt extracted from plants such as Coptis chinensis and Phellodendronchinensis is currently used in the treatment of diabetes hyperlipidemia and PCOS [] Recentstudies have found that berberine has good hypoglycemic and hypolipidemic effects and is aneffective insulin sensitizer Berberine reduces the synthesis of steroid hormones and theexpression of ovarian aromatase through the action on the hypothalamuspituitaryovarianaxis HPOA improves the insulin resistance status of PCOS patients reduces body weightinduces ovulation and regulates menstruation thereby increasing pregnancy rate and livebirth rate [] Clinical observations have demonstrated that even with longterm use of berberine its side effects are transient and mild suggesting that BBR is safe to use in PCOSpatients and a very promising plantbased compound for treating PCOS patients []Patients with PCOS have been found to be under a chronic lowgrade inflammation statusincluding high levels of leukocytes and disorder of the proinflammatory cytokines [] Interleukin IL6 is a multipotent cytokine that mediates inflammatory response by controllingcell differentiation migration proliferation and apoptosis thus playing a role in the development of insulin resistance [] IL17a is the signature cytokine secreted by the Th17 CD4ve T cell subset Activation of Th17type responses is important not only for host immunecontrol of extracellular bacterial and fungal infections but also associated with chronic inflammation and autoimmunity [] The IL1RA protein is a naturally occurring antagonist of proinflammatory cytokines These proinflammatory cytokines are involved in the underlyingmechanism of various chronic inflammatory conditions [] Therefore we hypothesize thatinflammatory factors are one of the important factors influencing the formation of PCOS andberberine may be an important drug that regulates PCOS inflammatory factorsPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayAntiMu¨llerian hormone AMH is an indicator of a patients ability to respond quicklyand efficiently to ovarian reserve In women AMH is produced by the granulosa cells of theovarian follicles and its secretion begins at puberty and lasts until menopause [] PCOS ischaracterized by hyperandrogenism and follicular blockade These two characteristics may bedue to an imbalance between AMH and follicle stimulating hormone [] Circulating insulinlevels in patients with PCOS increase thereby inducing follicular developmental disorderswhich in turn lead to ovarian polycystic ovary formation and higher than normal AMH HighAMH is one of the indicators of stubborn anovulation [] Research has shown that serumAMH level is relatively stable throughout and between menstrual cycles [] More and morestudies have used AMH as a biomarker for PCOS []In the present study we examined the effect and mechanism of serum inflammatory cytokines including IL6 IL17a IL1RA etc on insulin sensitivity of PCOS In addition weassessed whether berberine can improve insulin sensitivity of PCOS by antagonizing the proinflammatory effect of serum inflammatory cytokines Materials and methods SampleSerum samples and granulosa cells were collected from infertile women who were to betreated with In Vitro Fertilization IVF Intracytoplasmic Sperm InjectionEmbryo Transfericsiet Among them patients had PCOS infertility and were nonPCOS patientswhose infertility resulted from fallopian tube and male factors The nonPCOS women wererequired to have regular menstrual cycles The essays were analyzed by Guangdong ProvincialKey Laboratory of Reproductive Medicine at the first affiliated hospital of Sun YatSen University Data collection time ParticipantsInclusion criteria age Diagnosis of PCOS according to Rotterdam StandardRotterdam Diagnostic Criteria for PCOS Ovarian Ovulation Disorder Manifests Oligomenorrhea or Amenorrhea Clinically or biochemically determined androgen level increasesmore than 2nmolL or clinically manifested hirsute acne excluding Kaohsiung caused byother diseases Ovarian morphology showed polycystic changes Only of the above items can be met IVFET treatmentExclusion criteria Having orally taken drugs in the past three months that affect the results such as contraceptives or other hormone drugs insulin sensitizers and lipidlowering drugs Suffering from other androgen excess related diseases including congenital adrenal hyperplasia with hydroxylase deficiency androgen secreting tumor excessive use of androgenproducing drugs Cushing syndrome severe insulin resistance thyroid dysfunction andhyperprolactinemia Having a history of anic diseases of heart lung liver kidney and other important ansor patients with mental diseases and other reasons that may interfere the present studyoutcomesPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway Ethics statement The study was approved by the ICE for Clinical Research andAnimal Trials of the First Affiliated Hospital of Sun Yatsen University reference [] No and was conducted in accordance with the ethical standards of the Declaration ofHelsinki All participants have signed written informed consent and there were no minors ReagentBerberine was provided by Yuanye BioTechnology Co Ltd Shanghai China2NBDG N13195 Invitrogen DMEMF12 Gibco and Fetal bovine serumSV3008702 Hyclone were purchased from Thermo Fisher Scientific Waltham USA Thefollowing is a list of used reagents and their manufacturers informationRealtime quantitative PCR kits ROCHE CAT Reverse transcriptase PROMEGA CAT M1701RIPA pyrolysis fluid yantian Bi CAT P0013BBcl2 abcam CAT ab196495BAD abcam CAT ab90435BAX abcam CAT ab182733ACTINβ abcam CAT sc70319 Methods Granulosa cell acquisition and grouping The follicular fluid was centrifuged at rpm for minutes at room temperature The supernatant was discarded and the precipitate after centrifugation was resuspended by phosphate buffer saline PBS The cell suspension was slowly centrifuged to percoll level at room temperature with 1800rpm for 10minAfter centrifugation the white floc was sucked out and washed with PBS three times at1200rpm for 5min at room temperature We discarded the supernatant and added typeIV collagenase and blew it evenly Digestion was performed at ËC for 1520min We addedthe same amount of culture solution to stop digestion and blew it evenly The filtrate was filtered by micron cell filter and centrifuged at room temperature with 1000rpm for 4minThe supernatant was discarded the cells were suspended again with PBS 3ml erythrocytelysate was added to mix well and the mixture was allowed to stand at ËC for 10min It wasthen centrifuged at 1000rpm for 4min at room temperature Some white precipitate whichwas verified as granulocytes were observed after centrifugation After the seed plate of PCOSwomen granulosa cells was seeded the fluid was changed once hours After hours solvent or berberine was added for continuous intervention for hours which led to the PCOScontrol group and PCOS berberine intervention group Detection of serum inflammatory cytokines The Merck luminex testing platformwas used to detect serum cytokines according to the manufacturers instructions Realtime quantitative PCR At the end of berberine treatment Trizol method wasused to extract total RNA in the cells which was then used to reverse transcription of mRNATotal RNA was isolated from cells using Trizol regent Takara BioInc Tokyo Japan and I mgmRNA was reverse transcribed to cDNA using a reverse transcription Takara Bio Inc TokyoJapan and subjected to quantitative PCR which was performed with the BioRad CFX96Touch TM RealTime PCR Detection System BioRad CA using iQ TM SYBR Green Supermix BioRad CA and threshold cycle numbers were obtained using BioRad CFX ManagerSoftware Real time PCR was carried out using the following conditions min at ËC min at ËC and cycles of s at ËC min at ËC using 1μl of cDNA reverse transcribed as mentioned above Quantifast SYBR green PCR kit Qiagen Cat No and500nM of forward and reverse primers The following primers were usedPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayβactin GCCGTTCCGAAAGTTGCCTT GAGCGCGGCGATATCATCAAMPK ACAGCCGAGAAGCAGAAACA TTGCCAACCTTCACTTTGCCmTOR CTTAGAGGACAGCGGGGAAG TCCAAGCATCTTGCCCTGAGSTAT3 CTGAAACGGGCTTCAGGTCA TCCAGGGAGAAAGGGAGTCASOCS3 TCTGTCGGAAGACCGTCAAC CCTTAAAGCGGGGCATCGTAIRS1 TCTCTTCCCACGGCGATCTA TGACACTGCGGAAGGAACTCWe used the 2ÎÎCT method to calculate the relative expression level of target genes Western blot experiment After the end of each cell culture the cells were lysedusing RIPA lysate in an ice bath and the protein lysate was collected After the total proteinconcentration was determined by BCA method polyacrylamide gel electrophoresis was carried out according to the standard of μg of total protein per group After the electrophoresisthe protein in the gel was transferred to the polyvinylidene fluoride PVDF membraneblocked with skim milk powder and then incubated with the corresponding dilutedprimary antibody at ËC overnight and finally labeled with the corresponding horseradish peroxidase After incubation for one hour at room temperature the ECL luminescentsolution was added and detected by autoradiography using a Biored gel imaging systemImmunoreactive protein bands were visualized with enhanced chemiluminescence ECL on aChemiDoc MP Imaging System Blots were scanned and quantified with the Image analysissoftware BioRad Image Lab All specific protein band densities were normalized to βactin amounts Detection of glucose uptake capacity in granulosa cells Granulosa cells of bothnonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granulosacells were randomly divided into two groups one group was cultured normally and the othergroup was added with berberine with the final concentration of 100μM After 24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of100nM were replaced at ËC After 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and the difference in fluorescence density between different groups was compared by using flowjov10 Statistical methodsData were represented by mean±standard deviation SD and SPSS statistical softwarewas used for analysis Analysis of variance ANOVA was used to test betweengroup differences and the pvalues were corrected by Bonferroni p value less than was considered statistically significant Results Analysis of clinical baseline indicators of PCOS group and nonPCOSgroupAs shown in Table compared with the nonPCOS group the PCOS group had significantlyhigher BMIP LHP00001 LHFSHP00001 FPGP AMHP00001 the number of follicles obtainedP00001 ICSI mature eggsP and thenumber of normal fertilization P FSH levels were significantly lower in the PCOSgroup than those in the nonPCOS group P Detection of serum inflammatory cytokinesAs shown in Table IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group IL10 P IL13PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Comparison of clinical indicators between PCOS and nonPCOS groupsnonPCOS n PCOS n PvalueAge years oldMenarche ageSBP mmHgDBPmmHgPulse sminBMI kgm2FSH mIUmlLH mIUmlLHFSHE2 pgmlPRL ngmlT nmolLFPG mmollAMHngmleggs obtainedICSI mature eggsNormal fertilized eggsMeanSDMedianMeanSDMedian101371journalpone0235404t001Table Comparison of serum inflammatory cytokines between the PCOS and nonPCOS groupsIL10 pgmlIL13 pgmlIL17a pgmlIL1Ra pgmlIL1α pgmlIL1β pgmlIL2 pgmlIL6 pgmlIL8 pgmlTNFα pgml101371journalpone0235404t002nonPCOSn ±±±±±±±±±±±PCOSn ±±±±±±±±±PvalueP IL1α P IL1β P IL8 P and TNFα P inthe two groups was not statistically significantly different Correlation analysis between serum inflammatory cytokines anddiagnostic clinical indicatorsWe examined the correlation between serum inflammatory cytokines and clinical serum hormones and other levels in PCOS and nonPCOS patients In the nonPCOS group AMH levelwas negatively correlated with inflammatory cytokines IL17ar 0819P IL1ar 0716P IL1br 0678P IL2r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantlyPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaycorrelated with the levels of these inflammatory cytokines Among the diagnostic clinical indicators except AMH level there was no significant correlation between other clinical indicatorsand the expression level of inflammatory cytokines in the nonPCOS group and the PCOSgroup Tables Table Correlation analysis of BMI FSH and other inflammatory cytokines in the nonPCOS groupnonPCOSBMIIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarP101371journalpone0235404t003FSHPrLHLHFSHrPrTable Correlation analysis of E2 PRL and other inflammatory cytokines in the nonPCOS groupnonPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarPrPrPr101371journalpone0235404t004Table Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the nonPCOS groupnonPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t005PPPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Correlation analysis of BMI FSH and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαBMIFSHLHLHFSHrPrPrPr101371journalpone0235404t006Table Correlation analysis of E2 PRL and other inflammatory cytokines in the PCOS groupPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrPrPrPr101371journalpone0235404t007PPTable Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t008 Detection of protein imprinting in granulosa cellsIt can be seen from Fig 1A that there was no significant difference in the expression levels ofAMPK P PAMPK P and ACC P protein in PCOS groupPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Granule cell protein expression level B Gray expression analysis of protein expression A Representativewestern blot images and B Summary of expression changes among relative genes Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than wasconsidered statistically significant101371journalpone0235404g001PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Fluorescence density map of sugar uptake capacity of granular cells B Fluorescence density analysis Granulosa cells ofboth nonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granule cells were randomly divided into twogroups one group was cultured normally and the other group was added with berberine with final concentration of 100μM After24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of 100nM was replaced at ËCAfter 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and thedifference in fluorescence density between different groups was compared by using flowjov10 Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than was considered statisticallysignificant101371journalpone0235404g002compared with the nonPCOS group while the expression levels of mTORP00001 PmTORP00001 and STAT3P00001 were significantly increased At the same time theprotein expression level of IRS1P was significantly decreased Detection of granulated cell sugar uptake capacityAs shown in Fig 2A and 2B compared with granulosa cells obtained from the nonPCOSgroup the fluorescence intensity of granulosa cells in PCOS group was significantly reducedP00001 and the fluorescence density in the cells increased significantly after berberineP00001 Detection of expression of key factors of glucose metabolism ingranulosa cellsAs shown in Fig 1B the increased expression level of AMPK mRNA after the addition of berberine was significantly different from that in the PCOS group P Compared with thenonPCOS group the level of mTOR in PCOS granule cells was significantly increasedP and the expression level of IRS1 mRNA was significantly reduced P Berberine significantly reduced the expression level of mTOR mRNA in PCOS granule cellsP and increased the expression level of IRS1 mRNA in PCOS granule cellsP Discussion The correlation between selected serum inflammatory cytokines andPCOSThis study finds that IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group Studies have shown thatPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayPCOS associated with lowgrade chronic inflammation interleukin17A IL17A is one of themajor members of proinflammatory cytokines and is mainly involved in the development ofinflammatory and autoimmune diseases Within the immune process the genetic factors ofIL17A play a major role in the susceptibility of PCOS [] The level of IL1Ra is significantlyincreased in patients with PCOS which can lead to decreased insulin resistance and blood glucose metabolism causing obesity and metabolic syndrome in PCOS patients [] Tarkun [] studied the serum levels of IL6 in patients with PCOS higher than the normal control group and IL6 was significantly associated with insulin resistance and fasting blood glucose The increase of IL6 level is an important inflammatory factor inducing the occurrenceof PCOS AMH levels and inflammatory cytokines in PCOS patientsThis study finds that in the nonPCOS group AMH level was negatively correlated withinflammatory cytokines such asIL17a r 0819P 0004IL1a r 0716P 0002IL1b r 0678P 0031IL2 r 0765P 001IL8 r 0705P However in thePCOS group AMH levels were not significantly correlated with the levels of various inflammatory cytokineStudies have found that AMH plays a key role in the anovulatory mechanism of PCOS []AMH can promote the growth of preantral follicles to the small sinus stage in vitro whileincreasing the production of steroid hormones and paracrine factors as well as oocyte maturation AMH is a key follicular paracrineautocrine factor that has a positive effect on preantralfollicle survival and growth [] The alienation of AMH function affects the regulation of follicles and promotes the increase of small follicles in the body Due to the abnormal expressionof the number and function of follicles the regulation of the ovary on the matrix is weakenedand the follicular irregular growth is induced without follicular atresia The expression of normal levels of inflammatory cytokines may promote normal cell apoptosis Elevated inflammatory cytokines IL17aIL1aIL1bIL2IL8 may disrupt the ovarian follicle atresiaweakening the ability of apoptosis and inhibiting the maturation of oocytes In turn the AMHfunction is out of control and the correlation between AMH and inflammatory factor levels isinducedIt can be concluded that AMH levels under normal conditions can effectively regulate thelevel of inflammatory factors and promote the bodys own metabolism and reproductive function The expression of high AMH levels in PCOS patients causes a loss of correlation withinflammatory factors The abnormal level of AMH increases the level of inflammatory factorsresulting in a continuous low concentration of systemic inflammatory state in the humanbody leading to metabolic diseases such as insulin resistance and glycolipid metabolism andreproduction disfunction The effects of Berberine on granulosa cells insulin resistanceOur study finds that berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients Berberine can increase insulin sensitivity in patients reduce blood sugar improveinsulin resistance and achieve the therapeutic effect of treating PCOS Berberine is the mainactive ingredient of Chinese herbal medicine Coptis Cork and Mink and has been used totreat diarrhea metabolic disorders and infertility []Berberine regulates glucose metabolism through a variety of mechanisms and signalingpathways such as increased insulin sensitivity activation of the adenosine monophosphateactivated protein kinase AMPK pathway regulation of the intestinal microbiota andPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayinhibition of liver sugar Exogenous stimulates peripheral cell glycolysis promotes intestinalglucagon like protein1 GLP1 secretion upregulates liver lowdensity lipoprotein receptormRNA expression and increases glucose transporter []PCOS is a multifactorial endocrine disease that affects reproduction and metabolism Studies in dheainduced PCOS mouse models have found that mTOR and pmTOR serine2448are highly expressed in the ovary In normal mice in another study mTOR protein levels inthe corpus luteum of PCOS patients were the same as in healthy women However comparedwith healthy patients receiving insulin stimulation the expression of mTOR protein in the corpus luteum of PCOS patients is less so another link between PCOS and mTOR is metabolicdisorders during PCOS [] The combination of activation of the mTOR pathway and oxidative damage to DNA that causes replication stress is a particularly effective factor in promoting aging and aging [] Berberine inhibits the level of DNA damage signals [] It canreduce the expression and activation of γH2AX including tumor A and TK6 cells as wellas normal WI38 cells or mitogenic human lymphocytes [] It can also reduce intracellularreactive oxygen species and mitochondrial transmembrane potential which is a sign of mitochondrial activation [] However these drugs also significantly reduce phosphorylationlevels of Ser235 of ribosomal S6 protein RpS6 Ser2448 of mTOR and Ser65 of 4EBP1These data indicate that the reduction of mTOR S6K signal which in turn reduces the translation rate and is accompanied by a reduction in oxidative phosphorylation which leads to areduction in ROS and a reduction in oxidative DNA damage [] The mechanism by whichBRB exerts these effects may be by targeting mitochondriaThe protective effect of berberine on islet function may involve two pathways On the onehand berberine can improve insulin sensitivity in patients with PCOS with IR as the core []On the other hand berberine can promote insulin secretion from islet cells of PCOS patientsand protect islet cells through antioxidant activity [] Kong [] found that themolecular mechanism of berberine on insulin resistance by upregulating the expression ofinsulin receptors confirmed that berberine can reduce fasting blood glucose and fasting seruminsulin At the same time they also found that berberine activated its promoter through protein kinase CPKCdependent increasing insulin receptor mRNA and protein expressionIn fat and muscle cells berberine may stimulate cells by upregulating glucose transporter type GLUT1 expression and inhibiting retinol binding protein4 RBP4 At the same timeberberine also has a certain effect on the phosphorylation of IRS1 which can finally alleviateinsulin resistance []In other words berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients The mechanism by which berberine regulates the metabolism of glycolipids is not tostimulate the patient to increase insulin secretion but to increase the glucose consumption ofthe patients cells and improve glucose tolerance At the same time by increasing insulin sensitivity in patients further lowering blood sugar and improving insulin resistance and achievingthe effect of treating PCOS ConclusionIn this study we found that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 caused women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors altered their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroying thenormal ovulation and fertilization system of women and leading to polycystic ovary syndromecharacterized by menstrual thinning and abnormal ovulation Berberine can improve thePLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaysensitivity of insulin by regulating the signal pathway of mTOR mRNA and IRS1 mRNA inPCOS patients and achieve the therapeutic effect of treating PCOSSupporting informationS1 Raw ImagesPDFAuthor ContributionsConceptualization Yuwei Duan Wei LiData curation Wei LiFormal analysis Wei LiFunding acquisition Hongying KuangSoftware Manqi ShaoWriting original draft Hongying Kuang Yuwei Duan Wenxia AiWriting review editing Dan Li Yanwen Xu Wenxia Ai Wei Li Ying Wang Sha LiuMushan Li Xiaoqiu Liu Manqi ShaoReferencesTeede H Deeks A Moran L Polycystic Ovary Syndrome A complex Condition with PsychologicalReproductive and Metabolic Manifestations That Impacts on Health Across the Lifespan BMC Med ZHANG F MA T CUI P Diversity of the Gut Microbiota in DihydrotestosteroneInduced PCOSRats and the Pharmacologic Effects of Diane35 Probiotics and Berberine [J]Frontiers in microbiology IMENSHAHIDI M HOSSEINZADEH H Berberine and barberry Berberis vulgaris A clinical review [J] LI M F ZHOU X M The Effect of Berberine on Polycystic Ovary Syndrome Patients with Insulin Resistance PCOSIR A MetaAnalysis and Systematic Review [J] LI W LI D KUANG H Berberine increases glucose uptake and intracellular ROS levels by promoting Sirtuin ubiquitination [J] Biomedicine pharmacotherapy Biomedecine pharmacotherapie RONDANELLI M INFANTINO V Polycystic ovary syndrome management a review of the possibleamazing role of berberine [J] Benson S Obesity Depression and Chronic Lowgrade Inflammation in Women with PolycysticOvary Syndrome Brain Behav Immun 101016jbbi200707003PMID Rehman K Akash MSH Liaqat A Role of Interleukin6 in Development of Insulin Resistance andType Diabetes | Thyroid_Cancer |
"widely usedcancerspecific questionnaire assessing domains of healthrelated quality of life HRQoL Our aim was to facilitatethe interpretation of scores on this questionnaire by providing Austrian normative data based on a general populationsampleMethods The calculation of normative data was based on the EORTC QLQC30 data collected from an Austriangeneral population sample that was part of an international online panel study on the development of Europeannormative data Data reported herein were stratified and weighted by age and sex Normative data were calculated forall HRQoL domains of the EORTC QLQC30 For precise predictions of EORTC QLQC30 scores a regression modelbased on sex age and the presence of health conditions was builtResults The Austrian sample comprised Austrian participants female when weighted by age andsex based on United Nation statistics The mean age was years weighted years and weighted reported at least one health condition Men reported better physical Cohens d and emotional Cohens d functioning as well as less fatigue Cohens d and insomnia Cohens d compared with womenYounger individuals years reported less dyspnea Cohens d and pain Cohens d whereas olderindividuals ¥ years reported better emotional functioning Cohens d Conclusions We present Austrian normative data for the EORTC QLQC30 Differences by age and sex are mostly inline with the findings of other European normative studies The Austrian population sample shows higher HRQoL andlower morbidity compared with other European countries The normative data in this study will facilitate theinterpretation of EORTC QLQC30 scores in oncological practice and research at a national and international levelincluding crosscultural comparisonsKeywords Normative data EORTC QLQC30 Quality of life Oncology Patientreported outcome measures AustriaGeneral population Correspondence jenslehmannimedacat1University Hospital of Psychiatry II Medical University of InnsbruckAnichstrasse Innsbruck AustriaFull 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 0cLehmann Health and Quality of Life Outcomes Page of BackgroundPatientreported outcomes PROs are the gold standardof evaluating the impact of cancer and its treatment fromthe patients perspective The importance of PROs isreflected in their widespread use as study endpoints incancer clinical trials [ ] and in the steadily increasing integration of routine PRO assessments in daily clinicalpractice [ ] Regulatory authorities such as the UnitedStates Food and Drug Administration [] and the European Medicines Agency [] have published guidance documents to foster the collection of highquality PRO datain clinical trials Guidelines from international associationssuch as the European anisation for Research andTreatment of Cancer EORTC [] and the InternationalSociety for Quality of Life Research [] deal with the integration of PRO measures into clinical routine and provideinformation on how to collect process and use PRO dataIn the present study we calculate reference values basedon data assessed with EORTC QLQC30 [] a questionnaire that is among the most widely used PRO measuresin cancer clinical trials [] This questionnaire coversimportant functional health domains eg physical andemotional functioning and key cancer symptoms such asfatigue pain and nauseaIn the literature different methods have been proposedto support the interpretation of results from PRO measuresanalyzing not only individual patients and patient groups ata single time point but also group differences and changesover time One approach is to use minimally important differences to assess or compare changes in PRO results at thegroup or patient level [] To interpret scores obtained ata single time point thresholds for clinical importance canbe used [] Another important approach to interpretPRO data are normative data if comparisons are to bemade with for example a specific disease group or the general population on an individual or grouplevel Whenusing normative data it is important to note that these canvary considerably between countries [] Focusing oncountryspecific values allows for more precise interpretation which becomes even more accurate when regressionmodels that take age and sex distributions into account areused to generate PRO predictionsThe EORTC Quality of Life Group conducted a largescale international panel study that collected normativedata for the EORTC QLQC30 for European countriesCanada and the United States [] While the EORTCQLQC30 has widely been used in studies in Austria eg[] no normative studies have so far been publishedIn the current study we present age and sexspecific normative data for the health domains of the EORTCQLQC30 from the Austrian general populationInaddition we provide a regression model defined by agesex and the presence of health conditions to calculate normative data for specific groupsMethodsSamplingAs part of an international study conducted by Nolte [] data from the Austrian general populationwere collected by GfK SE wwwgfkcom apanel research company which contacted panel members who had voluntarily registered and agreed to participate in panelbased studies An equal number ofparticipants was recruited for each sex and age groupmalefemale ¥ yearsOnce a quota was met for a specific group the recruitment for this group was stopped Response rates topanel studies conducted by GfK are between and as participants are registered voluntarily and usually participate when contactedEORTC QLQC30The EORTC QLQC30 consists of items covering fivefunctioning scales physical social emotional role andcognitive nine symptom scales fatigue nauseavomiting pain dyspnea sleep disturbances appetite loss constipation diarrhea and financial impact and a globalhealth status scale [] Referring to a recall period ofoneweek except for physical function which does notrefer to a recall period at all patients indicate their answers on a 4point Likert scale Linear converted scalescores range from to Higher scores on the functioning scales and on the global health status scale indicate better functioning whereas higher scores on thesymptom scales indicate greater symptom burden fordetails on the scoring and scale structure see [] TheEORTC QLQC30 has been validated in a large European samples and has been widely used in Germanspeaking patients with cancer and the general populationin Germany [ ]Statistical analysisWeights were established following Nolte [] andwere based on official population distribution statisticspublished by the United Nations [] We weighted theresponses to correct for over or underrepresentation ofsex and agestratified subgroups [] We report normative values as means and standard deviations for each subgroup In addition we established a regression model topredict EORTC QLQC30 scores with the following independent predictors sex age age2 a twoway interactionage by sex and the presence of health conditions none orat least one health condition We chose the predictors asthey are linked to HRQoL and have been applied in previous studies [] The basic model can be expressed asfollows Intercept Sex male female Age Age2 Interaction of Age and Sex age sex Presence ofHealth Conditions none at least one 0cLehmann Health and Quality of Life Outcomes Page of ResultsSampleIn April and May we collected online survey datafrom individuals from the Austrian general population with an equal distribution of participants in the predefined age and sex groups Participants were on average years old years when weighted and ofparticipants when weighted reported at least onehealth condition Some participants reported more thanone health condition when weighted reported two and when weighted reportedthree or more health conditions The most frequently reported health conditions were chronic pain when weightedand arthritis whenweighted Regarding relationship status the percentageof respondents in our sample who said that they were ina longterm relationship was and whenweighted The majority of respondents or when weighted reported at least some postcompulsoryeducation and or when weighted obtained auniversity degree ie bachelors or higher The full descriptive data are reported in Table Normative data for the EORTC QLQC30Table shows a summary of mean scores and standarddeviations for the EORTC QLQC30 across all analyzedsexage groups Men and women differed in several domains the three largest differences were observed forthe scales measuring insomnia points for womenvs points for men Cohens d fatigue points for women vs points for men Cohens d and emotional functioning points for womenvs points for men Cohens d The three largest differences between age groups wereobserved for the scales measuring dyspnea pointsfor those aged years vs points for those aged¥ years Cohens d pain points for thoseaged years vs points for those aged ¥years Cohens d and insomnia points forthose aged vs points for those aged years Cohens d See Table for the normativedata stratified by age groupFor functioning scales ceiling effects ie achieving themaximum score were lowest for emotional functioning n and most prevalent for social functioning n Floor effects ie achieving theminimum score were observed most frequently in nauseavomiting n and least common for fatigue n Regression model predicting EORTC QLQC30 scoresTable shows the regression model predicting individual EORTC QLQC30 scores using the weighted normative data This model predicts EORTC QLQC30 scoresusing the individuals sex age and presence of healthconditions An easy to use spreadsheet for the calculation of predicted values for individuals and groups isavailable online see Supplementary For example fora woman aged years with at least one health condition the predicted physical functioning score is calculated as follows Intercept Sex Age Age2 Interactionof Age and Sex Presence of HealthConditions DiscussionIn this study we present age and sexspecific normativedata for the EORTC QLQC30 from a sample of the Austrian general population Men and women differed in several domains most notably insomniafatigue andemotional functioning In general men reported a higherfunctioning except for social functioning and less symptom burden except for diarrhea than women did Olderparticipants ¥ years reported higher symptom burdeneg pain and dyspnea and lower physical and role functioning compared to younger participants We observedboth ceiling effects for functioning scales and floor effects for symptom scales for some scales of the EORTCQLQC30 However floor and ceiling effects were not unexpected considering that we administered a cancerspecific questionnaire to a general population sampleA potentiallimitation of panel studies is that thismethod of data collection may be prone to underrepresenting specific groups of individuals eg those whoare older or less educated Therefore it should be considered whether the assessment of general population datacollected via online surveys are truly representative Acomparison of our data with Statistics Austrias report suggests that the data obtained from the online survey are representative in terms of most basic individualcharacteristics age sex and marital status [] For example regarding the relationship status in our sample of participants stated to be in a longterm relationship Statistic Austria reported a similar rate with ofAustrian adults being in a longterm relationship The unemployment rate of the Austrian sample was whilethe unemployment rate in the report of the StatisticAustria institute was for individuals older than years Furthermore the prevalence rates for commonhealth conditions found in our data match other data onthe Austrian population The prevalence of selfreportedchronic pain in our sample is close to the prevalence of chronic pain in Austria [] as diagnosed by adoctor which is for chronic back pain and for chronic neck pain The percentage of participants withdiabetes in our sample is in line with the prevalence rate of diagnosed diabetes among adults estimated in the latest Austrian diabetes report published by 0cLehmann Health and Quality of Life Outcomes Page of Table Sample characteristics N Sex N Age yearsFemaleMaleM SDMedian IQREducationa N Less than compulsory no or some primary educationCompulsory about years of schoolingSome postcompulsory above years of schooling withoutreaching university entrance certificatePostcompulsory below universityCollege bachelors or equivalent levelUniversity degree Master Doc or equivalentPrefer not to answer aMarital statusa N SingleEmployment statusa N Health statusab N Married or in a steady relationshipSeparated divorced widowedPrefer not to answer aEmployed full timeEmployed part timeHomemakerStudentUnemployedRetiredSelfemployedOtherPrefer not to answer aNo health conditionAt least one health conditionChronic painHeart diseaseCancerDepressionCOPDArthritisDiabetesAsthmaAnxiety disorderObesityDrugalcohol use disorderOtherPrefer not to answerMissingM mean SD standard deviation IQR interquartile rangea For calculating percentages the category prefer not to answer was treated as missing datab The sum of all health conditions is larger than the sample size as respondents could choose multipleUnweighted data Weighted data 0cLehmann Health and Quality of Life Outcomes Page of Table EORTC QLQC30 normative data for the Austrian general population stratified by age and sex N Physical functioningM TotalFemalesAll ¥MalesAll ¥SD Role functioningM SD Emotional functioning M SD Cognitive functioning M SD Social functioningM SD Global healthQoLM FatigueNauseavomitingPainDyspneaInsomniaSD M SD M SD M SD M SD M SD Appetite lossM SD ConstipationM SD DiarrheaM SD Financial difficultiesM M Mean scores SD standard deviation QoL quality of lifeSD the Austrian Health Ministry [] For cancer the prevalence rate was which is only slightly lower than the prevalence rate found in the latest Statistics Austriacancer report [] Notable differences of our data to theStatistics Austria data [] were observed only regardingthe level of education More than onethird of individualstaking part in the online survey reported at least auniversitylevel education while in the Statistic Austriadata only of the sample report a university or comparable degree In our publication of international normative data for the EORTC CAT CORE based on theinternational dataset the relationship between educationand HRQoL scales was investigated in depth [] Highereducation some postcompulsory vsless than postcompulsory education was linked to more favorableHRQoL scores However differences were of low practicalrelevance as indicated by the small effect sizes alleta2 ¤ A strength of our study is the consistent data collectionmode used by Nolte [] which allows comparisonwith other European normative data Comparisons ofHRQoL ratings across country borders can be made providing insight into international differences [ ] Atthe nationaltwo differentstudy offerslevel our 0cLehmann Health and Quality of Life Outcomes Page of Table EORTC QLQC30 normative data for the Austrian general population stratified by age N Physical functioningMTotalRole functioningEmotional functioningCognitive functioningSocial functioningGlobal healthQoLFatigueNauseavomitingPainDyspneaInsomniaAppetite lossConstipationDiarrheaFinancial difficultiesSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMSDMM Mean scores SD standard deviation QoL quality of lifeSD¥approaches for interpreting EORTC QLQC30 data Firstthe normative data allow interpretation and comparisonfor different age and sex groups Second the regressionmodel permits the calculation of expected EORTC QLQC30 normative scores using sex age and the presence ofhealth conditions This regression model can be used to generate more precise predictions than those made throughcomparing different age and sex groups using normative dataCompared with other score interpretation approachesnormative data in particular when relying on regressionmodels offer the advantage of not reducing the amount ofinformation For examplein an alternative approachthresholds for clinical importance are used to condense theinformation into severity categories eg clinically important vs not clinically important which can ease interpretation but also decreases the amount ofinformationconveyed The data we present in this study can be used tocompare HRQoL among patients with cancer and HRQoLin the general population matched by sex and age groupSuch comparisons can be useful at any stage of the cancerjourney as patients HRQoL is likely to be compromised atthe time of diagnosis [] and they can be used to determine whether cancer survivors return to population levelsor whether problems and impairments persist [ ]Ideally such comparisons use countryspecific normativedata which most accurately reflect the average level of 0cLehmann Health and Quality of Life Outcomes Page of Table Regression model for predicting EORTC QLQC30 scores using age sex and the presence of health conditionsPhysical functioningRole functioningEmotional functioningCognitive functioningSocial functioningGlobal healthQoLFatigueNauseavomitingPainDyspneaInsomniaAppetite lossConstipationDiarrheaFinancial difficultiesInterceptSexAge in yearsAge in years squared Agebysex Health conditions yesnoCoding is sex male female age in years above age by sex age in yearssex health conditions no health conditions at least onehealth condition QoL quality of lifeHRQoL within a particular national context Of coursenormative data need a certain amount of currentness to berelevant For the years to come this publication can serveas a reference but data should be updated in due timeSeveral observed results merit in depth discussionThe sex differences observed across various domains especially physical functioning are in line with other normative studies on the EORTC QLQC30 that havereported differences by sex In studies in Germany [] Slovenia [] Denmark [] Sweden [] and theNetherlands [] men tended to report higher functioningand lower symptom burden on most scales than womendid A similar pattern can be observed in the presentedAustrian normative data though both point differencesand effect sizes were rather small Our data allow for better interpretation of these differences among patients withcancer through the comparison of the magnitude of impairment in both groups of individuals patients with cancer and members of the Austrian general population Ashas been discussed previously [] sex differences amongpatients with cancer do not necessarily reflect a sexspecific impact of disease or treatment rather these differences may reflect more general factors including sexspecific response styles that also affect the general population as we observed in the present sampleRegarding age a European sample [] as well assinglecountry studies in Denmark Sweden and Slovenia[ ] report similar deterioration with lower physical and role functioning in older people ¥ or ¥ years compared to younger people or years asfound in our sample Those studies also found emotionalfunctioning to be independent of age [ ] or to evenincrease with age ¥ years [ ] showing a similarpattern of emotional functioning and age as observed inour sample However our sample differed from two studysamples in Germany which did not show an increase inemotional functioning with age and reported much higherdisparity between age groups on the global health statusscale with a difference of up to points oldest vs youngest group in favor of younger people [ ]Compared with the European sample reported byNolte [] fewer respondents in our sample reported health conditions or diseases in the European sample vs in our sample reported having nohealth conditions and our sample showed higher functioning and lower symptom burden on most scales According to the EU Statistics on Income and LivingConditions the Austrian life expectancy is slightly abovethe EU average years vs years and ofthe Austrian adult population report a good or verygood perceived health which ranks higher than mostother European countries measured [] Both of thesefindings support our result of generally high HRQoL interms of fewer health conditions and high functioningamong the Austrian general populationConclusionsThe normative data for the EORTC QLQC30 generatedin this study will ease and foster a more meaningful interpretation of scores obtained from patients with cancer 0cLehmann Health and Quality of Life Outcomes Page of and cancer survivors allowing comparisons of patientlevel and grouplevel data with the sex and agematched general population sample from Austria Usingour regression model precise predictions for individualsbased on sex age and presence of health conditions canbe generatedSupplementary informationSupplementary information accompanies this paper at doi101186s12955020015248Additional file AbbreviationsEORTC European anisation for Research and Treatment of Cancer GfKSE Gesellschaft für Konsumforschung Societas Europaea HRQoL HealthRelated Quality of Life PRO PatientReported OutcomesAcknowledgementsThe authors thank the European anization for Research and Treatment ofCancer for permission to use the data from an EORTC study grant number for this research We thank Jennifer Barrett PhD from EdanzGroup enauthorservicesedanzgroupcom for languageediting adraft of this manuscriptAuthors contributionsJL analyzed the data interpreted the data and wrote the manuscript JMGinterpreted the data and helped to draft the manuscript LMW and MSinterpreted the data and edited the manuscript SN GL and MR generatedthe data and edited the manuscript BH analyzed and interpreted the dataassisted in generating the data and edited the manuscript All authors readand approved the final manuscriptFundingThis research was partly funded by the European anisation for Researchand Treatment of Cancer Quality of Life Group grant number Availability of data and materialsThe datasets analyzed in the study at hand are available upon reasonablerequest from the EORTC Please use the Data Sharing form available throughthe EORTC website wwweortcdatasharingEthics approval and consent to participateNo ethics approval was sought as the study is based on panel dataAccording to the NHS Health Research Authority and the EuropeanPharmaceutical Market Research Association EphMRA panel research doesnot require ethical approval if ethical guidelines are followed The survey wasdistributed via the GfK SE member of EphMRA and obtained informedconsent by each participant before the study All data were collectedanonymously and identification of the respondents through the authors isimpossibleConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1University Hospital of Psychiatry II Medical University of InnsbruckAnichstrasse Innsbruck Austria 2Division of PsychosomaticMedicine Medical Department Charité Universitätsmedizin BerlinCorporate Member of Freie Universität Berlin HumboldtUniversität uu Berlinand Berlin Institute of Health Berlin Germany 3School of Health and SocialDevelopment Population Health Strategic Research Centre DeakinUniversity Burwood VIC AustraliaReceived June Accepted July ReferencesBlazeby JM Avery K Sprangers M Pikhart H Fayers P Donovan J Healthrelated quality of life measurement in randomized clinical trials in surgicaloncology J Clin Oncol Cella D Grunwald V Nathan P Doan J Dastani H Taylor F Bennett BDeRosa M Berry S Broglio K Quality of life in patients with advancedrenal cell carcinoma given nivolumab versus everolimus in CheckMate a randomised label phase trial Lancet Oncol LeBlanc TW Abernethy AP Patientreported outcomes in cancer care hearing the patient voice at greater volume Nat Rev Clin Oncol Austin E LeRouge C Hartzler AL Segal C Lavallee DC Capturing the patientvoice implementing patientreported outcomes across the health systemQual Life Res US Food and Drug Administration PatientReported 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Quality of Life Outcomes Page of consecutive annual assessments of approximately representativeDutch men and women Acta Oncol Giesinger J Kemmler G Mueller V Zabernigg A Mayrbaeurl B Thaler JOfner D Pegger C Rumpold G Weber B Are genderassociateddifferences in quality of life in colorectal cancer patients diseasespecificQual Life Res Eurostat EU Statistics on Income and Living Conditions EU SILC eceuropaeueurostatdatabrowserviewsdg_03_20defaultmaplangen Accessed Mar Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsSchwarz R Hinz A Reference data for the quality of life questionnaire EORTCQLQC30 in the general German population Eur J Cancer Nolte S Liegl G Petersen MA Aaronson NK Costantini A Fayers PMGroenvold M Holzner B Johnson CD Kemmler G General populationnormative data for the EORTC QLQC30 healthrelated quality of lifequestionnaire based on persons across European countriesCanada and the unites 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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 |
Human pyruvate dehydrogenase phosphatase PDP1 plays an important physiological role in energy metabolism however its expression and function in human pancreatic adenocarcinoma PDAC remain unknown This study aimed to investigate the expression pattern and mechanisms of action of PDP1 in human PDACMethods The expression pattern of PDP1 in PDAC was determined and its correlation with patient survival was analyzed Ectopic expression or knockdown of PDP1 was performed and in vitro proliferation and migration as well as in vivo tumor growth of PDAC were measured The mechanism was studied by biochemical approachesResults PDP1 was overexpressed in human PDAC samples and high expression of PDP1 correlated with poor overall and diseasefree survival of PDAC patients PDP1 promoted the proliferation colony formation and invasion of PDAC cells in vitro and facilitated orthotopic tumor growth in vivo PDP1 accelerated intracellular ATP production leading to sufficient energy to support rapid cancer progression mTOR activation was responsible for the PDP1induced tumor cell proliferation and invasion in PDAC AMPK was downregulated by PDP1 overexpression resulting in mTOR activation and cancer progressionConclusion Our findings suggested that PDP1 could be a promising diagnostic and therapeutic target for antiPDAC treatmentKeywords Pancreatic adenocarcinoma Pyruvate dehydrogenase phosphatase ATP mTOR AMPKIntroductionPancreatic adenocarcinoma PDAC which accounts for of pancreatic cancer cases is among the top lifethreatening cancers with a very high death rate and a survival rate of only approximately [] The malignancy of PDAC has been increasing in recent years Correspondence zhaojiangang78163com lianyu_chenhotmailcom Ye Li and Jia Shen contributed equally to this work Department of Integrated Oncology Fudan University Shanghai Cancer Center Shanghai China Department of Oncology Shaoxing Central Hospital Zhejiang ChinaFull list of author information is available at the end of the and this disease is estimated to be the second leading cause of cancerrelated death in the USA [] The dismal prognosis of PDAC could be due to the lack of sensitive detection at its early stage although many biomarkers have been suggested as indicators of PDAC in various experimental studies [] Effective treatments for PDAC are unavailable Although surgical resection is recommended as the only curative treatment [] only a very low number of patients who have small tumors that are detected early are suitable for surgery [] Chemotherapy as the firstline treatment is applied to nonsurgical PDAC patients [] however the response rate is low and these drugs can only prolong survival by a few months even in 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 0cLi a0et a0al Cell Biosci Page of responding patients [] Identifying new prognostic and therapeutic biomarkers for PDAC patients is importantThe human PDP1 gene encodes pyruvate dehydrogenase phosphatase PDP one of the two PDP isoforms in mammalian cells [] Physiologically PDP1 serves as a critical regulator of the pyruvate dehydrogenases complex PDC PDP1 positively regulates the catalytic activities of PDC by the removal of phosphates from the serine sites on E1α of the complex [] Upon activation by PDP1 the PDC irreversibly triggers oxidative decarboxylation of pyruvate into acetylCoA which is the main substrate for cellular energy production [] Mutation of PDP1 in some patients may cause PDC deficiency a genetic disorder characterized by neurodegeneration and abnormal metabolism [] Loss of PDP1 may also cause intolerance to exercise and mild developmental delay in patients [ ] and may produce a lethal phenotype in infants [] The expression of PDP1 in muscle cells of obese and diabetic subjects was reduced and could be reversed by endurance training [ ] The loss of PDP1 in obesity was found to signal insulin resistance that could be reversed by plasma insulin supplementation [] Overexpression of PDP1 was also found in human prostate cancer and could promote cell proliferation and tumor growth [] The expression pattern and function of PDP1 in PDAC remain unclearIn this study we attempted to identify the expression and functional role of PDP1 in human PDAC The expression pattern of PDP1 in human PDAC samples was illustrated by extracting data from the GEO database The correlation between PDP1 expression and patient survival was profiled to evaluate its prognostic value The functional role of PDP1 in the proliferation growth and invasion of PDAC cells was then evaluated in cellular and animal models The signaling pathway involved in the regulation of PDP1 was elucidated We believe this study will shed light on the prognostic and therapeutic value of PDP1 in PDACMaterials and a0methodsReagents plasmids and a0antibodiesSodium acetate and compound C were obtained from SigmaAldrich USA Human and murine ORF clones of PDP1 and shRNA against human PDP1 were purchased from Origene USA Antibodies against PDP1 phosphomTOR mTOR phosphoAMPKα AMPKα PCNA and actin were purchased from Cell Signaling Technologies USACell line and a0cell cultureThe PDAC cell line KP3 was purchased from the Japanese Collection of Research Bioresources Cell Bank Cells were cultured in RPMI medium supplemented with fetal bovine serum FBS and penicillinstreptomycin under humid conditions of CO2 and a0°C Panc2 cells were obtained from the Frederick National Laboratory for Cancer Research Frederick MD USA Cells were cultured in DMEM supplemented with FBS and penicillinstreptomycin under humid conditions of CO2 and a0°CAnimal studyThe animal study was performed in accordance with the approved protocol by Fudan University Ref No 2019FUSCCJS017 In brief Panc2 cells tagged with a luciferase reporter were mixed with Matrigel Matrix BD Bioscience USA A a0μl mixture containing Panc2 cells was injected into the pancreas of C57BLJ mice Measurement of orthotopic tumor size was performed once per week from a0week postinjection using the IVIS Spectrum live animal imager PerkinElmer USA with luciferin a0mgkg ip as a substrate At the end of the study the mice were sacrificed and the pancreas was dissectedCell proliferation and a0colony formation assaysCell proliferation was measured by cell counting In brief cells were seeded for growth and were counted at and a0days of cultivation The formation of colonies was measured by colony formation assays A total of cells were seeded for growth for a0days Cells were fixed with paraformaldehyde and stained with crystal violet The number of colonies formed was countedCell invasion assayThe invasiveness of PDAC cells was measured by chamber assays In brief cells were seeded onto the upper chamber of inserts precoated with Matrigel matrix BD Bioscience USA The receiving chamber was filled with DMEM supplemented with FBS Fortyeight hours later the medium on the upper chamber was removed and cells that migrated through the Matrigel matrix were dissociated with calceinAMcontaining buffer The fluorescence intensity representing the number of invaded cells was measured by a fluorescence microplate readerImmunoblottingTotal protein was extracted from the cell pellets using RIPA buffer and separated by SDSPAGE Protein was then transferred onto the PDVF membrane which was blocked in BSA blocking buffer for a0h at room temperature Then the membrane was incubated with primary antibodies overnight at a0°C followed by secondary 0cLi a0et a0al Cell Biosci Page of antibody incubation for a0 h at room temperature The membrane was visualized with chemiluminescence BioRad USA using ECL Select as a substrate GE Healthcare GermanyIntracellular ATP measurementIntracellular ATP measurement was performed with a commercial kit Biovision USA In brief cells were lysed with a0μl of ATP assay buffer followed by deproteinization through a a0 kDa spin column Then a0μl of flow through was mixed with a0μl of ATP assay buffer a0μl of ATP probe a0μl of ATP converter and a0μl of developmental reagent for reaction in the dark for a0min at room temperature The absorbance was then read at a0 nm The concentration of ATP in the cell samples was calculated with a standard curve plotted using an ATP standardHistologyParaffinembedded pancreatic sections were prepared and 5mm slices were generated For histological measurement of PDAC the slides were stained with hematoxylin eosin HE The image was captured under a light microscopeStatistical analysisData are presented as the mean ± SD Data were analyzed by oneway ANOVA and p was considered statistically significant All experiments were performed in triplicate unless stated otherwiseResultsPDP1 was a0overexpressed in a0PDAC and a0overexpression of a0PDP1 predicted poor prognosis of a0patientsThe expression and function of PDP1 in PDAC have not been reported A recent study showed that the expression of PDP1 was elevated in prostate cancer in which ectopic expression of PDP1 promoted cancer growth and progression [] Another study however observed that phosphorylation of PDP1 at Tyr94 suppressed its activity and reduced tumor growth [] These divergent observations indicated that PDP1 may have different functions in particular types of cancers To determine the role of PDP1 in PDAC we first examined its expression in human PDAC samples Data retrieved from two published datasets of PDAC GSE15471 and GSE28735 revealed that PDP1 was overexpressed in human PDAC tissues compared with the nonmalignant normal pancreatic tissues Fig a01a b Immunohistochemical examination of the tissue blocks confirmed that the protein expression of PDP1 was substantially elevated in the PDAC tissues Fig a01c To determine the clinical significance of this elevation we retrieved patient survival data from TCGA database We found that the expression of PDP1 predicted poor overall survival and diseasefree survival of the PDAC patients p and p respectively which indicates that PDP1 may be a poor prognostic factor in PDAC Fig a0 1d e Furthermore PDP1 may be a marker for patient mortality as its expression was significantly higher in patients who died than those who survived p Fig a01f These findings suggested that PDP1 was overexpressed in PDAC tissues and its overexpression can predict a poor prognosis for patientsPDP1 regulates the a0in a0vitro proliferation and a0invasion of a0PDAC cellsTo further elucidate the functional role of PDP1 in PDAC cells1 we first identified its in a0 vitro function in a set of PDAC cells The expression of PDP1 in different PDAC cells including MiaPaca2 Panc1 Panc2 KP3 and Capan1 was screened to select suitable cell lines for functional studies data not shown Panc2 and KP3 were selected due to their moderate level of PDP1 expression among the cell lines We then overexpressed PDP1 by transfecting a plasmid encoding the ORF of the genes while knocking down its expression by shRNA against PDP1 and established stably transfected cell lines by selection Fig a0 2a A proliferation assay was performed to examine whether PDP1 expression could alter cell growth in PDAC We found that in both cell lines PDP1 overexpression significantly promoted the growth rate of the cells while PDP1 knockdown had the opposite effects Fig a0 2b suggesting that PDP1 expression may promote tumor cell proliferation in PDAC This finding was further confirmed by colony formation assays which showed that PDP1 overexpression potently increased the number of colonies formed by PDAC cells while suppression of PDP1 reduced the ability to form colonies Fig a02c In addition as the in a0vitro invasiveness of tumor cells indicates the in a0vivo locoregional and distant spread of cancer we examined the invasion of PDAC cell lines with a chamber assay Overexpression of PDP1 significantly increased the number of cells invading through the extracellular matrix Fig a0 2d These observations suggested that PDP1 expression could promote the in a0vitro proliferation and invasion of PDAC cellsPDP1 overexpression accelerated the a0in a0vivo tumor growth of a0PDACTo further elucidate the in a0vivo functional role of PDP1 we established an orthotopic model of PDAC by injecting Panc2 cells into the pancreas of C57BL6J mice For noninvasive monitoring of tumor growth Panc2 cells with or without PDP1 overexpression were stably infected with a luciferase reporter and luciferin could be catalyzed to 0cLi a0et a0al Cell Biosci Page of Fig PDP1 was overexpressed in PDAC Data were retrieved from the GEO database and PDP1 was found to be overexpressed in PDAC tissues compared with normal pancreatic tissues in a GSE15471 and b GSE28735 c Immunohistochemical staining of PDP1 was retrieved from the Human Protein Atlas which suggested that PDP1 protein expression was upregulated in PDAC tissues Survival of PDAC patients was retrieved from TGCA database and patients with PDP1 expression above the median level had poor d overall survival and e diseasefree survival f Surviving PDAC patients had lower expression of PDP1 than the censored patientsFig PDP1 regulated PDAC cell proliferation and migration in vitro a Transfection of the PDP1 ORF clone or shRNA regulated endogenous expression of PDP1 in PDAC cells b Overexpression of PDP1 promoted PDAC cell proliferation while PDP1 suppressed PDAC cell proliferation c Overexpression of PDP1 promoted colony formation of PDAC cells while PDP1 suppressed PDAC cell colony number d Overexpression of PDP1 promoted PDAC cell migration while PDP1 suppressed PDAC cell migration p 0cLi a0et a0al Cell Biosci Page of emit bioluminescent signals Under an in a0vivo imager we observed that overexpression of PDP1 could significantly increase the signal intensity of tumor cellderived luciferase indicating a larger tumor formed by PDP1overexpressing PDAC cells than the control cells Fig a0 3a Endpoint measurement of the dissected tumors showed that tumor growth was accelerated by PDP1 overexpression Fig a03b Histological analysis by HE staining showed that the PDP1overexpressing PDAC cells had a more aggressive phenotype eg an unclear border between pancreatic tissues and the tumor tissues than the control cells Fig a03c This result was further proven by the immunoblotting of PCNA acellular marker of proliferation in PDAC tissues the expression was significantly higher in the PDP1overexpressing PDAC tumors than the control tumors Fig a03d These findings suggested that PDP1 overexpression could substantially accelerate the in a0vivo tumor growth of PDACPDP1 overexpression accelerated ATPassociated tumor cell growth in a0PDACMitochondrial PDP1 is an important regulator in energy production and metabolism in mammalian cells PDP1 catalyzes the dephosphorylation and concomitant reactivation of the α subunit of the E1 component of the pyruvate dehydrogenase complex PDC whose activity is critical for energy metabolism through the TCA cycle and oxidative phosphorylation [] A previous study suggested that suppression of PDP1 expression in embryonic stem cells is associated with reduced production of ATP the product of energy metabolism that is essential for many cellular processes [] Therefore we examined the cellular content of ATP in PDAC cells Overexpression of PDP1 could significantly increase the cellular ATP content in PDAC cells while knockdown of the protein showed the opposite effects Fig a04a To determine whether cellular ATP content plays an important role in mediating PDP1induced cell proliferation we supplied acetate as a substrate for ATP production by alternative mechanisms [] in PDAC cells with PDP1 knockdown The reconstitution of cellular ATP by acetate supplementation significantly reversed tumor cell proliferation in the cells with PDP1 knockdown Fig a0 4b Colony formation and cell invasion assays proved that the suppressive effect of PDP1 knockdown was attenuated by ATP supplementation in PDAC cells Fig a0 4c d These observations suggested that PDP1regulated cell proliferation and invasion in PDAC cells may be associated with the alteration of cellular ATP content and its related cellular activitiesmTOR is a0a a0downstream signaling pathway that a0mediates PDP1driven tumor cell proliferation in a0PDACmTOR signaling is often aberrantly hyperactivated in various types of cancer cells in response to the abundant cellular ATP to promote tumor cell proliferation [] In our study we examined the expression and activity of the mTOR pathway in PDAC cells with PDP1 knockdown Fig PDP1 overexpression promoted PDAC growth in vivo a PDP1 overexpression significantly increased the luciferase signal in the orthotopic tumors of PDAC in mice b PDP1 overexpression increased the tumor size of the orthotopic PDAC model c HE staining suggested that more cells were undergoing mitotic proliferation in the PDP1overexpressing tumors than the control tumors d PDP1overexpressing tumors expressed a higher level of PCNA than the control tumors p 0cLi a0et a0al Cell Biosci Page of Fig PDP1 induced PDAC cell proliferation and migration by inducing intracellular ATP a PDP1 overexpression induced intracellular ATP content while its knockdown reduced ATP levels Supplementation of an alternative substrate of ATP production acetate restored cell proliferation b colony formation c and migration of PDP1 knockdown PDAC cells d p Upon knockdown of PDP1 mTOR phosphorylation was significantly downregulated without notable changes in total protein expression Fig a0 5a To identify whether mTOR activity is crucial for PDP1mediated tumor cell proliferation we transfected a plasmid encoding the protein with a mutation that leads to constitutive activation of mTOR in PDAC cells with PDP1 knockdown Fig a05b Constitutive activation of mTOR restored the proliferation rate in the PDAC cells with PDP1 knockdown Fig a05c suggesting that mTOR activation is essential for PDP1driven tumor cell growth in PDAC In addition reactivation of mTOR in PDAC cells with PDP1 knockdown reconstituted the capacity of PDAC cells to form colonies and pass through the ECM Fig a0 5d e These findings indicate that mTOR activation mediates PDP1driven cell growth and invasion in PDAC as the downstream effectorAMPKα inhibition mediates PDP1induced mTOR activation in a0PDAC cellsWhen the cellular content of ATP is insufficient cells tend to activate AMPKα as a stress response pathway to limit growthrelated signaling [] Activation of AMPKα in PDAC was found to restrict cancer cell proliferation and aggressiveness in previous studies [ ] As PDP1 inhibition reduced ATP content as we observed in the present study we investigated whether AMPKα activity could be altered by PDP1 Overexpression of PDP1 potently suppressed the basal level of phosphorylated AMPKα without changing its total protein level while repressing PDP1 could activate AMPKα Fig a0 6a Inhibition of AMPKα in PDAC cells by its pharmacological inhibitor compound C CC restored mTOR activity Fig a0 6b suggesting that AMPKα acts as the upstream regulator of mTOR in the PDAC cells with PDP1 knockdown Furthermore inhibition of AMPKα significantly reactivated the proliferation colony formation and invasion of PDAC cells Fig a06ce A previous study showed that an increased AMPATP ratio would activate AMPK activity [] We then added AMP to the PDP1overexpressing cells and found that an increased AMPATP ratio led to the restoration of AMPK in the these cells Fig a0 6f An opposite trend in mTOR activity was observed as mTOR is a downstream effector of AMPK These observations together with the other findings in this study indicated that PDP1 regulates PDAC growth by modulating the ATPAMPKαmTOR pathwayDiscussionIn this study we observed that PDP1 was overexpressed in human PDAC Overexpression of PDP1 in PDAC cells promoted cell proliferation and migration in a0 vitro and stimulated tumor growth in a murine model of PDAC which could be related to the increase in intracellular energy production Fig a0 Expression of PDP1 was reported to be associated with ATP generation in previous studies for instance PDP1 expression and activity were shown to contribute to a higher aerobic capacity 0cLi a0et a0al Cell Biosci Page of Fig mTOR was responsible for PDP1mediated PDAC cell proliferation and migration a The phosphorylation of mTOR in PDAC cells representing its activity was measured by immunoblotting PDP1 overexpression activated mTOR signaling while its knockdown suppressed mTOR phosphorylation b Transfection of constitutively activated mTORC reactivated mTOR signaling in PDP1 knockdown PDAC cells mTORC represents a plasmid encoding a mutated mTOR with a change from arginine to proline which leads to constitutive activation of mTOR regardless of intracellular signaling Reactivation of mTOR restored cell proliferation c colony formation d and migration of PDP1 knockdown PDAC cells e p of the muscle suggesting a sustainable level of oxidative phosphorylationrelated ATP production [] Another study also suggested that activation of PDP1 in cancer cells could induce a switch from cellular glycolysis to oxidative phosphorylation the more efficient method of ATP production [] The depletion of cellular ATP abolished PDP1induced PDAC cell proliferation migration and invasion suggesting that ATP generation was the essential step by which PDP1 triggers these cellular processes PDP1 expression was also found to be critical in some other cellular processes such as recovery from injury and cell differentiation [ ] Given the important regulatory role of PDP1 in the physiological function of PDC our observations may suggest that overexpression of PDP1 in PDAC cells functions to hyperactivate the PDC system overloading the cellular substrate of the tricarboxylic acid cycle which in turn accelerates the oxidative phosphorylation process that can generate more ATP essential for cellular processesWe used acetate as an alternative carbon source in tumor cells with PDP1 knockdown PDP1 is a critical enzyme involved in the pathway of ATP production by consuming glucose PDP1 is involved the reaction of pyruvate to acetylCoA the direct source of the TCA cycle This facilitates the production of reducing equivalents in the form of NADH FADH2 etc by TCA cycles which can be used by oxidative phosphorylation for the production of ATP Acetate has been reported as an additional source for ATP production in cancer cells especially when glucoseglutamine is depleted [] The direct ligation of acetate to CoA by acetylCoA synthetases can yield acetylCoA in the TCA cycle [] The TCA cycle can therefore produce reducing equivalents in the form of NADH FADH2 etc which were used for ATP production by oxidative phosphorylation in PDP1 knockdown cells As we assumed that suppression of ATP production in PDP1 knockdown cells is responsible for inhibition of tumor growth we used acetate as an alternative source of ATP production in these cells and our observations proved that ATP restoration in PDP1 knockdown cells recovered tumor cell proliferation and growth supporting our claims We also observed that the addition of acetate had a minimal effect on tumor growth in mock tumor cells Mock cells can consume glucose to 0cLi a0et a0al Cell Biosci Page of Fig AMPK inactivation was responsible for PDP1mediated PDAC cell proliferation and migration a PDP1 overexpression suppressed AMPK signaling while its knockdown induced activation via phosphorylation b The presence of compound C mM inactivated AMPK signaling in PDP1 knockdown PDAC cells which in turn reactivated mTOR signaling Reactivation of mTOR restored cell proliferation c colony formation d and migration of PDP1 knockdown PDAC cells e f AMP was supplemented in the PDP1overexpressing cells to adjust the intracellular AMPATP ratio Increased AMPATP ratio in the PDPoverexpressing cells resulted in the restoration of AMPK activity but mTOR repression p Fig Schematic regulation of PDP1 in PDAC Overexpression of PDP1 may activate PDH which in turn accelerates the TCA cycle which provides reducing equivalents in the form of NADH FADH2 etc for the ATP production by oxidative phosphorylation The abundance of cellular ATP inhibits AMPK activation restoring the mTOR signaling that can promote proliferation and invasion of PDAC cellsproduce acetate which is then directly ligated by acetylCoA synthetases in the TCA cycle The reason for these results after addition of acetate to cells with an intact pathway of glucose metabolism is not fully understood however as the TCA cycle has a strict regulatory mechanism that involves production of reducing equivalents as 0cLi a0et a0al Cell Biosci Page of sources of ATP production this complicated process has ratelimiting steps even when the acetylCoA amount is overwhelming In this regard the addition of acetate may not further accelerate cell proliferation and growth in mock cells which can normally consume glucose to produce ATPWe observed that AMPKmTOR signaling was involved in PDP1 regulation of PDAC progression As an energy sensor AMPK is activated when the intracellular ATP level is insufficient and triggers a series of downstream responses that inhibit rapid cell proliferation hence AMPK has been frequently identified as a potential target in anticancer treatment [] PDP1 overexpression could induce activation of PDC which in turn generates more substrate for the production of ATP In this case overexpression of PDP1 in our study resulted in sustained repression of AMPK signaling due to the abundance of intracellular ATP that led to suppression of multiple downstream proliferationassociated mechanisms mTOR is a key molecule that mediates rapid protein synthesis during cancer progression [] Phosphorylation of mTOR signaling activates translational control by initiating the formation of the ribosome complex as well as the association of mRNA at its cap [] This process was halted under nutrientdeprived conditions in which mTOR was dephosphorylated and could not initiate capdependent translation [] The activation of AMPK as a nutrient sensor in some previous studies has been found to primarily downregulate mTOR activity in cancers [ ] PDP1mediated AMPK repression therefore could be a possible mechanism to facilitate mTORassociated PDAC progression which was confirmed by our observation that suppression of AMPK in PDP1 knockdown cells revoked mTOR signalingAMPK signaling which in turn suppressed mTOR activity Repression of AMPK in PDP1 knockdown cells restored the proliferation and migration of PDAC Our study suggested that PDP1 may be an attractive target for the diagnosis and treatment of PDACAcknowledgementsNot applicableAuthors contributionsLC and JZ conceived the idea designed the experiments analyzed the data and drafted the manuscript YL and JS performed the experiments and analyzed the data CC performed the experiment HG revised the manuscript All authors read and approved the final manuscriptFundingThis work was financially supported by the National Natural Science Foundation of China and Wu JiePing Medical Foundation Availability of data and materialsThe datasets used andor analysed during the current study are available from the corresponding author on reasonable requestEthics approval and consent to participateThe animal study was performed in accordance with the approved protocol by Fudan University Ref No FUSCCJSConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Integrated Oncology Fudan University Shanghai Cancer Center Shanghai China Department of Oncology Shanghai Medical College Fudan University Shanghai China Department of Oncology First Peoples Hospital of Pinghu Zhejiang China Department of Oncology Shaoxing Central Hospital Zhejiang China Received April Accepted July ConclusionIn summary in this study we identified the expression pattern and function of PDP1 in PDAC PDP1 was overexpressed in PDAC samples compared with normal pancreatic samples and high expression of PDP1 was correlated with a poor prognosis of PDAC patients Overexpression of PDP1 promoted the in a0 vitro proliferation colony formation and migration of PDAC cells while knockdown of PDP1 had the opposite effects Overexpression of PDP1 stimulated the in a0 vivo growth of orthotopic PDAC tumors in a murine model PDP1 regulated the production of intracellular ATP and supplementation with ATP recovered tumor cell proliferation and migration in PDP1 knockdown PDAC cells This result could be related to the alteration of mTOR activity in PDAC cells and recovery of mTOR activity in PDP1 knockdown PDAC cells restored cell proliferation and migration Mechanistically PDP1 knockdown activated References Collaborators GBDPC The global regional and national burden of pancreatic cancer and its attributable risk factors in countries and territories a systematic analysis for the Global Burden of Disease Study Lancet Gastroenterol Hepatol Rahib L Smith BD Aizenberg R Rosenzweig AB Fleshman JM Matrisian LM Projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the United States Cancer Res Kim J Bamlet WR Oberg AL Chaffee KG Donahue G Cao XJ Chari S Garcia BA Petersen GM Zaret KS Detection of early pancreatic ductal adenocarcinoma with thrombospondin and CA19 blood markers Sci Transl Med Hackert T Surgery for Pancreatic Cancer after neoadjuvant treatment Ann Gastroenterol Surg Satoi S Yamamoto T Yamaki S Sakaguchi T Sekimoto M Surgical indication for and desirable outcomes of conversion surgery in patients with initially unresectable pancreatic ductal adenocarcinoma Ann Gastroenterol Surg Chandana S Babiker HM Mahadevan D Therapeutic trends in pancreatic ductal adenocarcinoma PDAC Expert Opin Investig Drugs 0cLi a0et a0al Cell Biosci | Thyroid_Cancer |
case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies PLGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors 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 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patients glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic 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"Insulin shares a limited physiological concentration range with other endocrine hormones Not onlytoo low but also too high systemic insulin levels are detrimental for body functionsMain body The physiological function and clinical relevance of insulin are usually seen in association with its rolein maintaining glucose homeostasis However insulin is an anabolic hormone which stimulates a large number ofcellular responses Not only too low but also excess insulin concentrations are detrimental to the physiologicalbalance Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening theefficiency of insulin signaling insulin resistance this is not the case for most other hormonal actions of insulinincluding the promotion of protein synthesis de novo lipogenesis and cell proliferation the inhibition of lipolysisof autophagydependent cellular turnover and of nuclear factor E2related factor2 Nrf2dependent antioxidativeand other defense mechanisms Hence there is no general insulin resistance but selective impairment of insulinsignaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthaseeNOS Because of the largely unrestricted insulin signaling hyperinsulinemia increases the risk of obesity type diabetes and cardiovascular disease and decreases health span and life expectancy In epidemiological studieshighdose insulin therapy is associated with an increased risk of cardiovascular disease Randomized controlled trialsof insulin treatment did not observe any effect on disease risk but these trials only studied low insulin doses up to IUday Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes fromMendelian randomization studies comparing individuals with genetically controlled low or high insulin productionConclusions The detrimental actions of prolonged high insulin concentrations seen also in cell culture argue infavor of a lifestyle that limits circadian insulin levels The health risks associated with hyperinsulinemia may haveimplications for treatment regimens used in type diabetesKeywords Hyperinsulinemia Insulin resistance Nrf2 Autophagy eNOS Obesity Type diabetes mellitusInflammation Oxidative stress Cardiovascular morbidity and mortalityBackgroundMost endocrine hormones exhibit a window of optimalphysiological concentrations with compromised function of the anism at levels below or above that rangeFor instance subnormal levels of thyroid hormone define the clinical condition of hypothyroidism above normalrepresent hyperthyroidism which usuallyrequires therapy Addisons disease is characterized bylevels Correspondence kerstinkempfwdgzde2WestGerman Centre of Diabetes and Health Duesseldorf Catholic HospitalGroup Hohensandweg Duesseldorf GermanyFull list of author information is available at the end of the insufficient cortisol production while excess synthesis isseen in Cushing syndromeFor insulin we argue here that not only hypoinsulinemiabut also hyperinsulinemia is detrimental to body functionsHypoinsulinemia causes insulindeficient diabetes and thehormonal actions of insulin are necessary for the life of complex anisms [] On the other hand permanently elevatedlevels of insulin may cause disturbance of normal cellularphysiology and an function We describe the molecularbasis of these undesired insulin actions and consequences ofhyperinsulinemia for healthrelevant endpoints such as obesity or cardiovascular diseases 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 0cKolb BMC Medicine Page of transformingproteins345trisphosphateMain textInsulin signaling pathwaysBinding of insulin to its cognate cell surfacebound receptor causes a conformational change which initiatesa cascade of signaling events Autophosphorylation bythe insulin receptor tyrosine kinase is accompanied bytyrosine phosphorylation of receptor substrates suchas insulin receptor substrate IRS and Src homology domaincontainingSHCproteins Phosphorylation of IRS allows binding ofphosphatidylinositol3kinase PI3K and synthesis ofphosphatidylinositolPIP3which eventually leads to the phosphorylation and activation of the serinethreoninespecific protein kinaseB AKT Upon activation AKT interacts with severalsubstrates which mediate anabolic effects of insulinthese include glucose uptake glycogen synthesis denovo lipogenesis and protein synthesis [] Additionalpathways triggered by the activated insulin receptorcomprise phosphorylation of SHC followed by activathe Rat sarcoma Rasrapidly acceleratedtion offibrosarcoma Rafmitogenactivated protein kinasesignalregulated kinasekinaseERK pathway Theamitogenactivated kinase promoting cell proliferationand further cellular activities including protein synthesis [] Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase and subsequent hydrogen peroxidemediated inhibition of phosphatase and tensin homolog PTENwhich is an important negative regulator of PI3Ksignaling [] Fig terminal kinase ERK isMEKextracellularInsulin secretionInsulin secretion by pancreatic islet cells responds tothe level of circulating nutrients such as glucose aminoacids and free fatty acids Sweeteners may further increase carbohydrateinduced insulin secretion A largenumber of endogenous factors contribute to the regulation of cell activity either stimulatory inhibitory orboth contextdependent These include hormones neurotransmitters and immune mediators [] Insulin isessential for maintaining glucose homeostasis primarilyby facilitating the postmeal uptake of glucose intomuscle and fat cells via translocation of the glucosetransporter [] In the absence of dietary glucose supply and after depletion of glycogen stores glucose in circulation primarily comes from gluconeogenesis in theliver If circulating insulin levels are below the concentrations required for stimulating glucose uptake fromthe blood endogenous stores of fat and protein must beused for energy production For the maintenance of lifein the fasting state circulating insulin levels range between approx and pmoll percentile asdetermined for healthy adult persons in the NationalHealth and Nutrition Examination Survey NHANES[] In response to meals with varying carbohydratecontent insulin levels may rise to the range of approx pmoll []Insulin promotes obesityAlmost years ago insulin injections were one of theoptions of therapy in nondiabetic persons suffering fromundernutrition in the context of various diseases Insulindoses were in the range of those applied in type Fig Metabolic signaling of insulin is anabolic Insulin signaling through the insulin receptor engages several pathways and results in ananabolic state of metabolism The canonical pathway via phosphokinases PI3K and AKTPKB promotes glucose uptake and glycogen and lipidsyntheses whereas lipolysis is inhibited in adipocytes as well as hepatic gluconeogenesis In addition AKT kinases activate mTORC1 whichsupports de novo lipogenesis and protein synthesis The insulin signaling pathway via SHC and the MAP kinases MEK and ERK promotes cellproliferation and protein synthesis Another insulin signaling pathway involves NOX4 and the inhibition of PTEN an inhibitor of the PI3KAKT pathway 0cKolb BMC Medicine Page of diabetes and led to increased appetite and weight gain[] Indeed one major function of insulin as an anabolic hormone is to favor energy storage over usageThis is reflected by the finding that insulin infusion mUkgmin significantly inhibits lipolysis in the skeletalmuscle about and even more effective in adiposetissue about [] Doubling fasting insulin levelssuffices to inhibit lipolysis by approx and to promote lipogenesis for both mean insulin concentrationfor effect EC50 of approx pmoll [] At thisinsulin level gluconeogenesis is still ongoing For halfmaximal inhibition of gluconeogenesis insulin concentrations must rise to approx pmoll in arterial circulation In order to stimulate glucose uptake to halfmaximum insulin levels must rise to even higher levelsapprox ten times the fasting insulin concentrations percentiles for stimulating glucose uptake approx pmoll [] Thus a modest rise doubling offasting insulin levels will already substantially inhibit lipolysis and promote lipogenesis while gluconeogenesis isnot yet inhibited Since such small increases of systemicinsulin concentrations are enough for favoring adipogenesis fasting and diurnal insulin levels are a determinantof obesity risk Indeed several data support the obesitypromoting role of insulin for a detailed review see []Fig These include epidemiological studies which foundhigh fasting insulin levels and concomitant insulin resistance in children and adolescents to be associatedwith higher weight gain in later years [] Studies inadults are less consistent [] Pharmaceutical interventions that lower insulin secretion such as treatment withdiazoxide or octreotide led to significant body weightloss [] This fits with the observation that insulintherapy promotes weight gain [] One probable reasonis that insulin levels in the high normal range are closeto EC50 concentrations for inhibition of lipolysis []In mice modest lowering of circulating insulin concentrations by genetic manipulation ofinsulin genescaused resistance to weight gain despite a highfat diet[] Decreasing insulin gene expression in adult micevia partial gene ablation reversed dietinduced obesity[] In men the Hph1 T polymorphism in the insulingene region was found to be associated with higher fasting insulin levels and a more rapid weight gain in obesepersons[] A Mendelian randomization analysisshowed that persons with genetically determined higherinsulin secretion to oral glucose exhibited a higher bodymass index BMI [] supporting a causal relationshipbetween insulin and obesity riskTaken together moderate to high normal levels of insulin in metabolic healthy persons appear to be a riskfactor for the development of obesitytransientElevated insulin concentrations impair cellularfunctionsinsulin toxicityThere is ample evidence thatincreases ofmetabolic or immune mediator levels are benign physiological responses to biochemical challenges such as therise of systemic glucose or cytokines following mealsHowever chronic elevations of such mediators evenwhen modest in amplitude are usually detrimental tocellular functions [] In the case of glucose the termglucose toxicity was coined to describe this phenomenon[] Prolonged conditions of elevated glucose concentrations cause dysfunction of numerous cell types in thebody including beta cells neurons and the endothelium via several pathways including increased oxidative stress and activation of the sorbitol pathway [] As described below there seems to be a similardetrimental outcome oflongterm elevated insulinconcentrations on cellular functions a correspondingterm would be insulin toxicityFig Insulin promotes obesity Several independent types of observations support the conclusion that insulin promotes adipogenesis andobesity For details see description in the general text 0cKolb BMC Medicine Page of When cells are exposed to continuously elevated insulin levels there is a partial downregulation of insulin signaling The resulting insulin resistance is not primarilydue to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as aresult of receptor dysfunction In response to prolongedhyperinsulinemia there is diminished autophosphorylation of the insulin receptor compared to that observedafter shortterm exposure to insulin and subsequentsteps of the PI3KAKT signaling pathway are affected[ ] Consequently in muscle and fat cells there isless AKTstimulated translocation of GLUT to the cellsurface Fig Thus insulin resistance can be seen as aprotective mechanism for preventing excess activation ofglucose transport from the blood despite chronically elevated insulin levels for maintaining glucose homeostasisin vivo and for mitigating metabolic and oxidative stressdue to excess glucose influx [] Limiting glucoseexportfrom the blood does not necessarily requiredampening of insulin signaling During the early weeksof feeding with a high caloric diet mice show decreasedinsulindependent glucose uptake despite unperturbedinsulinstimulated AKT phosphorylation [ ] Fig An interesting aspect is that the partitioning of insulinreceptor isoforms A and B and of hybrid insulininsulinlike growth factor1 receptors among cell types maycontribute to insulin resistance in some tissues but thepathophysiological relevance is unknown []The phenomenon of insulin toxicity partly arises fromthe fact that there are additional cellular responses to elevated insulin levels which are not toned down duringrole ofinsulin resistance Fig These comprise the upregulation of protein synthesis and the accumulation of ubiquitinated or otherwise modified proteins probably dueto insufficient degradation of these polypeptides [] Amajorinsulin signaling via the canonicalmitogenactivated protein MAP kinase pathway RasMEKERK as well as via activation of NADPH oxidase has been observed [] Even some AKTdependentpathways do not appear to be suppressed by insulin resistance such as de novo lipogenesis in hepatocytes orthe upregulation of mechanistic target of rapamycincomplex mTORC1 [] Enhanced activity ofmTORC1 leads to increased protein synthesis and to deteriorated cell functions largely because of suppressedautophagy []Hence chronic exposure of cells to high ambient insulin concentrations causes an imbalance of cellular responses because of the downregulation of some insulinsignaling pathways insulin resistance but not ofothers The resulting functional state of cells is characterized by an unbalanced anabolic activity of insulin favoring protein synthesis while suppressing autophagyThe latter inhibits autophagic removal and turnover ofproteins and lipids which favors cell senescence [] Inshortterm experiments of exposure to high insulinlevels a protective cellular stress response is observedthe unfolded protein response probably due to the accumulation of derivatized proteins in the absence ofenough disposal In experimentally induced or diabetesassociated chronic insulin resistance and hyperinsulinemiathesuch a protectivestressresponse ofFig Signaling of insulin during insulin resistance During insulin resistance signaling through AKT kinases is partially impaired Not all AKTdependent pathways are affected as well as other signaling pathways indicating that insulin resistance is selective Therefore hyperinsulinemiain the presence of insulin resistance promotes anabolic cell activities via the MEKERK pathway and via mTORC1 Although the PI3KAKT pathwayis impaired during insulin resistance and provides only insufficient translocation of GLUT4 for glucose uptake and deficient activation of eNOSthere appears to be a normal activation of mTORC1 In addition to the anabolic consequences of signaling via the MEKERK pathway depicted inthe figure there is enhanced expression of ET1 and PAI1 not shown as well as inhibition of autophagy and of the nuclear factor Nrf2 whichcompromises cell constituent turnover and cell defense mechanisms to radical stress respectively Hyperinsulinemia downregulates glucoseuptake not only via dampening the PI3KAKT pathway insulin resistance but also via as yet unknown other pathways 0cKolb BMC Medicine Page of endoplasmic reticulum to high insulin levels is diminished or absent []Another activity of insulin is the suppression of transcription of the nuclear factor Nrf2 via induction of heterogeneous ribonucleoproteins F and K [] Nrf2 is thecentral regulator ofthe protective response of cellsagainst oxidative and other types of electrophile stress[] Suppression of Nrf2 expression is expected to impair the antioxidant and cytoprotective defense capacityof cells Insulin signaling required for Nrf2 inhibition occurs via the MAP kinase pathway and thus is not mitigated by insulin resistance [] Fig It therefore canbe assumed that hyperinsulinemia increases the susceptibility of cells against oxidative or other electrophilestress caused by environmental insults Prolonged exposure of cells to high insulin concentrations can thereforebe regarded as toxic Indeed exposure to nmoll insulin has been found to cause DNA damage in a numberof cell types including human lymphocytes [ ] Atthe only concentration tested nmoll insulin impairs oxygen radical defense and sensitizes apoptosispathways in human islets [] In the brain of micehyperinsulinemia impairs electrophysiological functionsof neurons and protein turnover causing a transition toa senescent cell state and an accompanying cognitive decline [] The direct toxic property of insulin deservesfurther studyChronically elevated insulin concentrations impair bodyfunctionsLongevityThe above list of detrimental cellular responses to highambientinsulin concentrations suggests concomitantfunctional impairments at the level of the anism Thisfits with the observed impact of insulin on longevityStudies in nonvertebrate model systems such as thenematode Caenorhabditis elegans or the fruit fly Drosophila melanogaster find that moderate to high insulinactivity shortens lifespan [ ] A consistent findingfrom mouse model studies is that decreased signaling ofanabolic hormones like insulin insulinlike growth factor or growth hormone results in a prolonged lifespan[] Disruption of the insulinreceptor substrate genecaused insulinresistance with defects in insulin signaling[] and led to an extension of lifespan by []A knockout of the insulin receptor in adipose tissue ofmice resulted in an increase of lifespan [] Disruption of the Ins1 gene and one of the two mouse Ins2alleles lowered insulin levels by Ins2 miceversus Ins2 controls in aged female mice without altering circulating insulinlike growth factorIGF1levels These aged experimental mice exhibited lowerfasting glucose improved insulin sensitivity and lifespan extension across[]two different dietsConcomitantly the proteome and transcriptome indicated a profile associated with healthy aging An important aspect is that this study selectively addressed insulinOther interventions for promoting longevity or extending healthspan such as caloric restriction not only lowercircadian insulin levels but several additional hormonesincluding IGF1 are also affected []InsulinIGF1 and hybrid insulinIGF1 receptorsshare signaling via PI3K and AKT The subsequent activation of the protein kinase mTORC1 is a major pathway for supporting somatic growth protein synthesisand fertility while impeding autophagy and lifespanSuppression of mTOR signaling by treatment with rapamycin prolongs life in model anisms and mice []In humans hyperinsulinemia in pre type diabetes isassociated with increased mTORC1 activity which mayhave a negative impact on beta cell survival healthspanand longevity []In the Leiden Longevity Studyfollowup of nonagenarians for years showed a strongassociation of low insulin and glucose levels with healthyaging []Since both IGF1 and insulin employ PI3K and AKTfor signal transduction it is difficult to disentangle thecontribution of insulin versus IGF1 to the modulationof longevity In animal models selective downregulationof circulating insulin levels improved the lifespan ofmice and in elderly persons of the Leiden LongevityStudy only insulin and glucose but not IGF1 consistently met all four predefined criteria of healthy aging[ ] Therefore it may be concluded that low circulating insulin concentrations are not only a marker oflongevity but are causally involved in promoting healthspan or lifespan extensionDetrimental combination of hyperinsulinemia with insulinresistanceInsulin resistance is defined as an attenuated effect of insulin on blood glucose homeostasis primarily by less efficient export of glucose from the blood into skeletalmuscle adipose and liver tissue Permanently elevatedinsulin concentrations in the blood are often consideredas an attempt to overcome insulin resistance Indeed induction of insulin resistance by genetic disruption of insulin signaling as well as by increased growth hormonelevels or an inflammatory milieu causes hyperinsulinemia [] The opposite causality is of more relevanceHyperinsulinemia during insulin infusion in humansleads to systemic insulin resistance [] while in vitrohigh ambient insulin concentrations cause an increase ininsulin resistance in isolated adipocytes [] A summaryanalysis of nine studies in rodents and seven trials inhumans confirmed that the first detectable change in thefasting state after feeding a high caloric diet for severaldays is an increase of basal insulin concentrations but 0cKolb BMC Medicine Page of not of blood glucose concentrations or insulin resistance[] Both increased secretion of insulin by à cells anddecreased insulin clearance in the liver contribute to elevated insulin levels postmeal the latter being of primaryimportance in the case of carbohydraterich food []functionincluding relaxation ofThe combination of hyperinsulinemia and insulin resistance appears to promote hypertension and atherogenesis Fig One important molecule for maintainingvesselthe arterialsmooth muscle layeris nitric oxide NO which isgenerated by endothelial NO synthase eNOS Insulinincreases NO production via posttranslational modification of eNOS via PI3KAKT activity howeverthismechanism is suppressed during insulin resistance [] Decreased local NO production impairs arterialsmooth muscle relaxation and concomitant vasodilatation An important factor in this context is the calciumion homeostasis of vascular smooth muscle cells Underphysiological conditions insulin promotes both calciuminflux into the cytoplasm of smooth muscle cells via several ion channels including Ltype and storeoperatedCa2 channels and counterregulatory NOmediated efflux of Ca2 and K ions which prevents calcium ioninduced myosin lightchain phosphorylation andFig Hyperinsulinemia insulin resistance and cardiovasculardisease High insulin concentrations in the blood may occur due togenetic predisposition overnutrition or highdose insulin treatmentof type diabetes Hyperinsulinemia induces insulin resistance as adefense response to maintain glucose homeostasis Converselyinsulin resistance may be directly induced such as by growthhormone or proinflammatory cytokines Hyperinsulinemia andinsulin resistance enhance the risk of cardiovascular disease byinducing endothelial dysfunction suppression of endothelial nitricoxide synthase eNOS and activation and promotion of calcium ioninflux into smooth muscle cells resulting in increased vascular toneenhanced reabsorption of sodium ions in renal tubules adhesion ofmacrophages to the vessel wall and development of arterial lesionswith increased lipoprotein lipase activity and cardiovascular diseaseconcomitant vascular contractility During insulin resistance NO production is impaired while the supportiveeffect of insulin on calcium ion influx via PI3K deltaand possibly the MEKERK pathway and vasoconstriction is still present Fig [ ]At the same time insulin signals through the mitogenactivated protein MAP kinase pathway to upregulatethe expression of endothelin1 ET1 plasminogen activator inhibitor1 PAI1 adhesion molecules and proinflammatory cytokines [ ] The reninangiotensinsystem is activated in the context of endothelial dysfunction and contributes together with decreased NO production and increased ET1 secretion to vascularstiffening and upregulation of vascular tone [] Inthe absence of hyperinsulinemiainsulin resistance thelower insulin levels exert less potential proatherogenicactivities which are counteracted by insulinstimulatedlocal NO production [ ]Elevated insulin levels also increase the risk of hypertension by enhancing renal reabsorption of sodium ionsby several transport systems in different segments of thenephron Fig Signaling of insulin occurs via insulinreceptor substrate IRS2 and is not suppressed duringinsulin resistance while signaling via IRS1 for counterregulatory mechanisms including local NO production isimpaired [ ] These detrimental actions may be mitigated during chronic hyperinsulinemiainsulin resistance [] However a metaanalysis of prospectiveepidemiological studies showed that the pooled relativerisk of hypertension was when comparing the highest to the lowest category of fasting insulin levels and for comparing highest to lowest selective insulinresistance categories calculated as homeostasis modelassessment of insulin resistance HOMAIR []As a consequence of endothelial dysfunction duringprolonged treatment with insulin arterial lesions rich inlipids are formed [] The progression of early fattystreak lesions to plaques is accompanied by the adhesionand proinflammatory activity of macrophages whicheventually develop into foam cells This process is drivenby endothelial and macrophage lipoprotein lipase activity as demonstrated by the observation of less atherosclerosis in mice with inactivated lipoprotein lipase gene[] Lipoprotein lipase activity in macrophages isenhanced with higher insulin levels in vivo but there isno direct stimulatory effect of insulin on isolated macrophages []The concern that hyperinsulinemia might promote arterial disease in diabetic persons developed in the late1960s due to the steady increase of incidences of atherosclerosis in diabetic persons despite improved glycemiaand decreased risk of ketosis due to insulin therapy []Since then a wealth of data supports the observationthatis ainsulin resistance and hyperinsulinemia 0cKolb BMC Medicine Page of marker of increased risk of cardiovascular disease in thegeneral population and in patients with diabetes [] Although observational studies suggested an approximatelylinear relation between the severity of hyperglycemiaand vascular damage severallarge randomized controlled trials have shown that intense glycemic controlper se does not decrease the risk of macrovascularcardiovascular events [] indeed insulin therapy may evenincrease the risk [ ] However these trials werenot randomized for insulin treatment and treatment ofCVD risk factors was not kept similar between patientsubgroups In the United Kingdom Prospective DiabetesStudy UKPDS hyperinsulinemia and insulin resistancewere not mitigated by insulin treatment and fastingplasma insulin levels even rose [] By contrastinUKPDS and other trials [ ] oral treatmentwith the biguanide metformin reduced the risk of cardiovascular events and in parallel decreased insulin resistance and hyperinsulinemiaIn epidemiological studies of type diabetesit hasbeen consistently observed that the addition of insulin tothe treatment regimen or the intensification of insulintreatment result in a higher rate of cardiovascular events[] Fig Indeed it has been shown that therisk increases with increasing insulin dosage [ ]These epidemiological studies may suffer from residualFig Hazard ratio of insulin medication versus different reference medications Shown are adjusted hazard ratios HR for each study with confidence interval Moderate insulin exposure high insulin exposure moderate insulin dose to units per day §high insulin dose units per day 0cKolb BMC Medicine Page of confounding since it is difficult to account for the possibly more advanced disease stage of patients receivinginsulin A higher rate of hypoglycemic events may be anadditional confounder However covariates consideredin the statistical analyses cover a broad range of potential risk factors from different categories SupplementTable Large randomized controlled trials such asUKPDS [] or the Outcome Reduction With InitialGlargine Intervention ORIGIN Trial [] did not observe an increased incidence of cardiovascular diseasewith insulin therapy but these trials focused on lowdose insulin therapy of up to a median of IUday or IUkgday respectively Similar randomized trials ofhigherdose insulin therapy as typicalfor realworldconditions have not been conducted Recent studies ofrealworld clinical settings report mean daily basal insulin doses of close to IUkg in the Canadian REALITY Study for insulinexperienced patients with type diabetes [] and of IUkg in a physician survey inNew York [] In the European multicentre EUTREAT Study mean baseline insulin doses were between and U per day depending on the type of insulin therapy regimen applied [] It can be concludedthat under realworld conditions the majority of insulinexperienced patients with type diabetes receive higherinsulin doses per day than those tried in UKPDS orORIGINIn the absence of randomized controlled trials aMendelian randomization is an appropriate approach oftesting for a causal relationship in humans Mendelianrandomization studies made use of the finding that somegenotypes are associated with high or low fasting insulinlevels When comparing individuals carrying ¥ allelesthat raise fasting insulin levels with those exhibiting genetically determined low fasting insulin levels an increasedrisk of elevated blood pressure cardiovascular disease andtype diabetes was observed [] In two large recentMendelian randomization studies a genetic profile predicting high insulin levels in the blood after adjustmentfor BMI was also associated with increased systolic bloodpressure and risk of myocardial infarction []ConclusionsAs discussed aboveinsulin signaling engages at leastthree different pathways and modifies a large number ofcellular responses Table Transient elevations of systemic insulin concentrations are physiological responsesto dietary stimuli or other challenges such as environmental toxins [] In case of prolonged upregulationof insulin levels such as in response to overnutritionglucose homeostasis is maintained by mitigating insulinsignaling via PI3KAKT for glucose export from theblood into tissues Consequently insulin resistance hasbeen considered as a defense response in order to avoidTable Key messagescid129 Insulin employs at least three different pathways of signal transductionOne pathway involves phosphorylation steps via IRSPI3KAKT anotheris the MAP kinases RasMEPERK and third leads to the activation ofNOX4cid129 Insulin resistance is selective because it partially mitigates the PI3KAKTpathway for limiting glucose uptake and eNOS activation despitehyperinsulinemia but many other hormonal actions of insulin are notsuppressedcid129 Signaling via mTOR and the MEPERK pathway causes suppression ofautophagy and NRF2 leading to deficient turnover and impaired celldefensecid129 Moderate to high normal insulin levels inhibit lipolysis and promotelipogenesisobesitycid129 Insulin resistance and hyperinsulinemia are interdependent Dietinduced hyperinsulinemia precedes insulin resistancecid129 In epidemiological studies insulin therapy of type diabetes isassociated with a higher risk of cardiovascular events or deathcid129 Randomized trials of insulin therapy and associated risks only studieddosages up to IUdaycid129 Mendelian randomization studies found that genetically determinedhigh insulin levels lead to cardiovascular diseasecid129 Suppression of hyperinsulinemia and concomitant insulin resistanceprovides substantial health benefitshypoglycemia [] However other hormonal actions ofinsulin via the MAP kinase MEKERK pathway and inpart via PI3KAKT are no | Thyroid_Cancer |
"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu HawaiiParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conï¬ict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR conï¬dence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conï¬ict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulï¬lling ï¬lial obligations in order to ensure equitable access to ACP AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals values goals and preferences regardingendoflife EOL care12 Contemplation of individuals EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conï¬icts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610 AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the beneï¬ts ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proï¬ciencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships uence individuals ACP engagement To ï¬ll thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawaiiwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentiï¬ed asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was afï¬liated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andï¬nancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conï¬ict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conï¬ict was measured using the 5item Family CulturalConï¬ict scale which assesses cultural and intergenerational conï¬ictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation ï¬nancial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conï¬ict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with conï¬dence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconï¬ict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not signiï¬cantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proï¬ciency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conï¬ictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and dont want tonever but want toreluctant to yes never and dont want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conï¬icte10 most cultural conï¬ict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents life which maysometimes be in opposition to their parents own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this populations lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The ï¬nding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conï¬ict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The ï¬nding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential beneï¬t and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved onesdeath and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the signiï¬cantrelationship between family conï¬ict and ACP engagement could bethat higher levels of family conï¬ict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These ï¬ndings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizeï¬ndings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conï¬ict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conï¬ictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education ï¬nancial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulï¬lling ï¬lial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Deï¬nition and recommendations foradvance care planning An international consensus supported by the EuropeanAssociation for Palliative Care Lancet Oncol 201718e543ee551 Sudore RL Lum HD You JJ Deï¬ning advance care planning for adults Aconsensus deï¬nition from a multidisciplinary Delphi panel J Pain SymptomManage 201753821e832e821 Sudore RL Schickedanz AD Landefeld CS Engagement in multiple steps ofthe advance care planning process A descriptive study of diverse older adultsJ Am Geriatr Soc 2008561006e1013 Sudore RL Knight SJ McMahan RD A novel website to prepare diverseolder adults for decision making and advance care planning A pilot studyJ Pain Symptom Manage 201447674e686 Dahlin C Giansiracusa D Communication in palliative care In Ferrell BCoyle N editors Textbook of Palliative Nursing New York NY Oxford University Press p 67e96 KataokaYahiro MR Conde FA Wong RS Advance care planning amongAsian Americans and Native Hawaiians receiving haemodialysis Int J PalliatNurs 20101632e40 Bischoff KE Sudore R Miao Y Advance care planning and the quality ofendoflife care in older adults J Am Geriatr Soc 201361209e214 Kale MS Ornstein KA Smith CB Kelley AS Endoflife discussions with olderadults J Am Geriatr Soc 2016641962e1967 Harrison KL Adrion ER Ritchie CS Low completion and disparities inadvance care planning activities among older Medicare beneï¬ciaries JAMAIntern Med 20161761872e1875 Pew Research Center Key facts about Asian Americans a diverse and growingpopulation Available at wwwpewresearchfacttank20170908keyfactsaboutasianamericans Accessed November Gao X Sun F Ko E Knowledge of advance directive and perceptions ofendoflife care in ChineseAmerican elders The role of acculturation PalliatSupport Care 2015131677e1684 Lee MC Byon HD Hinderer K Alexander C Beliefs in advance care planningamong Chinese Americans Similarities and differences between the youngerand older generations Asian Pac Isl Nurs J 2017283e90 Parks SM Winter L Santana AJ Family factors in endoflife decisionJ Palliat Med making Family conï¬ict and proxy relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difï¬cult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawaii Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspeciï¬c psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratiï¬cation of older adults preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c" | Thyroid_Cancer |
Pathwayspecific model estimation for improved pathway annotation by network crosstalkMiguel CastresanaAguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new networkbased method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gn gncid30 S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P S Notice that S Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iQcid31jPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP Q P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 pcid30 respectively This means that µ ¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n k logBα βk cid28 logŴα k logŴβ n klogŴα β n logŴα logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactomes specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the BenjaminiHochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A Betaalanine metabolism B Prostate cancer and C Alzheimers disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the Betaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway Prostate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the Prostate cancer pathway with a dispersion in the second quartile and Fig a03C shows the Alzheimers disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the Prostate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the Prostate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for Prostate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the Alzheimers disease and Parkinsons disease pathways share of their genes The Alzheimers disease the Parkinsons disease and the Huntingtons disease pathways have of the genes in common from the union between them Further the Oxidative phosphorylation the Nonalcoholic fatty liver disease the Alzheimers disease the Parkinsons disease and the Huntingtons disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each methods ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stabletheir variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU | Thyroid_Cancer |
Glioblastoma GBM is the most aggressive and common form of primary brain cancer Survival is poor and improved treatment options are urgently needed Dual specificity phosphatase6 DUSP6 is actively involved in oncogenesis showing unexpected tumorpromoting properties in human glioblastoma contributing to the development and expression of the full malignant and invasive phenotype The purpose of this study was to assess if DUSP6 activates epithelialtomesenchymal transition EMT in glioblastoma and its connection with the invasive capacityResults We found high levels of transcripts mRNA by qPCR analysis in a panel of primary GBM compared to adult or fetal normal tissues At translational levels these data correlate with high protein expression and long halflife values by cycloheximidechase assay in immunoblot experiments Next we demonstrate that DUSP6 gene is involved in epithelialtomesenchymal transition EMT in GBM by immunoblot characterization of the mesenchymal and epithelial markers Vimentin NCadherin ECadherin and fibronectin were measured with and without DUSP6 overexpression and in response to several stimuli such as chemotherapy treatment In particular the high levels of vimentin were blunted at increasing doses of cisplatin in condition of DUSP6 overexpression while NCadherin contextually increased Finally DUSP6 per se increased invasion capacity of GBM Overall our data unveil the DUSP6 involvement in invasive mesenchymallike properties in GBMKeywords Dualspecificity phosphatase DUSP6 Glioblastoma Epithelialtomesenchymal transition EMTIntroductionDUSP6 plays a prooncogenic role in cancers such as human glioblastoma thyroid carcinoma breast cancer and acute myeloid leukemia [] Particularly DUSP6 is upregulated in human glioblastoma where its overexpression induces reduction in proliferation rate Cell morphology exhibits a more flattened appearance lower Correspondence samanthamessinauniroma3it samanthamessinauniroma1it Department of Science Roma Tre University Viale Guglielmo Marconi Rome ItalyFull list of author information is available at the end of the levels of cellular detachment after stimulation with EGF and an increased pr sity to form colonies in soft agar Surprisingly mouse xenograft tumors expressing DUSP6 grew significantly faster than controls thus reflecting these changes in cell adhesion and morphology [] Moreover overexpression of DUSP6 has also been identified in a subset of mouse melanoma cell lines where it is associated with enhanced anchorageindependent growth and invasive capacity [] and its overexpression in papillary thyroid carcinoma PTC is associated with increased cell migration and invasion [ ] Finally in acute lymphoblastic leukemia ALL DUSP6 acts as prooncogenic phosphatase in preB cell transformation [] 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 0cZuchegna a0et a0al BMC Res Notes Page of Glioblastoma GBM is the most common and malignant type of primary brain tumor with relevant invasive and resistant properties [] The involvement of epithelialtomesenchymal transition EMT has been extensively investigated in glioblastoma although the real relevance of this program in malignant glioma is still controversial A number of preclinical studies have been launched to target the process considering the critical role played by EMT on GBM dissemination resistance to apoptosis and cancer stemness maintenance [] Of note DUSP6 is involved in epithelialtomesenchymal transition EMT in epithelial cancers such as breast and endometrial adenocarcinoma [ ] Moreover GBMs mesenchymal subtype are characterized by an elevated invasive potential and of note the most commonly used glioma cell lines ie U87MG and U251 also present a predominant mesenchymal signature [] with elevation in mesenchymal markers [] The aim of this study was to assess the importance of DUSP6 gene in epithelialtomesenchymal transition in GBM in correlation with its invasive capacityMain textMethodsCell culturesAstrocytoma primary WHO grade IV GBM1 GBM10 etc were established from tumor specimens of patients and cultured as described [] NHA Normal Human Astrocytes and NSC Neural Stem Cells were purchased from Cambrex Corporate NJ USA and grown according to the manufacturers instructions Normal Human Astrocytes were used as reference because of the presumed similarity between astrocytes and the celloforigin from which glioblastoma develops both adult NHA and foetal Primary Fetal Normal Neural Stem Cells from SVZ U87MG human GBMastrocytoma cell line was purchased from the bank of biological material Interlab Cell Line Collection Genova Italy Human breast cell cultures MCF7 MCF10A and MDA231 cell lines were purchased from the American Type Culture Collection ATTC LGC Standards srl Italy Cells were cultured at a0°C in CO2 in DMEM with high glucose plus vv fetal bovine serum FBS Euroclone Milan Italy penicillinstreptomycin a0 UmL Euroclone and lglutamine a0 mM Euroclone according to manufacturers instructions Primary glioblastomas cell lines WHO grade IV were established from tumor specimens of patients and cultured as described [ ] Cells were plated at of confluence on a0mm dishes and the day after infected with recombinant adenovirus as previously described with the amounts according to the scheme indicated in the figures [] In cycloheximidechase assay experiments the cells were treated with a0µgmL CHX in complete medium and then lysed at the indicated times as described in the figures legendsWestern Blot analysisCultured U87MG and U251MG cells were washed with PBS and lysed for a0min in icecold Radioimmunoprecipitation RIPA buffer Triton X100 deoxycholateDOC sodium dodecyl sulphate SDS a0mM Tris pH a0mM NaCl a0mM phenylmethylsulfonyl fluoride PMSF a0mgmL aprotinin leupeptin and pepstatin Cell lysates were clarified at rcf for a0 min at a0 °C and the cytosolic fraction was immediately subjected to protein determination using a Bradford colorimetric assay BioRad Laboratories Inc Hercules CA USA DUSP6 was detected with a homemade rabbit polyclonal specific antibody against DUSP6 Lennartsons lab Monoclonal antiαtubulin as loading control antiphosphoERK and antiFibronectin were purchased by SigmaAldrich St Louis MO USA AntiVimentin was purchased from Millipore and antiERK was purchased from Cell Signaling Technology Anti NCadherin and antiECadherin were purchased from Santa Cruz Biotechnology Goat antiMouse IgG H L Highly CrossAdsorbed Secondary Antibody HRP A16078 e Goat antiRabbit IgG H L Highly CrossAdsorbed Secondary Antibody HRP A16119 Thermofisher Scientific ECL detection kit from Amersham GEHealthcareRealtime quantitative PCRA quantitative assay for Human DUSP6 mRNA seq ref nePlus¢ RealTime PCR System Applied Biosystems¢ NM_0019464 expression was established using StepOusing PowerUp¢ SYBR¢ Green Master Mix Applied Biosystems¢ using the following program a0°C10 a0min cycle a0°C15 a0s a0°C90 a0s cycles a0°C15 a0s a0 °C90 a0 s cycles All reactions were normalized with the housekeeping gene for 18S PCR oligoprimers were Human DUSP6 forward primer ²CgAggACCgggACCgCTT CAC C3² and reverse primer ²CCgAgATggggATTTgCTTgTATT3² generating a a0 bp fragment Human 18S forward primer ²gACCgATgTATATgCTTgCAgAgT3² and reverse primer ²ggATCTggAgTTA AAC TggTCCAg3² The two transcripts of mRNA DUSP6 were detected by PCR using the following primers Forward primer ²CgAggACCgggACCgCTT CAC C3² Reverse primer ²AgTTAggggATATgTTggATTTT3² The expected size of the fragment was a0 bp for the transcript variant NM_0019464 and a0bp for the transcript variant NM_0226524 0cZuchegna a0et a0al BMC Res Notes Page of Cell invasion assayTranswell inserts Corning® FluoroBlok¢ Plate Permeable Support with a0µm Colored PET Membrane for trex¢ Matrix Gibco by Life Technologies U87MG cells 24well plates were used Inserts were coated with Gelwere transduced with TRK or DUSP6 expressing adenoviral vectors and cells were seeded in serumfree Dulbeccos modified Eagles medium in the upper chamber on the top of the matrigel Dulbeccos modified Eagles medium FBS a0μL was dispensed in the lower chamber as a chemoattractant After a0h the apparatus was washed with PBS and cells that did not migrate were removed with a cotton swab then inserts were fixed paraformaldehyde for a0min at room temperature The results were quantified by counting all the cells of the inserts in duplicate from two independent experiments using the objective Data were tested for normal distribution of variables using the ShapiroWilks test and statistical significance between groups was determined using Students ttestResultsQuantitative transcriptional analysis of DUSP6 was assessed by RTqPCR measuring high mRNA levels in a panel of twenty primary glioblastomas GBM WHO grade Fig a01b by quantifying the mRNA foldinduction over Normal Human Astrocytes NHA and Neural Stem Cells NSC which specify distinct glioblastoma subtype [] We measured increased expression in primary samples approximatively sevenfold enrichment in GBM15 GBM53 and GBM176 compared to controls In addition human longterm cultures U87MG U251MG and T98G displayed high mRNA levels compared to primary samples of GBM their values differ by several orders of magnitude Fig a0 1c Furthermore mRNA levels in breast cancers lines MCF7 MCF10 were undetectable but not in MDAMB231 a model for triplenegative breast cancer expressing aberrantly high levels and 6000fold enrichment Fig a01c Interestingly the gene contains three distinct introns Fig a0 1a producing short and longPCR products sized a0bp and a0 bp respectively with a marked prevalence of the longtranscript in primary glioblastomas personal observation ie samples GBM11 and GBM15 Fig a01eFurther we used cycloheximidechase assay after singletime point western blotting as a measurement of halflife endogenous DUSP6 protein Fig a0 Unstimulated primary GBM Panel a and longterm cultures Panel b show high protein levels in both primary and longterm glioblastoma as assayed by semiquantitative Western blot analysis Fig a0 2a b The ERKMAPK cascade activation was sustained in both primary and longterm cultures U87MG U251 and T98G while overexpression adenovirusmediated CTRL completely abrogate pERK and ERK ½ signals Fig a0 2a De novo protein synthesis was measured by timecourse cycloheximide CHX experiment in both cell lines Fig a0 2c d We found that acutely inhibiting protein synthesis diminished DUSP6 diminished expression in both U87MG and U251MG but did so with different kinetics In contrast to previously published data [] we report long halflife in both cell lines U87MG and U251 respectively more than a0h and up to a0h These results agree with the stable endogenous protein exerting oncogenic properties in cancersThen we examined the expression of mesenchymal markers associated with EMT in glioblastoma cultures in na¯ve condition and in DUSP6overexpression Firstly we assayed protein endogenous levels of Vimentin NCadherin ECadherin Fibronectin in U87MG upon several stimuli serum addition serum deprivation EGF and cisplatin by Western Blot with specific antibodies Fig a03a Immunoblot analysis for pERKERK ratio shows different kinetics of ERKMAPK kinases phosphorylation upon EGF and cisplatin CDDP treatmentsInterestingly cisplatin treatment reduces the amount of NCadherin ECadherin and Fibronectin but not Vimentin Fig a0 3a NCadherin and Fibronectin were slightly upregulated in adenovirusmediated DUSP6 overexpression Fig a03b Otherwise the epithelial marker ECadherin was almost absent in both na¯ve and adenoviralexpressing cells Next in DUSP6overexpressing U87MG cells increasing doses of cisplatin ranging from to a0 µLmL affected the EMT markers in opposite fashion Vimentin protein expression is completely blunted upon treatment whereas high levels are shown in adenoviralexpressing control vector CTRL and in adenoviralexpressing DUSP6 untreated lane Fig a0 3b Finally we assayed invasive ability of both na¯ve glioblastoma cells compared to DUSP6adenoviralexpressing U87MG cells by Transwell invasion assay see Methods We here report that DUSP6 increased the invasion capacity of the glioma U87MG cells Fig a03c compared to MOCK cells na¯ve U87MG and adenoviral empty vector as negative control TRKin epithelialtomesenchymal DiscussionThis study was designed to explore DUSP6 involvement in glioblastoma We previously showed that DUSP6 is upregulated in human glioblastoma and in a0vitro adenovirusmediated overexpression results in a transformed phenotype [] Here we extended our transcriptional analysis to include a new set of primary cell cultures transition 0cZuchegna a0et a0al BMC Res Notes Page of Fig DUSP6 mRNA is overexpressed in human glioblastomas a Schematic grey boxes denote protein coding region of DUSP6 gene and colored arrows represent the approximate locations of the primers annealing sites in DUSP6 gene b Bar diagram shows relative quantification of total DUSP6 mRNA across a panel of human glioblastoma samples grade IV astrocytoma Normal Human Astrocytes NHA and Neural Stem Cells NSC were used as controls because of the presumed similarity between astrocytes and the celloforigin from which glioblastoma develops both adult NHA and foetal NSC Relative expression was normalized to housekeeping gene 18S expression c Upper panel relative DUSP6 mRNA expression quantified by qPCR on human glioblastoma cultures U251MG U87MG and T98G Lower panel human breast cell cultures MCF7 MCF10A and MDA231 were assayed as positive controls d Qualitative PCR for DUSP6 mRNA in primary glioblastomas Expression of differentsize transcripts was detected using primers shown in the upper panel Schematic denotes blackgrey boxes exons and the relative location of the forward and reverse primer annealing sites in DUSP6 mRNA gene Ethidium stained agarose gel of endpoint products from the different amplicons DUSP6 using Normal Human astrocytes NHA Neural Stem Cells NCS and two samples of primary glioblastoma mRNA The two alternative transcripts of DUSP6 mRNA are shown transcript variant NM_0019464 and transcript variant NM_0226524of glioblastoma and longterm cultures By using qRTPCR we found high levels of mRNA DUSP6 across the glioblastoma samples Moreover we assayed breast cancer cell lines and found that the mesenchymal MDAMB231 showed the highest levels of mRNA expression compared to normal epithelial MCF10 and MCF7 cell lines Interestingly DUSP6 is involved in maintaining the mesenchymal state in breast cancer [] Moreover here we report high protein expression and long halflife values by cycloheximidechase assay in longterm cultures U87MG and U251 in line with previous data on fibroblasts [] We demonstrate that DUSP6 gene is involved in epithelialtomesenchymal transition EMT in GBM Whereas we clearly show that DUSP6 per se increases invasion capacity of GBM the evidence on epithelialtomesenchymal transition in GBM does not lead to a firm conclusion We report immunoblot characterization of the mesenchymal and epithelial markers with and without DUSP6 overexpression Vimentin is clearly downregulated 0cZuchegna a0et a0al BMC Res Notes Page of Fig DUSP6 protein is overexpressed in human glioblastomas a Western Blot analysis of DUSP6 pERK and ERK in primary GBM samples In both panels CTRL indicates positive immunereactive control obtained with U87MG infected with the adenovirus DUSP6 b Western Blot analysis of DUSP6 pERK and ERK in U87MG U251MG T98G and MCF7 cell lines The corresponding bar graphs show relative expression of proteins normalized to αtubulin c d Posttranslational regulation of DUSP6 protein levels U87MG and U251MG cell lines were treated with cycloheximide CHX µgmL for the time indicated and the decay of the target proteins over time was determined by Western Blot with specific antibodies The corresponding bar graphs show relative expression of proteins normalized to αtubulin 0cZuchegna a0et a0al BMC Res Notes Page of Fig Differential regulation of epithelial and mesenchymal markers by DUSP6 a Immunoblot analysis of proteins DUSP6 pERK ERK vimentin NCadherin ECadherin fibronectin in longterm cultures U87MG treated with different stimuli serum starvation EGF ngmL cisplatin CDDP µgmL for h left lane or h right lane b Immunoblot analysis of mesenchymal and epithelial markers in U87MG adenovirusexpressing DUSP6 compared to TRK empty vector The cells were treated with increasing doses of cisplatin CDDP at different concentrations respectively μgmL and protein expression assayed by western blot on total lysates at h c Invasion assay Bar graph reports the mean of total number of invaded cells per field compared to control MOCK ie U87MG not infected U87MG cells were transduced with adenovirusexpressing DUSP6 or control vector TRK ie U87MG infected with empty vector and seeded on matrigelcoated transwell inserts The experiment was performed for h and the invading cells were counted objective in nine randomly chosen microscopic fields per transwell Data are presented as mean ± SD from two independent experiments performed in duplicatein cisplatintreated overexpressing DUSP6 cells but not in absence of cisplatin NCadherin is upregulated in cisplatintreated overexpressing DUSP6 whereas it was downregulated in na¯ve cells In addition our data on cadherin switch are in line with the inconsistency of the literature with some reports showing that GBM do not express ECadherin but others showing the occurrence of an E to NCadherin switch More importantly our data show that cisplatin treatment clearly downregulates Fibronectin and both Cadherins while Vimentin underwent no change in na¯ve cells in absence of DUSP6 overexpression Notably classical cadherin switch which is widely accepted as an EMT hallmark in carcinomas is a controversial matter in GBM [ ]Of note it was found that Ncadherin expression is inversely correlated with the invasive behavior of GBM and its ectopical expression reduces cell migration and restores polarity in GBM cells [ ] Conversely lower expression of NCadherin was recognized in a panel of GBM primary samples at mRNA and protein levels [] 0cZuchegna a0et a0al BMC Res Notes Page of Recently association of the EMT transition with chemoresistance has been reported [] Our results clearly show that mesenchymal markers are downregulated by cisplatin in na¯ve cells except for vimentin This is in line with vimentin increased levels in resistant GBM cultures compared to parental ones [] Moreover conflicting results are reported on DUSP6 role in chemotherapyresistance in epithelial cancers [ ] Here we report the first observation of EMT markers in response to a drug used in chemotherapy during DUSP6 overexpression Particularly the high levels of vimentin were blunted at increasing doses of cisplatin in a condition of DUSP6 overexpression while NCadherin contextually increased These data have implications on chemotherapy response in glioblastoma treatmentLimitationsLimitations of this study include i limited number of lowpassage serumfree cell lines cultured from patient tumor tissue GBM in the text ii the poorlyrepresentative cell line models U87MG and U251MG which are not exhaustive model for glioblastoma multiforme molecular subtypes classical proneural and mesenchymal [] Further this study reports only an in a0vitro characterization based on western blot analysis of the classical mesenchymal markers Additional file a0 and from the pointofview of phenotypic characterization the invasion assay does not exhaustively demonstrate that DUSP6 activates EMT in GBM by enhancing its invasive propertiesSupplementary informationSupplementary information accompanies this paper at https doi101186s1310 yAdditional file a0 Figure S1 Uncropped versions of the western blot used in this manuscript corresponding to Fig Original gels of Western Blot analysis of Fig panels A and B immunereactive bands corresponding to specific antibodies against DUSP6 pERK and ERK and αtubulin as specified in Methods section Original gels of Western Blot analysis of Fig panels C and D immunereactive bands corresponding to specific antibodies against DUSP6 pERK and ERK and αtubulin as specified in Methods section Figure S2 Uncropped versions of the western blot used in this manuscript corresponding to Fig Original gels of Western Blot analysis of Fig panels A and B immunereactive bands corresponding to specific antibodies against DUSP6 pERK ERK Vimentin NCadherin ECadherin Fibronectin as specified in Methods sectionAbbreviationsDUSP6 Dualspecificity phosphatase CHX Cycloheximide ERK12 Extracellular regulated kinases EMT Epithelialtomesenchymal transition ALL Acute lymphoblastic leukaemia GBM GlioblastomaAcknowledgementsWe are grateful to Prof Antonio Porcellini and Dr Luca Persano for their cheerful assistance with preliminary data of this workAuthors contributionsSM and BM conceived the project SM CZ and EDZ designed and performed the experiments CZ and EDZ analyzed the experiments and cowrote the paper All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsAll data presented or analyzed in this study are included in this Ethics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Biology Federico II University of Naples Naples Italy Department of Medicine and Health Sciences V Tiberio University of Molise Campobasso Italy Department of Science Roma Tre University Viale Guglielmo Marconi Rome Italy Received October Accepted July References Messina S Frati L Leonetti C Zuchegna C Di Zazzo E Calogero A Porcellini A Dualspecificity phosphatase DUSP6 has tumorpromoting properties in human glioblastomas Oncogene DeglInnocenti D Romeo P Tarantino E Sensi M Cassinelli G Catalano V Lanzi C Perrone F Pilotti S Seregni E et al DUSP6MKP3 is overexpressed in papillary and poorly differentiated thyroid carcinoma and contributes to neoplastic properties of thyroid cancer 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IFNBased Biotherapeutics toHarness the Host AgainstFootAndMouth DiseaseGisselle N Medina Teresa de los Santos and Fayna DiazSan Segundo Plum Island Animal Disease Center PIADC ARS USDA Orient Point NY United States Kansas State University Collegeof Veterinary Medicine Manhattan KS United StatesFootandmouth disease FMD is a highly contagious vesicular disease of clovenhoofedanimals that severely constrains international trade of livestock and animal productsCurrently disease control measures include broad surveillance enforcement of sanitarypolicy and use of an inactivated vaccine While use of these measures has contributedto eliminating footandmouth disease virus FMDV from a vast area of the worldthe disease remains endemic in three continents and outbreaks occasionally appearin previously declared FMDfree zones causing economic and social devastationAmong others a very fast rate of viral replication and the need for days to achievevaccineinduced protection are the main limitations in controlling the disease Newfastacting antiviral strategies targeted to boost the innate immunity of the host to blockviral replication are needed Here we review the knowledge on the multiple strategiesFMDV has evolved to block the host innate immunity with particularly focus on the pastand current research toward the development of interferon IFNbased biotherapeuticsin relevant livestock speciesKeywords footandmouth disease virus FMDVIFNλ IFNÏinterferon IFN antivirals biotherapeutics IFNα IFNÎINTRODUCTIONThe Disease FootAndMouth DiseaseFootandmouth disease FMD is one the most serious livestock diseases that aï¬ects clovenhoofedanimals including cattle swine sheep and goats as well as numerous species of wild species The disease displays high morbidity but is usually not lethal except when it aï¬ects young animalsthat may develop myocarditis Infected animals secrete copious amounts of virus ps beforethe onset of the clinical phase of the disease Typical FMD clinical signs include fever and theappearance of vesicular lesions on the tongue mouth feet and teats Among ruminants thatrecovered from the disease a relatively large number become asymptomatic virus carriers although it is not clear what is the contribution of these carrier animals to disease transmissionin nature The World anization for Animal Health OIE lists FMD as a reportable diseaseand therefore by law participating nations are required to inform the anization about all FMDoutbreaks OIE member nations with reported cases of FMD are forbidden to engage in tradingof FMDsusceptible animals or their products Thus the presence of FMD in a country can havesevere economic consequencesDiï¬erent interventions to control an FMD outbreak include restriction of susceptible animalmovement slaughter of infectedcontact animals decontamination of infected and surroundingEdited byMariano PrezFilgueiraNational Agricultural TechnologyInstitute ArgentinaReviewed byMargarita S¡izSevero Ochoa Molecular BiologyCenter CSICUAM SpainKenneth James GenoveseAgricultural Research ServiceUnited States Department ofAgriculture United StatesCorrespondenceGisselle N MedinagissellemedinausdagovTeresa de los SantosteresadelossantosusdagovFayna DiazSan SegundofaynadiazsansegundousdagovSpecialty sectionThis was submitted toVeterinary Infectious Diseasesa section of the journalFrontiers in Veterinary ScienceReceived April Accepted June Published August CitationMedina GN de los Santos T andDiazSan Segundo F Use ofIFNBased Biotherapeutics to Harnessthe Host Against FootAndMouthDisease Front Vet Sci 103389fvets202000465Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVpremises and vaccination Vaccination is an option used mostlyin countries in which FMD is endemic but diseasefree nationsprefer to abstain from such practice In general FMDfreecountries that occasionally opted to vaccinate to better containthe outbreak did slaughter all vaccinated animals to regaincommerce rights faster as occurred in the outbreak inthe UK and the Netherlands The current approvedFMD vaccine consists of puriï¬ed chemically inactivated virus[binary ethylenimine BEItreated] formulated with oilbased oraluminum adjuvants that induces serotypespeciï¬c protection inapproximately days and it is applied with a boosting protocolfor ensuring longterm protection While this vaccine hasbeen successfully used for many decades leading to diseaseeradication of a vast area of our planet challenges remainFMD is endemic in most of Africa and Asia and occasionallyepizootics appear in South America or in nations that havebeen diseasefree for many years as it happened in the UKthe Netherlands South Korea Taiwan and Japan Novelvaccine technologies have been developed but to this end noneof them has fully addressed the limitations of the commerciallyavailable vaccine or is currently approved for massive use Alternatives or additional therapeutics that could complementor in some instances substitute for vaccination protocols includethe use of antivirals and biotherapeutics that act quickly priorto induction of vaccineinduced immunity The development ofsuch molecules requires a thorough understanding of the biologyof the virus and its intricate interactions particularly with theinnate immune molecular and cellular mechanisms evolved bythe hostThe AgentFootandmouth disease virus FMDV is a member of theAphthovirus genus within the Picornaviridae family and it is theetiologic agent of FMD The virus contains a singlestrandedRNA of positive polarity Its genome of ¼ nucleotidesconsists of a long reading frame ORF ï¬anked by a ² anda ²untranslated region UTR The ORF encodes a polyproteinof about amino acids which is processed by virusencodedproteases Processing results in the generation of precursors andmature protein products including four structural [1A VP4 1BVP2 1C VP3 1D VP1] and ten nonstructural NS proteins[Lpro 2A 2B 2C 3A three distinct copies of 3B VPg 3Cproand 3Dpol] Due to high genetic variability FMDV is categorizedin seven distinct serotypes A Asia1 C O and Southern AfricanTerritories SAT and numerous subtypes or topotypesUpon infection the virus spreads very rapidly usually achieving morbidity Depending on the route of entry less than tissue culture infectious doses are required to infect andcause disease in animals In fact FMDV is one of thefastest replicating RNA viruses in nature taking as little as h to induce cytopathic eï¬ects in susceptible tissue culture cellsOne could envisage that during FMDV replication almost everycomponent of the virus must play a role in dampening interferingcellular responses to allow such rapid virus replicationInnate Immunity and Interferon ActivationEarly protection against viralfundamentallymediated by the action of interferons IFNs the pillar moleculesof the innate immune system Expression of IFN isinfection istriggered by the recognition of molecular signatures collectivelynamed pathogenassociated molecular patterns PAMPs viacellular receptors pattern recognition receptors PRRs that candistinguish self from nonself molecules Figure Bindingof PAMPs to PRRs triggers a series of signal transduction eventsand posttranslational modiï¬cations PTMs phosphorylationubiquitination ISGylation etc that ultimately activate latenttranscription factors to induce IFN transcription Subsequentlysecreted IFN proteins bind to speciï¬c receptors on the plasmamembrane to activate in an autocrine and paracrine mannerdiscrete and overlapping cellular signal transduction pathwaysDepending on the cell type and aï¬ected tissue over speciï¬cIFNstimulated genes ISGs may be induced many of whichdisplay antiviral activity to control the viral infection There are three families of IFNs based on the speciï¬c receptorusage types I II and III Table Type I IFNsie IFNα and IFN signal through a heterodimeric receptorcomplex formed by IFNAR1IFNAR2 type II IFN IFNÎsignals through the complex IFNÎR1IFNÎR2 and type IIIIFNs bind the receptor complex IL28RαIL10R Despitethe receptor diï¬erencesthe three IFN families transducesignals through the Janus kinase JAKsignal transducer andactivator of transcription STAT pathway and type I and typeIII IFNs induce redundant responses Figure Overall therapid production of IFN helps to limit viral replication whilemodulating other immune functionsFOOTANDMOUTH DISEASE VIRUSIMPAIRS INNATE IMMUNITY MOLECULARINTERACTIONSRecognition of FMDV RNA by the host cell results in theestablishment of a rapid antiviral state to limit and controlinfection This selective pressure has allowed FMDV to evolvemany strategiesto ensure enhanced virulence and rapidinfectivity In general RNA viruses can bypass the IFN responseby blocking i global cellular transcription and translationii IFN induction and iii IFN signaling Similarly to otherRNA viruses FMDV can also target IFNindependent antiviralresponses mostly associated with cellular metabolic functionsie autophagy apoptosis stress granule formation etc thathave been extensively described elsewhere In thissection we will summarize the current literature on studiesconducted in vitro that explain how FMDV counteracts the hostinnate immune response at the molecular level including RNAsensing activation of adaptoreï¬ector proteins and regulation ofsignaling pathways by speciï¬c PTMsBlock on Cellular Transcription andTranslationFMDV inhibition of cellular gene expression and proteinsynthesis during infection is mainly driven by the viralencoded proteases Leader Lpro and 3C FMDV Lpro isa papainlike protease PLP thatthetranslation initiation factor eIF4G including eIF4GI and eIF4GII to disable capdependent protein synthesis AlsoFMDV Lpro causes degradation of the transcription factorinduces cleavage ofFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Antiviral signaling pathways induced during viral infection Cellular detection of microbial molecules known as pathogenassociated molecular patternsPAMPs ie viral RNA is mediated by pattern recognition receptors PRRs including cytosolic RNA sensors ie RIGI MDA5 or LGP2 andor membraneboundTLRs PAMPPRR interaction activates signal transduction cascades black arrows that result in the production of IFN and inï¬ammatory cytokines RIGI and MDA5contain two caspase recruitment domains CARD and an RNA helicase domain In the case of RIGI ubiquitination green circles is required for its effective activationActivated signals from either RIGI or MDA5 are transmitted downstream via the mitochondrial adaptor MAVS resulting in the formation of MAVS ï¬laments At thisstage different PTMs such as ubiquitination or ISGylation black circles can regulate their functions Endosomal RNAs are detected by TLR3 or TLR78 which signalthrough adaptor proteins TRIF and MyD88 respectively MyD88 uses other adaptors IRAK14 to allow for interaction with TRAF proteins In addition to their role asadaptor proteins TRAFs also serve as E3 ubiquitin Ub ligases to regulate signaling TRAFmediated induction of polyUb is sensed by NEMO thus recruitingdownstream effector kinases such as TBK1 or IKK These proteins form different signaling complexes ie NEMOTBK1 and NEMOIKK leading to phosphorylationblue arrows of transcription factors IRF37 to a lesser extent IRF1 and IRF5 are also phosphorylated IRF phosphorylation triggers dimerization and translocationContinuedFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE orange arrows to the nucleus where they bind mainly to IFN promotersenhancers Alongside with this pathway TRAF6E3 ligases can activate MAPK3and other kinases including ERK12 and JNK which phosphorylate the components of the AP1 heterodimer allowing for translocation to the nucleus and binding tothe IFN promoterenhancer to activate transcription Activated IKK also phosphorylates IκB releasing NFκB which then translocates to the nucleus and binds at theIFN promoter AP1 activating protein CARD caspase activation and recruitment domain DUB deubiquitinase ER endoplasmic reticulum IκB inhibitor of KBkinases IKK IκB kinase IL interleukin IRAK interleukin1 receptorassociated kinase IRF IFN regulatory factor LGP2 laboratory of genetics protein MAPKmitogenactivated protein kinase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiationassociated gene MyD88 myeloid differentiationprimary response protein 88d NEMO NFκB essential modulator NFκB nuclear factorκB PKR protein kinase R PTM posttranslational modiï¬cation RIGIretinoic acidinducible gene I TANK TRAF family memberassociated NFκB activator TBK TANK binding kinase TLR Tolllike receptor TRAF TNF receptorassociated factor TRIF TIRdomaincontaining adapterinducing interferonTABLE Use of IFNbased therapies against FMDVTypeReceptSignalSubtypeSpeciesMilestoneType IIFNAR1IFNAR2JAK1 TYK2IFNαPorcinebovine ¢ Recombinant bacterial expressed IFNα is a potent biotherapeutic againstIFNαPorcine¢ Ad5 delivered poIFNα protects swine against different serotypes of FMDVFMDV in vitro IFNIFNδIFNÏ7IFNαÏIFNÏType IIIFNÎ R1 IFNÎ R2JAK1 JAK2IFNÎType IIIIFNλR1IL10R2JAK2 TYK2IFNλ1IFN΢ poIFNαprotection correlates with enhanced tissuespeciï¬c innate immune cellinï¬ltration in swine ¢ poIFNα protection correlates with upregulation of essential ISGs in vitro ¢ Ad5 delivered porcine poIFN protects swine against FMDV ¢ Bacterially expressed poIFNδ8 signiï¬cantly inhibits FMDV replication in vitro ¢ E coli produced poIFNÏ7 protects cells against FMDV ¢ Bacterially expressed IFNÎ±Ï added prior to infection resulted in a signiï¬cantreduction in FMDV replication in vitro ¢ Ovine IFNÏ has antiviral effect against FMDV in vitro ¢ Recombinant bovine IFNÎ reduced FMDV replication in BTY cell culture ¢ High dose of Ad5poIFNÎ protects swine against FMD ¢ Replication of FMDV in IBRS2 cells is inhibited by treatment with the puriï¬edrecombinant poIFNλ1 PorcinePorcinePorcinePorcineOvineBovinePorcinePorcineIFN CombosOtherIFNvaccinecombosIFNλ3Bovine¢ Inoculation with Ad5boIFNλ3 resulted in the induction of several ISGs in tissuesof the upper respiratory tract and protected cattle against challenge withFMDV PorcinePorcineIFNαIFN΢ Ad5poIFNλ3 protects swine against challenge with FMDV ¢ Use of a combination of Ad5poIFNÎ and Ad5poIFNα or Ad5poIFNαΠshowed an enhancement of the antiviral activity against FMDV in swinePoly ICPorcine¢ Double stranded ds RNA poly ICLC in combination with Ad5poIFNαprotected swine against FMDV siRNAPorcine¢ Combination of Ad5poIFNαΠwith Ad3siRNA targeting FMDV NS codingregions blocked replication of all serotypes of FMDV in vitro IRF73Porcine¢ Inoculation with Ad5IRF735D resulted in induction of IFNα and fully protectedmice and swine challenged with FMDV day after treatment IRESPorcine¢ Use of synthetic IRES in combination with adjuvanted typeO FMD improvedimmune response and protection against FMDV challenge IFNαPorcine¢ Use of a combination of Ad5poIFNα and Ad5A24 in swine resulted incomplete protection after challenge IFNαÎPorcine¢ Ad5poIFNαΠcoadministered with Ad5siRNA targeting NS regions of FMDVand a commercial inactivated FMD vaccine partially protected swine IFNλ3Bovine¢ Use of a combination of Ad5bovIFNÎ3 and AdtO1M in cattle resulted incomplete protection after aerosol challenge nuclear factor NFκB and results in blockage of speciï¬cdownstream signaling eï¬ectors Studies in porcine cellsdemonstrated that FMDV Lpro can promote its selfbindingto the transcription factor activitydependent neuroprotectiveprotein ADNP and negatively regulate the activity of the IFNα promoter In contrast chromatin changes that favor theupregulation of IFN and ISGs can inhibit FMDV replication Interestingly deletion or mutations in diï¬erent domainsof Lpro result in viral attenuation in vitro and in vivo Furthermore these studies have shown a strong type I IFNactivity upon infection with diï¬erent versions of FMDV Lpromutants Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Type I II and III interferon IFNmediated signaling All type I and type III IFN subtypes bind to respective receptors IFNAR1IFNAR2 and IFNLR1IL10R2These interactions trigger the phosphorylation of JAK1 and TYK2 kinases which in turn phosphorylate STAT1 and STAT2 JAK2 mediates type III IFNdependent STATphosphorylation Phosphorylated heterodimers of STAT1STAT2 bind to IRF9 forming the ISGF3G complex which then translocates to the nucleus and binds toIFNresponsive elements ISREs present in the promoters of over ISGs Type II IFN binds to the heterodimeric IFNÎR1IFNÎR2 receptor also inducingphosphorylation of JAK1JAK2 kinases In turn mostly STAT1 is phosphorylated Phosphorylated homodimers of STAT1 translocate to the nucleus and induce theexpression of genes controlled by gammaactivated sequence GASdependent promoter sequences IFNAR12 IFN alpha receptor12 IFNÎR12 IFNgammareceptor12 IFNALR1 IFNlambda receptor IL10R2 IL10 receptor ISGs IFNstimulated genes ISGF3G ISG factor gamma JAK12 Janus kinase STATsignal transducer and activator of transcriptionInterruption of cellular translation during infection can alsobe mediated by FMDV 3Cpro a chymotrypsinlike cysteineprotease that similarly to Lpro targets eIF4G and the capbindingcomplex eIF4A for cleavage although these events occur later inthe infection 3Cpro can also participate in the inhibitionof hostcell transcription by cleaving histone H3 upon FMDVinfection Block on Interferon InductionDuring infection the initial event that leads to the productionof IFN and proinï¬ammatory cytokines is the recognition ofviral RNA Figure Sensing of FMDVRNA is mediated byMDA5 a protein that belongs to a family of helicasesknown as retinoic acidinducible geneI RIGIlike receptorsRLRs Recent studies have shown that the interaction betweenRLRs RIGI and LGP2 and the FMDV proteins Lpro 2Band 3A interferes with the induction of type I IFN Indeed overexpression of either FMDV 2B or 3A resulted in thedownregulation of RIGI and MDA5 mRNA expression In contrast upregulation of LGP2 transcripts has been observedduring FMDV infection in porcine cells despite a detectablereduction of LGP2 protein levels presumably due to FMDVFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVLproinduced cleavage The apparent inconsistencybetween the levels of LGP2 mRNA and protein during FMDVinfection may be explained by LGP2s ability to serve as a positiveand negative regulator of RIGI and MDA5 signaling presumablyaï¬ecting multiple steps of the IFN induction pathway Inaddition to RLRs nucleotidebinding oligomerization domainNODlike receptors NLRs NOD1 and NOD2 also participatein the recognition of RNA A study by Liu et al describedthe association of NOD2 with FMDV 2B 2C and 3Cpro to blockinnate immunity activation Protein kinase R PKR is anotherrecognized PRR that acts as an RNA sensor Binding ofRNA to PKR induces a conformational change that leads toautophosphorylation and activation The primary target ofactivated PKR is the eukaryotic initiation factor α subuniteIF2α whose phosphorylation results in the blockage of cellularprotein synthesis a relatively common process during viralinfection Although no direct interaction between FMDVRNA and PKR has been demonstrated it has been reportedthat PKR activity modulates FMDV infectivity In fact in tissueculture experiments depletion of endogenous levels of PKR usingsiRNA resulted in increased FMDV titers Furthermoreit has been recently shown that overexpression of autophagyrelated ATG5ATG12 proteins induces transcription of PKR andsubsequent reduction of FMDV replication These resultssuggest that PKR has a complex role as an RNA sensor but also asan antiviral agent during FMDV infectionIt has been demonstrated that FMDV also targets DExDHbox RNA helicases formally accepted as PRRs and modulatorsof the antiviral signaling pathway In vitro experimentsintending to analyze proteinprotein interactions revealed theassociation between the RNA helicase DDX1 and FMDV 3D Interestingly these studies indicated that during FMDVinfection in porcine cells cleavage of DDX1 was detected whileoverexpression of DDX1 resulted in the upregulation of IFNand other ISG mRNAs which correlated with virus inhibition Other DExDHbox RNA helicases such as RNA helicaseH RHA are hijacked during FMDV infection and interact withFMDV UTR 2C and 3A to facilitate virus replication Signaling pathways downstream from RNA sensing involvethe activation of diï¬erent adaptor and eï¬ector proteins Oneof the pathways that lead to signal activation requires theformation of speciï¬c complexes such as NFκB essentialmodulator NEMO and the kinase IKK which bridges theactivation of NFκB and IFN regulatory factor IRF signalingpathways It has been demonstrated that FMDV 3Cpro interactswith NEMO and induces its cleavage resulting in impairedinnate immune signaling IRFmediated signals driven byIRF3 and IRF7 can also be targeted by FMDV proteinsSpeciï¬cally overexpression of Lpro in PK15 cells resulted inthe downregulation of IRF3 and IRF7 protein levels andinactivation of IFN and IFNλ1 promoter Other factors involved in the activation of IFN includeconventional PTMs such as phosphorylation and ubiquitinationwhich ensure eï¬ective regulation of these signaling pathways Also diï¬erent cellular deubiquitinases DUBs can reverseubiquitination to control the intensity of the immune signalingresponse Interestingly it has been shown that FMDV Lprocan remove ubiquitin Ub molecules from several proteinsrequired for IFN mRNA expression and those involved in theactivationrepression of the IFN loop This role became moreevident by the observation that during infection FMDV Lprocan cleave cellular substrates modiï¬ed with the Ublike moleculeISG15 Furthermore mutation of Lpro thatimpairsdeISGylaseDUB function results in viral attenuation Inthis regard identiï¬cation of FMDV targets for deubiquitinationand deISGylation may contribute to elucidate the role ofthose factors in counteracting the innate response and developnovel countermeasuresBlock on Interferon SignalingThe ligandmediated association of the speciï¬c IFN receptorspromotes a signaling cascade that results in the phosphorylationof the receptor by the action of JAKs These events result inthe generation of docking sites for downstream adaptor andeï¬ector proteins including signaltransducer and activatoroftranscription STAT proteins that associate with otherfactors and translocate to the nucleus inducing transcriptionof a plethora of ISGs described above and in Figure Although blockage of the JAKSTAT signaling pathway hasnot been reported during FMDV infection overexpressionof either FMDV 3Cpro or VP3 can inhibit this response Forinstance IFNtreated HeLa cells overexpressing FMDV 3Cprosuppressed IFNstimulated promoter activities and inducedproteasome and caspaseindependent protein degradationof karyopherin α1 KPNA1 the nuclear localization signalreceptor Thisinteraction inhibited the nuclear translocation of STAT1STAT2impeding maximal ISG promoter activity In another studyin HEK293T cells overexpression of VP3 followed by coimmunoprecipitation revealed the association between VP3 andJAK1 FMDV VP3 also inhibited virustriggered activation of theIFN promoter leading to the decrease in transcription of ISGspresumably due to lysosomalinduced degradation of JAK1 A yeast twohybrid screen identiï¬ed FMDV 2C in complex withNmyc and STAT interactor Nmi a protein known to augmentimmune function dependent on STATmediated transcriptionInterestingly such interaction resulted in the recruitment ofNmi to vesicular compartments followed by the induction ofapoptosis in BHK21 cells tyrosinephosphorylated STAT1forEvidently FMDV proteins can also target crosstalk pathwaysinduced by JAKSTAT signaling and due to this versatilityunderstanding of these signaling events during FMDV infectionis challengingFOOTANDMOUTH DISEASE VIRUSIMPAIRS INTERFERONMEDIATEDCELLULAR INNATE IMMUNE RESPONSESSimilarly to what happens in vitro FMDV manipulates theearly innate immune response in vivo to ensure a windowof opportunity that favors viral replication and spread beforeFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVthe onset of eï¬ective adaptive immunity required for virusclearance During infection FMDV interacts with a range of hostcells including natural killer NK cells dendritic cells DCsmonocytesMÏ and Îδ T cells All these cells play an importantrole in innate immune responses that trigger the productionof large quantities of IFN and other cytokines which serve asautocrine agents Shortly after FMDV infection in swinethe number ofcirculating NK cells transiently decreases and the remaining NKcells show a dysfunctional lytic activity against target cells and areduction of IFNÎ production In parallel FMDV blocks theability of porcine DCs to mature into conventional DCs cDCs dampening their response against Tolllike receptor TLRligands Another subset of porcine DCs plasmacytoid DCspDCs also referred to as the major professional systemic IFNαproducers are also aï¬ected by FMDV During infectionpartial depletion of pDCs in the peripheral blood has beendetected and the remaining pDCs are less capable of producingIFNα in response to ex vivo stimulation by TLR ligands or virus Similar to pDCs FMDV infection reduces the productionof IFNα on Langerhans cells LCs a distinct subset oftissueresident DCs of the skin It has also been suggestedthat porcine Îδ T cells and MÏ can serve as targets for FMDVinfection in swine although the interplay betweenthese cells and FMDV remains unclearComparably to swine FMDV infection in cattle triggersseveral early events in the innate immune system althoughthe eï¬ects are not exactly the same For instance bovine NKcells originated from FMDVinfected cows have an elevatedcytotoxic function against bovine target cells in vitro In addition some subsets of cDCs are signiï¬cantly decreasedduring the peak of viremia while the expression of majorhistocompatibility complex MHC class II molecules on allbovine cDCs is reduced and the processing of exogenous antigenis impaired Furthermore during FMDV infection thenumber of systemic mature bovine pDCs characterized bythe expression of CD4 and MHC class II is increasedpresumably to intensify a humoral response and T cell activationwhile levels ofimmature CD4 MHC class IIpDCs aredeclined Examination of bovine Îδ T cells revealed thatthese cells with the surface expression marker WC1 showa transient activated phenotype and increased expression ofIFNÎ FMDV also aï¬ects the innate immune response at the cytokinelevel in the natural host In vivo cytokine proï¬le analysis duringthe clinical phase of disease shows a systemic decrease of proinï¬ammatory cytokines [IL1 IL6 and tumor necrosis factorTNFα] and an increase of the antiinï¬ammatory cytokine IL and IFNα Most likely these changesare related to the early T cell unresponsiveness and lymph iadescribed in swine and cattle during FMDV infection Interestingly a signiï¬cant induction of inï¬ammatoryand antiviral factors at the local level is detected in cattle in sitesof abundant viral ampliï¬cation such as the nasaloropharynx orvesicular lesions A consistent upregulation of IFNα Î and λ mRNA in distinct microanatomical compartmentsof the nasopharyngeal mucosa concurrent with occurrence ofviremia has also been detected in cattle In contrast studiesin swine demonstrated that IFN expression in infected swineskin is inhibited These diï¬erences may be due to theanalysis of follicleassociated epithelium of the nasopharyngealmucosa in cattle vs skin in swine or to the speciï¬c samplingtechnique used in each experiment While in the cattle studylasercapture microscopy was used to focus only in areas of highFMDV replication in the swine study RNA was extracted froma piece of skin without discriminating between microanatomicalcompartments Evidently more studies are needed to elucidatethe intricate interactions between FMDV and the innate immunesystem of speciï¬c animal hostsEFFECTIVE USE OF INTERFERONAGAINST FOOTANDMOUTH DISEASEVIRUS IN VITROType I InterferonThe role of IFN in controlling FMDV replication was ï¬rstproposed in when Dinter and Philipson demonstrated thatcalf kidney cells exposed to FMDV could become persistentlyinfected and proposed this was a consequence of the inductionof an IFNlike inhibitor present in the supernatant of infectedcells Later studies also suggested that swine leukocytestreated with phytohemagglutinin produced an inhibitor ofFMDV replication with properties similar to IFN It wasnot until that new studies demonstrated that the abilityof FMDV to form plaques in cell culture correlated with thesuppression of type I IFN α protein expression Theseresults were further supported by detection of IFN protein andantiviral activity in the supernatants of primary porcine ovineand bovine kidney cells infected with an attenuated FMDVmutant leaderless as compared to the supernatants of cellsinfected with wildtype WT virus Later studies by the samegroup provided proof of concept on the use of recombinantbacterial expressed IFNα as a potent biotherapeutic againstFMDV This approach was further developed by deliveringrecombinant porcine IFNα using a replicationdefectivehuman Adenovirus vector Ad5poIFNα Infection ofIBRS2 cells with Ad5poIFNα resulted in secreted poIFNα IFN protein detected as early as h postinfection hpiand lasting for at least h Most important expressed IFNprotein displayed strong biological antiviral activity againstFMDV Followup studies by the same group showed that allFMDV serotypes are very sensitive to Ad5delivered poIFNαand sterile protection could be achieved in vivo highlightingthe potential of this approach for the development into abroad biotherapeutic strategy to control FMDV replicationIn the last years advancements is genomics have ledto the characterization of almost all type I IFN subtypes inthe porcine and bovine genome which are morenumerous than those identiï¬ed in primates and mice This hasrevealed diï¬erent functional genes and pseudogenes with diverseexpression proï¬les and antiviralfunctions against diï¬erentviruses mostly in swine In fact a recent studyFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVdemonstrated that poIFNÏ7 known for its ability to inducethe highest levels of antiviral activity when compared to otherpoIFNÏ subtypes elicits an antiviral state against FMDV inIBRS2 cells treated with the recombinant form of poIFNÏ7produced in Escherichia coli Other subclasses of type IIFN known to be produced in swine and cattle include IFNalphaomega IFNÎ±Ï also known as IFNµ and IFN delta IFNδ Signiï¬cant reduction in FMDV replication has been observedupon treatment of porcine cells with bacterially expressed IFNÎ±Ï or IFNδ8 prior to viral infection Recently another member of type I IFN family IFNÏ whichis only produced in ruminants has been evaluated as an antiviralagainst FMDV IFNÏ is a paracrine reproductive hormonesecreted constitutively by trophoblasts and endometrial cells toincrease the life span of the corpus luteum however productionis not induced upon viral infection While its secretionis restricted to ruminantsit has a broadspectrum activityagainst various crossspecies viruses Interestingly IFNÏ has homology with the amino acids of IFNα which allows forbinding to type I IFN receptors The property of IFNÏ thatmakes it an interesting therapeutic candidate for the treatmentof various viral diseases is its signiï¬cantly lower toxicity ascompared to other type I IFNsType II InterferonIn contrast to type I IFN the type II IFN family is composedof only one member IFNÎ which exerts its actions througha speciï¬c receptor IFNGR1IFNGR2 IFNÎ is weakly resistantto heat and acid and it is able to activate leukocytes suchas macrophages and granulocytes also exerting regulatoryfunctions on T and B lymphocytes Indeed productionof IFNÎ is used as a tool to measure cellmediated immuneresponses against FMDV in vaccinated cattle andin swine Interestingly IFNÎ responses as measured b | Thyroid_Cancer |
"attention has been paid to whether snoring frequency is associated with body composition inmenopausal women particularly in China This study objected to investigate the association between selfreportedsnoring and body composition in peripost menopausal Chinese women as well as metabolic indicatorsMethods This crosssectional study enrolled participants aged years from the Menopause Clinic in theShanghai Sixth Peoples Hospital Participants were categorized into four subgroups stratified by selfreportedsnoring frequency never rarely night per week occasionally nights per week regularly ¥ nights perweek while body composition was measured using bioelectrical impedance analysis BIA Besides blood samplewere collected to test the glycolipid indicatorsResults In our sample of investigation regular snoring ¥ nights per week was found to be an independent riskfactor for higher fat mass total upper limbs trunk with the highest risk of times for fat mass of trunk afteradjusting for metabolic confoundersp Meanwhile regular snoring was independently associated withhigher fat mass total and each segment only in menopausal transition p Conclusions We suggested that selfreported regular snoring may be taken as a simple alternative to predict higherfat mass ¥ kg upper quartile in menopausal women Similarly body composition should be attached to thegreat importance to those who in menopausal transition in order to help to prevent obstructive sleep apnea OSAKeywords Body composition Snoring Menopausal transitionBackgroundSnoring the manifestation of increased upper airway resistanceis commonly regarded as a reliable proxymarker of obstructive sleep apnea OSA [ ] Moreover regular snoring has been suggested to be correlatedwith obesity [] hypertension [] and diabetes mellitus[] OSA is supposed to be more prevalent in men than Correspondence yctengsjtueducn taomfsjtueducn Yang Zhou and Fei Liu contributed equally to this work1Department of Gynecology and Obsterics Shanghai Jiao Tong UniversityAffiliated Sixth Peoples Hospital Yishan Road Shanghai ChinaFull list of author information is available at the end of the women however the gap was narrowed when womenapproach menopause [ ] Women in menopause transition are more likely to report perspective poor sleepsnoring [] which largely affected quality life of menopausal women In addition previous studies have reported that menopause was an important risk factor forsnoring mainly due to the declining ovarian hormones[ ] Thus it is important to combat snoring in peripost menopausal womenMeanwhile menopause is a vital window for variationsin the body composition and rising in the body weightcaused by hormonal alterations [] However body 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 0cZhou BMC Women's Health Page of in menopausal women [] Changesmass index BMI is not a valid measure of true obesitystatusinmenopauserelated body composition may be coveredand underestimated by stable BMI since the counteractive effect of loss of lean mass and gain of fat masswhen aging Therefore body composition by bioelectrical impedance analysis BIA may be a more representative and precise instrument rather than BMI amongmenopausal Chinese women []So far current studies on the association of snoringand obesity have focused primarily on men and children[ ] while underrepresented women In addition anyassociation between snoring and body composition inmenopausal women has received little attention Sinceits possible that glycolipid metabolism may confoundthe association and whether snoring is associated withbody composition in menopausal women independentlyof glycolipid metabolism confounders remains unknownGiven the evidence of the cross interplay among snoringobesity and menopause we aim to explore the association with snoring and body composition in menopausalwomenMethodsStudy participantsThis crosssectional study enrolled participants who visited the Menopause Clinic in the Shanghai Sixth Peoples Hospital HanChinese woman aged yearspassing through the menopause were recruited Exclusion criteria were with rhinitis having severe internalsuch as myocardialinfarction stroke and cancer current smoking atleast once per week for the previous months excessive alcohol drinking at least one pack per month forthe previous months suffering from thyroid disease having tubercle and cachexy missing dataUltimately715 participants were recruited in this studyillnesses andor diseasesGeneral questionnaireBaseline sociodemographic information was collectedfrom a questionnaire through facetoface interviewwhich has been previously employed [] seen in supplementary file Variables included age marital statusemployment status education level income per monthmenopausal age menopausal status history of chronicdisease ie hypertension diabetes mellitus rhinitisother diseases besideslifestyle ie smoke alcoholconsumption were recorded Guiding by the Stages ofReproductive Aging Workshop STRAW []participants were divided into three different menopausalsubgroups namely menopausal transition group consecutive irregularities for over days of menstrual cycleearly postmenopausal group absence of menstrual periods for months years and late postmenopausalfor ¥ yearsgroup absence of menstrual periodsHypertension was defined by any prior diagnosis fromthe questionnaire or by the criteria recommended by theseventh report of the Joint National Committee on Prevention Detection Evaluation and Treatment of HighBlood Pressure JNC7 [] While diabetes mellitus wasidentified by FPG ¥ mmolL or received any treatmentfor diabetes according to the WHO criteria []Snoring frequency assessmentParticipants were asked by the question to assess thesleep snoring frequency which was applied previously[ ] Over the past weeks did you snore And ifdid how many times per week and the options for responses were never rarely occasionally and regularly corresponding to never night per week nights per week and ¥ nights per week respectively seen in supplementary file Anthropometric and lab testsWe measured and recorded participants weight heightBody mass index BMI was computed by dividingweight in kilograms by the square of their height in meters We took the blood pressure for all participants onthe right arm three consecutive times after 5min sittingsystolic blood pressure SBP diastolic blood pressureDBP Blood samples were collected for the detectionof serum concentration of triglyceride TG cholesterolTC highdensity lipoprotein HDL lowdensity lipoprotein LDL and fasting blood glucose FBG after anovernight fastBody compositionWe measured the body composition by BIA TBF418Banalyzer TANITA of lean mass LM fat mass FMand fatfree mass FFM and each segment includedupper lower limbs and trunk We also recorded basalmetabolic rate BMR concurrently [] The welltrained staff guided the participants to take off heavyclothes socks and shoes and hold the hand electrodesstanding barefoot in contact with footpad electrodes[] Fat mass total and each segment and lean masstotal and each segment were stated in the dichotomized form with a cutoff of the highest quartile as thehigher one comparing the highest to the lower two tertiles We defined ¥ kg ¥ kg and ¥ kg ashigher total fat mass higher fat mass of upper limbs andhigher fat mass of trunk respectivelyStatistical analysesAll statistical analyses were taken by SPSS IBMCorporation Armonk NY USA Data were tested fornormal distribution bythe Kruskal WallisHtestLevenes test of homogeneity of variance was also 0cZhou BMC Women's Health Page of performed Variables were presented as mean ± standarddeviation SD when they showed normal distributionswhereas medians inter quartile range or values Oneway ANOVA normal distributions the KruskalWallis Htest skewed continuous variables and Ï2 testcategorical variables were carried out to compare thedifferences among the four groups Snoring was analyzedas a categorical variable with never as the referencegroup Relationship between body composition andsnoring frequency was computed by multiple logistic regression analysis Covariates included TG TC HDLLDL FBG SBP DBP age marital status employmentstatus education level income per month menopausalage menopausal status hypertension diabetes mellitusTwosided p was considered significantResultsCharacteristics of the study participants based on snoringfrequencyA total of participants were finally entered into thestudy The basic characteristics among the four groupsdivided by the snoring frequency never rarely occasionally regularly were presented in Table Participants were on average ± years of age with amean weight of ± kg and the average BMIwas ± kgm2 The mean lean mass fat massand fat free mass were ± kg ± kgand ± kg respectively Compared with nonsnorers rare and occasional snorers regular snorerstended to be older showed higher triglyceridelowerHDLC and had less income p Moreover therewas an ascending trend in the incidence of hypertensionin different snoring frequency subgroupswith innonsnorers increasing to in regular snorersp However we did not observe the differenceamong three menopausal status respect to the snoringfrequencyBody composition of the study participants distributed bysnoring frequencyAs presented in Table compared with nonsnorersrare and occasional snorers regular snorers had higherfat mass upper limbs trunk lower limbs In additionwe found that there was an increasing trend in the fatmass of upper limbs trunk and lower limbs and also inlean mass of upper limbs with the increase of the sleepsnoring frequency p Odds ratio of snoring frequency for body composition bymultiple logistic regression analysisWe next investigated the odds ratio of snoring frequencyin predicting for body composition p in univariate analysis after adjusting for potential confounders Asdepicted in Fig compared with nonrare andoccasional snoring regular snoring was the risk predictor for higher total fat mass ¥ kg OR 95CI P higher fat mass of upperlimbs ¥ kg OR 95CI P higher fat mass of trunk ¥ kg OR 95CI P while other segmentsshowed no significance after adjustments In additionregular snoring increased the highest odds ratio OR of for fat mass of trunk among the other statisticallysignificant body compositionsIndependent determinants for regular snoring stratifiedby menopausal statusWe also investigated the independent roles of body composition for predicting regular snoring in multivariate logistic regression analysis in Table however we did notobserve any significance of body composition in predicting for regular snoring after adjusting confoundersInterestingly when the participants were stratified bydifferent menopausaltransitionearly postmenopause late postmenopause we observedthat fat mass segments were independently associatedwith regular snoring in menopausal transition but notpostmenopause TotalOR 95CI P fat mass of upper limbs OR 95CI P fat mass of trunkOR CI P fat mass oflower limbs OR 95CI P were independent indicators for regular snoring afteradjusting for confounders in menopausal transitionmenopausalstatusfat massDiscussionTo our knowledge this is the first study to document associations of snoring and body composition as well asmetabolic indicators in women with regard to menopausal status The main finding was that regular snoring¥ nights per week was an independent risk factor forhigher fat mass total trunk upper limbs in menopausalwomen after adjusting for wellestablished metabolicvariables Of special concern was that regular snoringhad a times significantly higher odds of higher fatmass of trunk which was the highest among other significant body composition This finding was in concordant with the previous study that OSA was more inclinedto a centralobesity phenotype than a wholeobesity pattern []Several mechanisms can interpretthis associationUpper airway resistance and collapsibility caused byregular snoring could result in intermittent hypoxia andsympathetic activation thus leading to the aggravationof obesity especially for abdominal fat [] In additionprotective role of progesterone and estrogen in respiratory control vanished after menopause which was associated with continuum from increased airway resistance 0cZhou BMC Women's Health Page of Table Body composition and characteristics of the women distributed by snoring frequencyVariablesRarelyn ± ± ± ± Occasionallyn ± ± ± ± Regularlyn ± ± ± ± Totaln ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Snoring FrequencyNevern ± ± ± ± ± ± Age yearsWeight KgHeight cmBMI Kgm2BMRTG mmollTC mmollHDLC mmollLDLC mmollFPG mmolLSBP mmHgDBP mmHgChronic disease n HypertensionDiabetesMarital status n MarriedSingleSeparatedDivorcedWidowedMenopausal status n Perimenopause Early postmenopause Late postmenopause Employment n Education n Junior or belowSenior highCollege or aboveIncome RMBmonth n Lean mass kgUpperTrunkLowerFat mass kgUpperTrunkLower ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Fatfree kg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P value 0cZhou BMC Women's Health Page of Table Body composition and characteristics of the women distributed by snoring frequency ContinuedVariablesRarelyn ± ± ± Occasionallyn ± ± ± Regularlyn ± ± ± Totaln ± ± ± P valueSnoring FrequencyNevern ± ± ± Upper limbsTrunkLower limbsHigher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsOR OR OR OR OR OR Higher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsRare snoringOR OR OR OR OR OR Occasional snoringHigher fat massHigher fat mass of upper limbsHigher fat mass of trunkHigher fat mass of lower limbsHigher lean massHigher lean mass of upper limbsOR OR OR OR OR OR Fig Odds ratios 95CI of snoring frequency for body composition in women analyzed by multivariate logistic regression Covariates age BMITG TC HDL LDL FBG SBP DBP hypertension diabetes mellitus menopause income education employment status means p0005Regular snoring 0cZhou BMC Women's Health Page of Table Odds ratio of body composition for regular snoring stratified by menopausal status by logistic regressionFat mass kgRegular snoringPerimenopause n Odds ratio CI Pvalue Fat mass of upper limbs kg Fat mass of trunk kg Fat mass of lower limbs kg manifested as snoring [] Taken together menopause make women lose protective effects against snoring and further augmentsnoreobesity associationespecially snorecentralobesity associationInterests in obesity and OSA as regards to which isthe chicken or the egg has existed since the dawn ofhistory [ ] Thus to identify the mutual effect ofsnoring and obesity we also assessed the role of bodycomposition in predicting the snoring We found thatfat mass was an independent risk factor for regular snoring only in menopausal transition not postmenopause ina multivariable model Taken together we suggestedthat the rise in obesity may serve as a key contributor tothe burgeoning prevalence of snoring in women whilemenopausal transition not postmenopause period maymark this relationshipThe reason can be explained by the fact that menopausal transition is more concerned with fluctuation ofsex hormone than postmenopause which predisposed tomodulate sleep regulation and breathing thus leading tothe snoringBesides other independent factors for regular snoringsuch as higher TG lower LDL were compatible with onestudy [] Although selfreported snoring was closelyrelated with hypertension and diabetes Unexpectedlywe did not find that hypertension was related with regular snoring after multiple adjustments These divergentfindings may be attributed to difference in sample sizeethnicity culture and the definition of hypertension anddiabetes etc Another possible explanation is that manyprevious studies did not consider menopause status in toaccount which may aggravate the snoreobesity association thus overshadow the snorehypertensiondiabetesassociationHowever our study should be interpreted in light ofthe following limitations First one limitation of thepresent study is that the crosssectional design does notpermit conclusion of causality further prospective studies are needed to verify the association between snoringfrequency and body composition Second selfreportedsnoring frequency but not polysomnography the goldstandard for diagnosing OSA which could bring aboutthe statistical error However precious study has suggestthat selfreport is a reliable measure []Early postmenopause n Odds ratio CI Pvalue Late postmenopause n Odds ratio CI Pvalue ConclusionsRegular snoring ¥ times per week may be an independent strong predictor for fat mass of trunk in menopausal women while fat mass in turn serves as a strongpredictor for regular snoring only in menopausal transition Taken together early detection and interventionsof participant showing regular snoring and higher fatmass in menopause could have important preventiveimplicationsSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12905020010252Additional file AbbreviationsBIA Bioelectrical impedance analysis OSA Obstructive sleep apneaTG Triglyceride TC Cholesterol HDL Highdensity lipoprotein LDL Lowdensity lipoprotein FBG Fasting blood glucose BMI Body mass indexFM Fat mass LM Lean mass FM Fat mass FFM Fatfree mass BMR Basalmetabolic rateAcknowledgementsThe authors would like to acknowledge all women who consented to takepart of this study We are also thankful for the support and cooperation fromstaff members of obstetrics and gynecology in Shanghai Jiao TongUniversity Affiliated Sixth Peoples HospitalAuthors contributionsMT conceived the study and YT designed the study [YZ] drafted andcritically revised the manuscript FL designed the questionnaire and analyzedthe data CL [YZ] JH [YZ] LG and SJ administered the questionnairesurvey and managed the data All authors read and approved the finalmanuscriptFundingThis study was supported by grants from the Science and TechnologyCommission of Shanghai Municipality The role of thefunding body in the design of the study and collection analysis andinterpretation of data and in writing the manuscript should be declaredAvailability 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 participants had provided verbal informed consent after full explanationbecause the study posed no threat to the health of patients This study wassubmitted to and approved by the ethics committees of Institutional ReviewBoard in Shanghai Sixth Peoples Hospital affiliated for Shanghai JiaotongUniversity No2016R07 0cZhou BMC Women's Health Page of Consent for publicationNot applicableCompeting interestsThe authors declare that they have no conflict of interestAuthor details1Department of Gynecology and Obsterics Shanghai Jiao Tong UniversityAffiliated Sixth Peoples Hospital Yishan Road Shanghai China2Reproductive medicine center Shanghai Jiao Tong University Affiliated SixthPeoples Hospital Yishan Road Shanghai ChinaReceived July Accepted July ReferencesLee YH Kweon SS Choi JS A GenderSpecific Association betweenSelfReported Snoring and Hemoglobin A1c Levels in a General Populationwithout Type Diabetes Mellitus Yonsei Med J httpsdoi103349ymj20175861152 PubMed PMID PubMedCentral PMCID PMCPMC5653480Song J Wang C Ma A Selfreported snoring is associated with chronickidney disease independent of metabolic syndrome in middleaged and elderlyChinese J Diabetes Investig httpsdoi101111jdi12855PubMed PMID PubMed Central PMCID PMCPMC6319474 engBiggs SN Tamanyan K Walter LM Overweight and obesity add to behavioralproblems in children with sleepdisordered breathing Sleep Med httpsdoi101016jsleep201709001 PubMed PMID Shivashankar R Kondal D Ali MK Associations of Sleep Duration andDisturbances With Hypertension in Metropolitan Cities of Delhi Chennaiand Karachi 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chicken or the eggDiabetes Care 200831Suppl 2S303 httpsdoi102337dc08s272PubMed PMID PubMed Central PMCID PMCPMC2453667 eng Behan M Kinkead R Neuronal control of breathing sex and stresshormones Comprehensive Physiology httpsdoi101002cphyc100027 PubMed PMID eng Behan M Zabka AG Thomas CF Sex steroid hormones and the neuralcontrol of breathing Respir Physiol Neurobiol PubMed PMID eng Bixler EO Vgontzas AN Lin HM Prevalence of sleepdisordered breathing inwomen effects of gender Am J Respir Crit Care Med Pt httpsdoi101164ajrccm16339911064 PubMed PMID eng Carneiro G Zanella MT Obesity metabolic and hormonal disordersassociated with obstructive sleep apnea and their impact on the risk ofcardiovascular events Metabolism httpsdoi101016jmetabol201803008 PubMed PMID eng Zhang N Chen Y Chen S SelfReported Snoring Is Associated withDyslipidemia High Total Cholesterol and High LowDensity LipoproteinCholesterol in Obesity A CrossSectional Study from a Rural Area of China Int JEnviron Rese Public 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"MiRNAs play important roles in the development of ovarian cancer activation of primitive folliclesfollicular development oocyte maturation and ovulation In the present study we investigated the specific role ofmiR23a in cov434 cellsResults Downregulation of miR23a was observed in serum of PCOS patients compared with the healthy controlsuggesting the inhibitory effect of miR23a in PCOS MiR23a was positively correlated with Body Mass Index BMI andnegatively correlated with Luteinizing hormone LH Testostrone T Glucose Glu and Insulin INS of PCOS patientsMiR23a mimic inhibited the proliferation and promoted apoptosis of human cov434 cells In addition flow cytometryassay confirmed that miR23a blocked cell cycle on G0G1 phase MiR23a inhibitor showed opposite resultsFurthermore double luciferase reporter assay proved that miR23a could bind to the UTR of FGD4 directly throughsites predicted on Target Scan FGD4 level was significantly suppressed by miR23a mimic but was significantlyenhanced by miR23a inhibitor We further proved that miR23a increased the expression of activated CDC42 GTPbround and pPAK1 suggesting that miR23a induced cell cycle arrest through CDC42PAK1 pathwayConclusions In our study reveals that miR23a participates in the regulation of proliferation and apoptosisof cov434 cells through target FGD4 and may play a role in the pathophysiology of PCOSKeywords miR23a Polycystic ovary syndrome FGD4 Binding site Cell cycleBackgroundPolycystic ovary syndrome PCOS is the most common reproductive endocrine and metabolic disorder disease inwomen characterized by ovulation disorders hyperandrogenism and insulin resistance [ ] PCOS affects about of women of childbearing age accounting for ofanovulatory infertility and usually a lifelong disease Itscommon clinical manifestations include menstrual disorders subfertility acne vulgaris alopecia seborrheia obesity hirsutism and acanthosis [] Women with PCOS havean increased risk of insulin resistance hypertension type Correspondence linjinet163com3Gynaecology Mindong Hospital in Ningde City No Heshan Road FuanFujian ChinaFull list of author information is available at the end of the diabetes oxidative stress dyslipidemia cardiovasculardisease and endometrial cancer [] Therefore understanding the molecular mechanism of metabolic diseases underlying the pathophysiology of PCOS will help to identify newdiagnostic and therapeutic strategies In addition althoughthe exact etiology of PCOS remains to be understood ithas been clear that the survival and proliferation of granulosa cells are closely related to the pathogenesis of PCOS[]In recent years the role of microRNAs miRNAs inovarian physiology and pathology has attracted muchattention Some studies have shown that miRNAs playimportant roles in the development of ovarian canceractivation of primitive follicles follicular developmentoocyte maturation and ovulation [] Several studies 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 0cLin Journal of Ovarian Research Page of have found a variety of differentially expressed microRNAs in ovarian granulosa cells of PCOS patients whichare closely related to the proliferation and apoptosis ofovarian granulosa cells and the production of progesterone estradiol and testosterone [ ]responsibleforinducing caspasedependentThe human miR23a gene is located on chromosome of the human genome and transcribed into a part of themiR23a27a242 cluster [] Mi23a27a242 clusterwhich encodes primicroRNA transcripts composed ofthree kinds of miRNAs miR23a miR27a and miR242isandcaspaseindependent apoptosis of embryonic kidney cellsHEK293Tthrough human cJun Nterminal kinasepathway [] In recent years more and more evidencehas shown that miR23a is essential for folliculogenesis Ithas been reported that the expression of circulating miR23a of patients with PCOS was downregulated comparedwith healthy women and proved that miR23a is a betterindicator for evaluation of PCOS than the miR23b []However as far as we know the specific role and mechanism of miR23a in PCOS have not been studiedStudies proved that miR23a issignificantly upregulated in premature ovarian insufficiency POI patients serum and poor ovarian response POR patientsovarian granulosa cells [] Compared with normalwomen miR23a was significantly upregulated in follicular cells of women receiving assisted reproductive technology ART due to oviduct and endometriosis []More critically miR23a can promote the apoptosis byaffecting the expression of multiple targetsincludingXIAP SMAD5 and Sirt1 [ ]Thereforein the present research we hypothesizedthat miR23a is involved in the development of PCOS byregulating downstream pathways related to cell survivalin ovarian cells The objective of this study was to confirm the regulatory effect and mechanism of miR23a onthe growth of cov434 cells We analyzed the expressionof miR23a in serum samples from PCOS patients andhealthy women and the correlation between miR23alevel and PCOS symptoms We focused on a new molecular mechanism by which miR23a induces apoptosisin granular cellsdisease smoking and using alcohol or drugs The serumof healthy women was collected as the control groupThe volunteers in the control group had normal menstruation normal ovaries and no history of reproductivesystem disease or appendicitis The control and PCOSgroup did nottake any medications in the past months including oral contraceptives or other hormonalmedications with no intrauterine devices or smokingPatients with reproductive system disease or appendicitishistory were excluded from the control group All volunteers had understood the purpose and requirements ofthis study and signed a written informed consent beforeparticipating in the study ml of elbow venous bloodfrom each sample was taken and stored in a refrigeratorat °C All the experiments involved in this studyhave obtained the ethical approval of Mindong hospitalin Ningde CityEvaluation of BMI and sex hormoneThe weight and height of the volunteers were measuredto calculate Body mass index BMIBMI weightheight2 Radioimmunoassay RigorBio Scientific andTechnology Co Beijing was used to measure the levelof total testosterone and other sex hormonesCell line and transfectionCell lines KGN derived from a granulosa cell tumorcov434 derived from a granulosa cell tumor and SVOGderived by immortalization of granulosaluteal cellsusing SV40 large T antigen were purchased from cellresource bank of Chinese Academy of Sciences cells were seeded into well plates MiR23a micmic nM miR23a inhibitor nM and negative control NC nM mimic NC and nM inhibitor NCRuibo Biotechnology Co Ltd Guangzhou China weretransfected into cov434 cells by Lipofectamine¢ Normal untreated cov434 cells were cultured as control Thesequence of siRNA used in this study is as follows miR23amimic ²CCTTTAGGGACCGTTACACTA3² mimicNC ²TTCTCCGAACGTGTCACGTTTC3² miR23ainhibitor ²TAGTGTAACGGTCCCTAAAGG3² inhibitor NC ²TTCTCCGAACGTGTCACGTTTC3²Materials and methodsSamplesThe serum of Chinese women with PCOS was collected in Mindong hospital Ningde City Fujian Provincefrom September to December According tothe revised PCOS diagnostic criteria published by theRotterdam consensus [] the PCOS group excluded patients with Cushings syndrome delayed congenital adrenal hyperplasia thyroid dysfunction hyperthyroidismhyperprolactinemia or androgen secreting tumor as wellas patients with diabetes hypertension chronic kidneyReal time fluorescence quantitative PCR qPCRTotal RNA were extract from samples or cells using Trizolreagent Related expression of target gene was calculatedusing 2ÎÎCt method This study involves the followingsequences miR23a3p Reverse transcription ² GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGAAAT3² miR242 Reverse transcription ²GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGTGT3² miR27a3P ²GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCGGAA3² U6 Reverse transcription ²AAAATATG 0cLin Journal of Ovarian Research Page of GAACGCTTCACGAATTTG3² miR23a3p forward primer ²GCGATCACATTGCCAGGG3² and reverse primer²AGTGCAGGGTCCGAGGTATT3² miR242 forwardprimer ²CGCGTGCCTACTGAGCTGAA3² and reverseprimer ²AGTGCAGGGTCCGAGGTATT3² miR27a3Pforward primer ²GCGCGTTCACAGTGGCTAAG3² andreverse primer ²AGTGCAGGGTCCGAGGTATT3² U6forward primer ²CTCGCTTCGGCAGCACATATACT3²and reverse primer ²ACGCTTCACGAATTTGCGTGTC² FGD4 forward primer ²CCTGCCTCTGCTTCTTGTGTCTC3² and reverse primer ²TGGTTGTCAATCCATGCCTTCCTG3²Cell proliferation assayAfter h of transfection cells were seeded into a well plate at the density of cells per well Eachgroup of cells was treated with replicates After incubation for the specified time and h μl of CCK8 reagent was added and incubated at °C for h The absorbance of each pore was measured at nm by an enzyme labeling instrumentFlow cytometry analysis for cell cycleAfter h of transfection the cell cycle was detected byflow cytometry The cells were fixed with ethanolovernight at °C The cells were resuspended with μl of binding buffer μl PI was added to the cellsuspension and incubated at room temperature for min The results were analyzed by ModFit and displayedby FL2w and FL2aFlow cytometry analysis for apoptosisAfter h of transfection the apoptotic cells were detected by flow cytometry μl of PI and FITC annein Vwere added into μl cell suspension and incubated atroom temperature for min Cell apoptosis was detected using a flow cytometerWestern blotThe total protein was extracted with RIPA buffer BCAmethod was used to detect the protein concentrationThe extracted protein was electrophoresis by SDSPAGEand transferred to PVDF membrane PVDF membranewas incubated in skimmed milk at room temperaturefor h and then primary antibody overnight at °Cfollowed by the secondary antibody at room temperaturefor h QUANTITY ONE software is used for resultanalysis The following antibodies were used in this research antiFGD4 Abcam ab97785 87KDaantiCDC42 Abcam ab155940 21KDa antiPAK1 Abcam ab223849 60KDa and βactinTransGen Biotech HC201 42KDaDouble luciferase reporting assayThe plasmids of wild type FDG4WT and mutant typeFDG4MUT luciferase reporter genes were constructedusing pcDNA31as the empty vector MiR23a mimicmimic NC FDG4WT and FDG4MUT plasmids were cotransfected into cov434 cells by LipofectamineTM Cells were divided into four groups FGD4WT ²UTR miR23a mimic NC FGD4Mut ²UTR miR23a mimicNC FGD4WT ²UTR miR23a mimic FGD4Mut ²UTR miR23a mimic After h of transfection FirelyLueiferase F and Renilla Luciferase R were detected byGLOMAX \\fluorescence detector and the relativeluciferase activity F R was calculatedStatistical analysesAll data were analyzed with SPSS SPSS Inc Chicago IL software and represented as mean ± SD Spearman method was used to analyze the relationshipbetween miRNA level and other indicators Independentsample ttest was used to evaluate the difference between two groups and Oneway ANOVA was used toanalyze the difference between three and more groupswith post hoc contrasts by Bonferroni test P wasconsidered statistically significantResultsMiR23a was downregulated in serum of PCOS patientsPeripheral blood was collected from local PCOS patients for the detection of miR23a level with healthywomens peripheral blood as the control Clinical information on age BMI and sex hormone levels of PCOSpatients and normal control samples are alllisted inTable As shown in Fig 1a the serum miR23a level inPCOS patients was significantly lower than that in thecontrol group P Then we detected the level ofmiR27a and miR242 using qPCR As shown in Fig 1amiR27a and miR242 also downregulated in peripheralblood of PCOS patients compared with healthy sampleTable The clinical information of PCOS and control groupsClinical indexPPCOSn ± Controln ± ± ± ± ± ± ± ± ± ± ± AgeE2 pgmLBMI Kgm2LH mIUmLFSH mIUmLPRL mIULT mIUmL ± ± ± Glu nmolmLINS μUmLE2 Estradiol BMI Body Mass Index LH Luteinizing hormone FSH Folliculestimulatinghormone PRL Prolactin T Testostrone Glu Glucose INS Insulin ± ± ± 0cLin Journal of Ovarian Research Page of Fig MiR23a was downregulated in serum of PCOS patients a qPCR was performed to detect the expression of miR23a in PCOS samplePCOS and healthy control Normal b miR27a and miR242 levels were detected using qPCR in PCOS and normal group Correlation betweenmiR23a level and BMI was analyzed in PCOS c and control d group Correlation between miR23a level and LH was analyzed in PCOS econtrol f group Correlation between miR23a and GLU level was analyzed in PCOS g and control h group Correlation between miR23a andINS level was analyzed in PCOS i control j group Correlation between miR23a and T level was analyzed in PCOS k and control l groupP P P The correlation between the expression of miR23a andclinical index of PCOS patientsWe further analyzed the correlation between the expression of miR23a and clinical index As shown in Table the BMI of PCOS patients were significantly higher thanthat of healthy controls P The correlation analysis showed that there was a positive correlation between serum miR23a level and BMI in PCOS patientsFig 1b P r but no correlation wasfound in healthy control group Fig 1c P r As shown in Table the serum LH concentration in PCOS patients was ± mIUmL whichwas significantly higher than that in healthy women ± mIUmL P Furthermore therewas a negative correlation between serum miR23a leveland LH concentration in PCOS patients Fig 1d P r but no correlation was found in healthy controlgroup Fig 1e P r The serum miR23alevel was also negative correlated with GLU Fig 1fP r INS Fig 1h P r and T Fig 1j P r concentration inPCOS patients but not in healthy control group GLUFig 1g P r INS Fig 1i P r and T Fig 1k P r MiR23a inhibits the proliferation of cov434 cellsIn this study the expression of miR23a in three humangranulosa cell lines was detected by qPCR As shown inFig 2a the expression level of miR23a was lowest incov434 cells and highest in KGN cells Therefore wechose cov434 cell line for subsequent experiments Subsequently miR23aspecificsiRNA or mimic was transfected into cov434 cells to explore the role of miR23aAs shown in Fig 2b the expression of miR23a in cells 0cLin Journal of Ovarian Research Page of Fig MiR23a inhibits the proliferation of human ovarian granulosa cells a The expression of miR23a in three human ovarian granulosa celllines KGN cov434 and SVOG was detected by qPCR b MiR23a was overexpressed by the transfection of miR23a mimics c MiR23a was knockeddown by the transfection of miR23a inhibitor d CCK8 was performed to detect the proliferation of cov434 cells P P was significantly increased by the transfection of miR23a mimic P Similarly the expression of miR23a in cells was significantly knocked down by the transfection of miR23a inhibitor Fig 2c P Then CCK8 assay was performed to detect the effectof miR23a on the proliferation of cov434 cells Asshown in Fig 2d compared with the control group thetransfection of miR23a mimic significantly inhibited theproliferation of cov434 cells P on the contrarythe transfection of miR23a inhibitor significantly promoted the proliferation of cov434 cells P Thesedata proved that the expression level of miR23a was involved in the regulation of cov434 cell proliferationMiR23a induced cell cycle arrest on G0G1 phase ofcov434 cellsNext flow cytometry was used to detect the effect ofmiR23a on the cell cycle of cov434 As shown in Fig cells stagnated in G0G1 phase after transfection ofmiR23a mimic P and the proportion of cells inS phaseand G2M phase decreased significantlyP The results were consistent with the inhibition of cell proliferation by overexpression of miR23asuggesting that miR23a induced cell cycle arrest andthus inhibit cell proliferation in cov434 cells On thecontrary the proportion of G2M phase cells increasedsignificantly in the miR23a inhibitor group P while that of G0G1 and S phase cells decreased P The results showed that low expression of miR23a promoted cell cycle progression and thus cell proliferationMiR23a promotes apoptosis of cov434 cellsFlow cytometry was performed to detect the effect of theexpression of miR23a on the apoptosis of cov434 cellsAs shown in Fig apoptotic cells increased significantlyP after the transfection of miR23a mimic anddecreased significantly P after the transfection ofmiR23a inhibitor These results suggested that overexpression of miR23a promoted apoptosis while low expression of miR23a inhibited apoptosisFGD4 is the bind target of miR23a in cov434 cellsThen we predicted six novel potential target of miR23avia the analysis on bioinformatics software Target ScanSubsequently the results of double luciferase reporterassay proved that only FGD4 could bind to miR23a directly through predicted sites The binding sites areshown in Fig 5a Cotransfection of miR23a mimicinhibited the luciferase activity of FGD4WT plasmidP but had no effect on the luciferase activity ofFGD4Mut plasmid Fig 5b The results showed thatmiR23a and FGD4 bind directly through predictive sitesThe effect of miR23a on the expression of FGD4 incov434 cells was investigated using qPCR and westernblot As shown in Fig 6a the expression of FGD4 wassignificantly decreased by the transfection of miR23amimic P whereas the transfection of miR23a 0cLin Journal of Ovarian Research Page of Fig MiR23a induced cell cycle arrest on G0G1 phase of cov434 cells a Flow cytometry was used to detect the effect of miR23a on the cellcycle of cov434 with transfection of miR23a mimics or inhibitor b Column diagram showed the analysis of cell cycle P inhibitor significantly increased the mRNA expression ofFGD4 in cov434 cells P As shown in Fig 6b andc the protein level of FGD4 was significantly decreasedby the transfection of miR23a mimic P whereasthe protein level of FGD4 was significantly increasedby miR23a inhibitor P Combining with thedouble Luciferase Report experiment these results indicated that miR23a physically bind to the ²UTRregion of FGD4 thereby regulating the level of FGD4in cov434 cellsMiR23a induces the activation of CDC42PAK1 signalingpathway in cov434 cellsCDC42 is a member of the Rho GTPase protein familyFGD4 is responsible for activating CDC42 through GTPexchange of GDP PAK1 a serinethreonine kinase wasinitially identified as a protein interacting with CDC42[] CDC42PAK1 signaling pathway involved in theregulation of cell proliferation apoptosis and cell cycle[] As shown in Fig 6d the protein expression of activated CDC42 GTP bround was significantly increasedby the transfection of miR23a mimic P and significantly decreased by the transfection of miR23a inhibitoreffect of miR23a on theexpression of pPAK1 protein was similar to that ofCDC42 protein Fig 6fP TheDiscussionIn this study we explored the differences in serum levelsof miR23a between PCOS patients and normal womenas well as the effects of miR23a on biological behaviorsuch as proliferation and apoptosis of cov434 cells andrelated specific molecular mechanisms in order to provide limited theoretical support and experimental datafor the application of miRNA in PCOS treatmentFirstly we found that compared with healthy womenthe serum level of miR23a in PCOS patients decreasedsignificantly According to previous reports the level ofmiR23a in patients with ovarian disease remains uncertain Yang reported that miR23a was highlyexpressed in the plasma from premature ovarian failure POF patients compared with controls with afold change [] However Dang et alfoundthat miR23a is downregulated in the plasma ofChinese patients with premature ovarian failure []This inconsistency may be caused by individual differences and low sample size MiR23a level in patientswith ovarian disease still needs to be verified in alarge number of samplesMoreover miR23a was positively correlated with BMIand negatively correlated with serum LH T Glu andINS concentration Hyperandrogenism and hyperinsulinemia in PCOS patients are the most important physiological changes exacerbating endocrine disorders [] 0cLin Journal of Ovarian Research Page of Fig MiR23a promotes apoptosis of human ovarian granulosa cells a Flow cytometry was used to detect the effect of miR23a on theapoptosis of cov434 with transfection of miR23a mimics or inhibitor b Column diagram showed the analysis of cell apoptosis P MiR23a is closely related to the changes of hormonelevels suggesting that it may be involved in the progression of PCOS and is a potential clinical treatment targetMurri also reported an inverse relationship betweenBMI and LH concentrations in patients with PCOS []Serum is composed of multiple components from a variety of tissues and ans Therefore the concentration ofmiR23a in serum is regulated by a variety of componentsand factors In addition the results also indicated that thedecrease in miR23a had a negative impact on the occurrence of PCOS and the increase in LHThen we investigated the role of miR23a in cov434cells We have found that miR23a can affect the proliferation of cov434 cells by regulating cell cycle and participate in the regulation of cell apoptosis through aseries of cell functional studies It has been shown thatmiR23a is closely related to apoptosis by inhibiting theexpression of Apaf1 and Bcl2 apoptotic proteins including Noxa Puma and Bax in neurons [] It hasalso been reported that miR23a protects differentiatedembryonic stem cells from apoptosis induced by bonemorphogenetic protein BMP4 by targeting SMAD5[] These data provide strong support for our resultssuggesting that miR23a may be closely related to granulosa cell apoptosis through a variety of pathwaysThese results suggest that miR23a may be closely related to the pathogenesis and development of PCOSTherefore we further study the molecular mechanism ofmiR23a involved in the proliferation and apoptosis ofcov434 cells The biological functions of miRNAs depend mainly on their effects on targets The same microRNAs may have hundreds oftarget proteins thosechange with cell type and cell state MiR23a can promote the apoptosis of cov434 cells by affecting the expression of multiple targets [ ] At presentmany targets have been found including Xlinked inhibitor of apoptosis protein XIAP SMAD5 and Sirt1 [] In this study we found FGD4 as a new target ofmiR23a The direct interaction between the ²UTR region of FGD4 mRNA and the expression of miR23awas demonstrated by double luciferase reporter assayThe results of qPCR and Western blot showed thatoverexpression of miR23a inhibited the expression ofFGD4 at the level of protein and mRNA while low expression of miR23a promoted the expression of FGD4at the level of protein and mRNAFGD4 is a Guanine Nucleotide Exchange Factor GEFspecific to CDC42 Rho GTPase and also an Factinbinding protein which is essential for maintaining myelin formation in Schwann cells [] FGD4 consists of N 0cLin Journal of Ovarian Research Page of Fig FGD4 binds to miR23a via the UTR in cov434 cells a the binding site of miR23a to UTR of FGD4 b Double luciferase reporter assaywas performed to confirm the binding between miR23a and FGD4s UTR P terminal Factin bindingFAB domain Dbl homologyDH domain two pleckstrin homology PH domainand FYVE domain [] FGD4 has many functions including binding to Factin through FAB domain activating Rho GTPasetransduction pathway byincreasing the concentration of CD42 binding to GTPsignalThe structure domain of FGD4 indicates that it acts as acrosslinker between membrane structure and actincytoskeleton therefore the functional deletion mutationof FGD4 coding gene may result in truncated FGD4 expression and lead to motor sensory neuropathy orCharcotMarieToothCMTtype [ ] TheFig MiR23a induces the activation of CDC42PAK1 signaling pathway in cov434 cells a The expression of FGD4 was detected using qPCR incov434 cells with transfection of miR23a mimics or inhibitor b Western blot was performed to detect the levels of CDC42 and pPAK1 incov434 cells with transfection of miR23a mimics or inhibitor c Column diagram showed the expression level of FGD4 d Column diagramshowed the expression level of CDC42 e Column diagram showed the expression level of pPAK1 0cLin Journal of Ovarian Research Page of mutation is mediated by inhibiting guanine nucleotideexchange leading to the decrease of CDC42 activity andthe demyelination of peripheral nerves ultimately However in this study mir23a expression and function wereonly studied by using patients peripheral blood and celllines cultured in vitro The expression and function ofmir23a in vivo and patients ovarian cells still need further verificationIn addition recent studies have shown that FGD4 expression in prostate cancer clinical samples is significantly upregulated compared with the normal groupand downregulation expression of FGD4 in prostatecancer cell lines can cause cell cycle arrest and proliferation reduction [] It seems that FGD4 is also involvedin the tumorigenesis of nasopharyngeal carcinoma dueto its activation of CDC42 [] Studies have shown thatactivated CDC42 regulates downstream signals such asPAK1 WASP and ACK PAK1 as a serinethreoninekinase was originally identified as a protein that interacts with CDC42 and was subsequently found to serveas a downstream node for various oncogenic signalingpathways Studies have shown that the CDC42PAK1signaling pathway involved in cell cycle proliferationand apoptosis regulation [] Our study found thatmiR23a affects the expression of FGD4 as well as theprotein levels of activated CDC42 GTP bround and pPAK1 Therefore we hypothesized that miR23 regulated CDC42PAK1 signaling pathway by targetingFGD4 expression ultimately affecting apoptosis ofcov434 cellsIn our study reveals that the serum level ofmiR23a is significantly downregulated in PCOS patients and that miR23a participates in the regulation ofproliferation and apoptosis of cov434 cells through target FGD4 which may have potential for clinical treatment of PCOS patientsAcknowledgementsNot applicableAuthors contributionsJL and HH mainly performed the experiments and analyzed the data JL was amajor contributor in writing the manuscript LL helped with the data analysisand carried out the experiment design WL and JH helped with theexperiments and analysis All authors read and approved the final manuscriptFundingThis study was supported by Ningde medical technology improvementprojectAvailability of data and materialsAll data generated or analysed during this study are included in thispublished Ethics approval and consent to participateThis research study was approved by the Institutional Review Board of FujianMedical UniversityConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Graduate School Fujian Medical University Fuzhou China 2The 900thhospital of the Joint Service Support Force of the Chinese PeoplesLiberation Army Fuzhou China 3Gynaecology Mindong Hospital in NingdeCity No Heshan Road Fuan Fujian ChinaReceived December Accepted July ReferencesUtiger RD Insulin and the polycystic ovary syndrome Diabetes Res ClinPract Polak K Czyzyk A Simoncini T Meczekalski B New markers of insulinresistance in polycystic ovary syndrome J Endocrinol Invest httpsdoi101007s4061801605238Trikudanathan S Polycystic ovarian syndrome Med Clin N Am American College of Obstetricians and Gynecologists' Committee onPractice BulletinsGynecology ACOG Practice Bulletin No PolycysticOvary Syndrome Obstet Gynecol 20181316e157 httpsdoi101097AOG0000000000002656Li X The role of androgen in autophagy of granulosa cells from PCOSGynecol Endocrinol OvidiuLeonard B miRNA expression profiling in formalinfixedparaffinembedded endometriosis and ovarian cancer samples OncoTargets Ther Maalouf SW Liu WS Pate JL MicroRNA in ovarian function Cell Tissue ResKim SH Paeonol inhibits anaphylactic reaction by regulating histamineand TNFα Int Immunopharmacol Naji M Differential Expression of miR93 and miR21 in Granulosa Cellsand Follicular Fluid of Polycystic Ovary Syndrome Associating with DifferentPhenotypes Sci Rep Bindu M miR122 Regulates LHR Expression in Rat Granulosa Cells byTargeting Insig1 mRNA Endocrinology Chhabra R Dubey R Saini N Gene expression profiling indicate role of ERstress in miR23a27a24 cluster induced apoptosis in HEK293T cellsRNA Biol Xiong W Circulatory microRNA 23a and microRNA 23b and polycysticovary syndrome PCOS the effects of body mass index and sex hormonesin an Eastern Han Chinese population J Ovarian Res Guo Y Sun J Lai D Role of microRNAs in premature ovarian insufficiencyReprod Biol Endocrinol Yang X Role of microRNAs in premature ovarian insufficiency ReprodBiol Endocrinol Dang Y MicroRNA223p is downregulated in the plasma of HanChinese patients with premature ovarian failure Fertil Steril 807e1 Nie M Yu S Peng S Fang Y Wang H Yang X miR23a and miR27apromote human granulosa cell apoptosis by targeting SMAD5 Biol Reprod httpsdoi101095biolreprod115130690 Alford C Toloubeydokhti T AlKatanani Y The expression of microRNAmiRNA mir23a and 23b and their target gene CYP19A1 aromatase infollicular cells obtained from women undergoing ART[J] Fertil Steril 88suppS1 Nie M miR23a and miR27a Promote Human Granulosa CellApoptosis by Targeting SMAD5 Biol Reprod S¸rensen AE MicroRNA Species in Follicular Fluid Associating WithPolycystic Ovary Syndrome and Related Intermediary Phenotypes J ClinEndocrinol Metab jc20153588 Wu C Exercise activates the PI3KAKT signal pathway by decreasingthe expression of 5αreductase type in PCOS rats Sci Rep Murri M Effects of polycystic ovary syndrome PCOS sex hormonesand obesity on circulating miRNA21 miRNA27b miRNA103 and miRNA expression J Clin Endocrinol Metab 20139811E1835Sabirzhanov B Downregulation of miR23a and miR27a followingExperimental Traumatic Brain Injury Induces Neuronal Cell Death throughActivation of Proapoptotic Bcl2 Proteins J Neurosci 0cLin Journal of Ovarian Research Page of Musto A miR23a miR24 and miR27a protect differentiating ESCsfrom BMP4induced apoptosis Cell Death Differ Nie MY Yang X Physiological and pathological effects of miR23a and miR27a in ovary Horn M Myelin is dependent on the CharcotMarieTooth Type 4Hdisease culprit protein FRABINFGD4 in Schwann cells Brain Kondo D A novel mutation in FGD4 causes CharcotMarieToothdisease type 4H with cranial nerve involvement Neuromuscul Disord Zis P A novel mutation in the FGD4 gene causing CharcotMarietooth disease J Peripher Nerv Syst Edwards D PRL3 increases the aggressive phenotype of prostatecancer cells inVitro and its expression correlates with highgrade prostatetumors in patients Int J Oncol Liu HP EpsteinBarr VirusEncoded LMP1 Interacts with FGD4 toActivate Cdc42 and Thereby Promote Migration of NasopharyngealCarcinoma Cells PLoS Pathog 201285e1002690Kumar R Gururaj AE Barnes CJ p21activated kinases in cancer Nat RevCancer httpsdoi101038nrc1892Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliat | Thyroid_Cancer |
emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic beneï¬t until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic proï¬les ofHCC contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufï¬ciently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPaciï¬c trial promoted the approvalof sorafenib as ï¬rstline targeted therapy for advanced HCC5ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel ï¬rstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predeï¬ned primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asï¬rstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPaciï¬c trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21 Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as ï¬rstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24 andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical beneï¬t of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer beneï¬ts when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbeneï¬t in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these ï¬ndings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42 which was supported by theï¬ndings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redeï¬ned PFS not conventional PFS buttime until unTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival beneï¬t compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival beneï¬t compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modiï¬cation ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classiï¬cation BCLCguidelines and although the results are difï¬cult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also beneï¬ted fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratiï¬cation would enable the identiï¬cation of thosepatients who will beneï¬t most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second ï¬rstlinetargeted agent for HCC emerged Lenvatinib was approved for theï¬rstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from realworld conditions conï¬rmed the efï¬cacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufï¬cient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88 With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneï¬cial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop ï¬rstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy ï¬nallyconï¬rmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival beneï¬tthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was conï¬rmed by Yoo et al99 in aretrospective study of safety and efï¬cacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefï¬cacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identiï¬ed regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconï¬rms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs and the incrementalcosteffectiveness ratio ICER mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112 compared with regorafenib QALY and ICER RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival beneï¬t in patientswith AFP of ngml or higher118 This was later conï¬rmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the ï¬rstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent ï¬ndingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and conï¬rms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefï¬cacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efï¬cacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efï¬cacy and safety in advanced HCCs as systemicï¬rstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespeciï¬ed statistical signiï¬cance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as ï¬rstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbeneï¬t regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on ï¬rstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efï¬cacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the ï¬rst combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136 Such results were conï¬rmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ammation and rheumatic and musculoskeletal phenotypesincluding ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a ï¬rstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145 where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153 Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as ï¬rstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as ï¬rst155 or secondline inadvanced HCCs156 but unfortunately the heterogeneousnature of the results precludes ï¬rm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufï¬ciently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentiï¬erPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentiï¬erPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164 especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyproï¬le169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneï¬cially employed to produce a therapeutic dualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efï¬cacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR ï¬broblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classiï¬cation of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassiï¬cationG1G6S1S3Msig iC1iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and lowMS1 SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell proï¬lingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespeciï¬ed criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identiï¬ed to be able to predict the effect of or responseto treatment with sorafenib172 Although the molecularclassiï¬cation of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul | Thyroid_Cancer |
"Pressure sores are sometimes refractory to treatment often due to malnutrition Small intestinalbacterial overgrowth SIBO obstructs absorption in the digestive tract and causes malnutrition However little isknown about the association between pressure sore wound healing and SIBO Here we report a case of a patientwith a refractory sacral pressure sore and SIBOCase presentation A 66yearold woman who was spinal cord injured years before visiting our hospitalpresented with the chief complaint of a sacral pressure sore cm in size which was refractory totreatment Physical examination showed abdominal distension and emaciation with a body mass index of Further examination revealed elevated serum alkaline phosphatase UL bilateral tibial fracture multiple ribfracture and osteoporosis We diagnosed the patient with osteomalacia with vitamin D deficiency Despite oralsupplementation serum levels of calcium phosphorous and vitamin D remained low Also despite concentrativewound therapy for the sacral pressure sore by plastic surgeons no wound healing was achieved Due to asuspicion of disturbances in nutrient absorption we performed bacterial examination of collected gastric andduodenal fluid which showed high numbers of bacteria in gastric content E coli Streptococcus speciesand Neisseria species and duodenal content E coli Candida glabrata Therefore we diagnosed thepatient with SIBO and started selective decontamination of the digestive tract using polymyxin B sulfate andamphotericin B After starting treatment for SIBO the sacral pressure sore began to heal and was nearly healed after days The patients serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins alsogradually increased after starting treatment for SIBOContinued on next page Correspondence 2m2hy4gmailcom1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba 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 0cKubota BMC Gastroenterology Page of Continued from previous pageConclusion We report a case of a patient with a refractory sacral pressure sore that healed after starting treatmentfor SIBO We conclude that SIBO may be an overlooked cause of malnutrition and poor wound healing in patientswith chronic pressure soresKeywords Pressure wound Small intestinal bacterial overgrowth Spinal cord injury Malnutrition Wound healingCase reportBackgroundPressure sores in patients with spinal cord injury SCIare sometimes refractory to treatment Chronic gastrointestinal symptoms are also frequently seen in patientswith SCI [ ] and malnutrition caused by decreasedgastrointestinal motility in SCI patients is a major causeand exacerbating factor of pressure sores Evaluation ofnutritional status in patients with pressure sores is essential [] as nutritional intervention can be a valuabletreatment option for pressure sores However small intestinal bacterial overgrowth SIBO is rarely consideredin the evaluation of malnutrition in SCI patients withpressure sores SIBO is defined as the presence of morethan colony forming units CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Here we report the case of an SCI patientwith a refractory sacral pressure sore that healed afterstarting treatment for SIBO To the best of our knowledge this is the first report of an association between apressure sore and SIBOCase presentationA 66yearold woman visited our hospital for the purpose of treating her sacral pressure sore day whichshe developed months prior due to bed rest duringtreatment of a left humeral fracture in another hospitalShe had paraplegia as well as bladder and rectal disturbance due to SCI at the fourth lumbar level L4 causedby a suicidal jump in response to paranoid delusions at years of age Spinal fusion surgery and cystostomywere performed early after SCI Otherwise she had ahistory of hysterectomy due to uterine cancer at yearsof age lymphaticovenular anastomosis as a treatment forposthysterectomy lymphedema in the bilateral lower extremities at years of age and cholecystectomy at years of ageWhen she visited our hospital she was taking the following oral medicines propiverine hydrochloride vitamin B12 etizolamflunitrazepam sodium bicarbonateanhydrous monobasic sodium phosphate mixture Clostridium butyricum tablets sodium risedronate hydraterebamipide sodium ferrous citrate fursultiamine hydrochloride alfacalcidol and potassium Lasparate She didnot take proton pump inhibitors PPI Her vital signswere as follows body temperature of °C low bloodpressure of mmHg pulse rate of bpm and respiratory rate of per min Physical examinationshowed abdominal distension emaciation with a bodymass index of and a sacral pressure sore cm in size including a pocket entrance of cmFig 1a Most of the surface of the pressure sore wascovered by granulation Our evaluation of the pressuresore with DESIGNR [] was D3 e3 s8 i0 g3 N3 P24with a total score of Table Bacterial culture examination ofthe pressure soreshowed Corynebacterium striatum and methicillinresistant Staphylococcus aureus Laboratory data showedan elevated serum alkaline phosphatase level of UL and low serum levels of hemoglobin gdL albumin gdL calcium mgdL and zinc μgdL Onday we observed a sudden decrease of hemoglobin to gdL with a positive fecal occult blood test bilateralpleural effusion on chest xray and serum albumin levelof gdL Upper gastrointestinal endoscopy showed agastric ulcer at H2 stageAs a result of searching for the cause of alkaline phosphatase elevation bilateral tibial fracture multiple ribfracture and osteoporosis were found Fig 1e and fFemoral bone density was of the young adult meanA low serum inanic phosphorous level was foundTable along with a low serum level of 25hydroxyvitamin D3 25OHVitD3 below the detection limit andelevated level of parathyroid hormone Table Levelsof other fatsoluble vitamins were also low vitamin A μIUdL vitamin K1 ngdL and vitamin E mgdL Examination using ultrasound and computedtomography showed normalthyroid and parathyroidglands Basing on these finding we diagnosed osteomalacia with vitamin D deficiencyOn day oral supplementation of calcium phosphorous and vitamin D was started Despite supplementationserum levels of calcium phosphorous and 25OHVitD3on day showed poor improvement calcium mgdLphosphorous mgdL and 25OHVitD3 below the detection limitOn day we performed bacterial examination ofcollected gastric and duodenal fluid with suspicion of adisturbance in absorption which showed elevated num E coli bers of bacteria in gastric contentStreptococcus species and Neisseria species and 0cKubota BMC Gastroenterology Page of Fig See legend on next page 0cKubota BMC Gastroenterology Page of See figure on previous pageFig Patient images a b c and d Sacral pressure sore a Day sore cm in size with an entrance of cm DESIGNR score wasD3 e3 s8 i0 g3 N3 P24 with a total score of b Day ie days after starting SDD for treating SIBO reduced size of sore DESIGNR scorewas D3 e3 s3 i0 g1 n0 p0 with a total score of c Day ie days after staring SDD healed sore DESIGNR score was d0 e0 s0 i0 g0 n0p0 with a total score of d Day ie days after staring SDD no recurrence of the sore e and f Osteoporosis and multiple fractures eXray showing left tibial fracture f Tc99 m bone scan showing accumulation in multiple ribs vertebrae and right ulna g h and i Endoscopicexamination and results of bacterial culture of the upper digestive tract All stomach duodenum and proximal jejunum samples were positive forE coli g Stomach Food residue can be seen Acid level was decreased to pH h Duodenum Food residue is evident i Proximal jejunum Flatvilli and a jejunal ulcer are observedTable DESIGNR assessment tool for pressure sore Reprinted with permission from John Wiley and Sons In Matsui et alDevelopment of the DESIGNR with an observational study an absolute evaluation tool for monitoring pressure ulcer woundhealing Wound Repair Regen Depthd No particular skin lesion and no rednessD Lesion extends into the subcutaneous tissuePersistent rednessLesion extends into dermisExudatee NoneSlight does not require daily dressing change Moderate requires daily dressing changeSizes NoneSmaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2Lesion extends to the muscle tendon and boneLesion extends into the articular or body cavityU It is impossible to measure the depthE Heavy requires dressing change more than twice a dayS cm2 or largerInflammationInfectioniNoneSigns of inflammation fever redness swelling and pain around thewoundIClear signs of local infection eg inflammation pus and foulsmellSystemic impact such as feverGranulation tissueg Granulation cannot be assessed because the wound is healed or tooshallowG Healthy granulation tissue occupies or more but lessthan Healthy granulation tissue occupies or moreHealthy granulation tissue occupies or more but less than Healthy granulation tissue occupies less than No healthy granulation tissue existsNecrotic tissuen NonePocketp NoneN Soft necrotic tissue existsHard and thick necrotic tissue is attached to the woundP Smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger but smaller than cm2 cm2 or larger 0cKubota BMC Gastroenterology Page of Table Laboratory data before starting supplementation withvitamin DWBCμL104μLgdL104μLgdLgdLULULULULULULULULmgdLmgdLmLmin173 m2mEqLmEqLmgdLmgdLmgdLμgdLμgdLμgdLμgdLμIUmLmgdLmgdLmgdLmgdLsecondpgmLμgdLμIUmLpgmLngdLof woundirrigationtreatmentdebridementstarting SDD the pressure sore was refractory to multiple methodsincludingdepressurizationointmentbasic fibroblast growth factor and negative pressurewound therapy After starting SDD the pressure sorebegan to heal On day ie days after startingSDD the pressure sore DESIGNR score was D3 e3 s3i0 g1 n0 p0 with a total score of Fig 1b Regardingthe nutritional status the serum albumin level increasedfrom gdL just before starting SDD to gdL at days after starting SDD Also the hemoglobin levelincreased to gdL and the serum zinc level increasedto μgdL Serum levels of calcium phosphorous vitamin D and other fatsoluble vitamins also gradually increased Fig and Table A repeat diagnosticbacterial examination of the upper digestive tract contents for SIBO was not performed because of obviousimprovements in most of the laboratory data Therewere no adverse effects of SDD such as antibioticassociated diarrhea In contrast the patient presentedwith constipation that was noted before starting SDDThe sacral pressure sore was completely healed on day ie days after starting SDD with a DESIGNRscore of d0 e0 s0 i0 g0 n0 p0 and total score of Fig1c In addition the patient showed improved nutritionalstatus and had a serum albumin level of gdL Wesuccessfully reduced the dose of polymyxin B from to million units daily similarly the dose of amphotericin B was reduced from to mg daily on day ie days after starting SDD without any signs ofSIBO recurrence There was no recurrence of the sacralpressure sore with a serum albumin level of gdL onday ie days after staring SDD Fig 1d Onday we successfully ended the use of amphotericinB however the use of polymyxin B at million unitsper day continued On day ie days afterstarting SDD while still using polymyxin B at million per day the serum albumin level was gdL thehemoglobin level was gdL and the serum zinc levelwas μgdL There were no signs of SIBO recurrenceor the sacral pressure soreDiscussion and conclusionsWe report the case of a patient whose sacral pressuresore and osteoporosis were improved by treatment forSIBO Although nutrition status is known to be important for the healing of pressure sores SIBO is rarelychecked as a cause of malnutrition in patients with pressure sores However SIBO is a potential cause of malnutrition in patients with SCI due to decreased intestinalmotility resulting from autonomic disturbances and reduced physical activity [] SCI is also a risk factor forpressure sores [] However to the best of our knowledge there are no previous reports of an associationRBCHbPltTotal ProteinAlbuminASTALTγGTPLDHALPChECKAmyBUNCreatinineeGFRNaKCaiPMgFeZnUIBCFerritinErythropoietinTotal CholesterolTriglycerideHDLCholesterolLDLCholesterolPTINRAPTTACTHCortisolTSHFT3FT4duodenal content E coli Candida glabrataFig 1g h and i Therefore we diagnosed SIBO Onday we started selective decontamination of the digestive tract SDD using oral administration of polymyxin B sulfate million units daily and oraladministration of amphotericin B mg daily Before 0cKubota BMC Gastroenterology Page of Table Vitamins and bone metabolism markers before starting supplementation of vitamin DVitamin AVitamin K1Vitamin K2Vitamin E125OH2 Vitamin D25OH Vitamin D3Retinol binding proteinVitamin B1Vitamin B12Nicotinic acidFolic acidTRACP5bNTxBone type ALPIntact P1NPOsteocalcinintact PTHPTHrPFGF23TRPTmPGFR Below the detection limitIUdLngmLngmLmgdLpgmLpgmLmgdLngmLpgmLμgmLngmLμUmLnmolBCELμgLμgLngmLpgmLpmolLpgmLmgdLnormal range between pressure sores and SIBO Thus our case drawsattention to the fact that SIBO can be an overlookedcause of poor wound healing during the treatment ofpressure soresSIBO was first reported by Vantrappen as an increased concentration of 14CO2 in a bile acid breath testfor patients with an absent interdigestive motor complex[] Today consensus diagnostic criteria for SIBO arethe presence of more than CFUmL of bacteriaor any amount of E coli in the proximal small bowelcontent [] Relatively little is known about commensalsinhabiting the small intestine mainly due to the limitedaccessibility of this environment for microbiological analysis [] In the healthy state the numbers of intestinalbacteria range from to CFUmL and mainly include gramnegative and grampositive aerobes such asStreptococcus Lactobacillus and Bacteroidesspecies[] Regarding the amount of bacteria in proximal jejunal aspiration Khoshini report that normal subexceed CFUmL and thereforejectsproposed more than CFUmL coliform bacteriaas the threshold for SIBO [] In our case CFUmLE coli existed in duodenal content and CFUmL Ecoli existed in gastric content which met the traditionaldiagnostic criteria of SIBOrarelyOther diagnostic methods for SIBO are breath testsusing hydrogen or hydrogen methane with lactulose lucose [] Breath tests have clinical utility for diagnosing SIBO because they are less invasive than obtaining proximal small bowel content However there areno standardized criteria for diagnosing SIBO usingbreath tests [] We did not perform a breath test inour case studyDespite no previous reports of an association betweenunhealed pressure sores and SIBO nutritional status isknown to be important for the healing of pressure sores []In our case the sacral pressure sore which was initially refractory began to heal after starting treatment for SIBOAmong intrinsic factors related to the healing of pressuresores blood levels of hemoglobin albumin and zinc are especially important [] Our patient had anemia hypoalbuminemia and a low zinc concentration which graduallyimproved after starting treatment for SIBOSIBO is caused by multiple factors including disturbances in defense mechanisms of the digestive tractanatomical abnormalities surgical interventions and disturbed gastrointestinal motility [ ] Bures described several endogenous defense mechanisms thatprevent bacterial overgrowth [] including secretion ofgastric acidintestinal motility a properly functioning 0cKubota BMC Gastroenterology Page of Fig Bone metabolism markers After starting SDD levels of bone metabolism markers gradually improvedTable Vitamins and trace elements before and after supplementation and selective digestive decontamination SDDVitamin K1 ngmLVitamin K2 ngmLVitamin E mgmL125OH2 Vitamin D pgmL25OH Vitamin D3 pgmLNicotinic acid μgmLMg mgdLFe μgdLBefore supplementationAt the time SDD started days after starting SDD days after starting SDDNANANA 0cKubota BMC Gastroenterology Page of ileocecal valve production of secretory immunoglobulinson the surface of the gastrointestinal mucous membraneand the bacteriostatic properties of pancreatic juice andbile In our case the patients history of cholecystectomyand hysterectomy were possible causes or exacerbatingfactors of SIBO Disturbed gastrointestinal motilitycaused by paraplegia below the L4 level due to SCI is another possible cause of SIBO in our case as well as decreased physical activity due to fracture ofthe lefthumerus and bilateral tibiaChronic gastrointestinalinvolvement is seen in of patients with SCI [ ] SCI patients lackcentral nervous system control over the gastrointestinalsystem [] Liu report that bowel problems in SCIpatients are related to high levels of cord injury completeness of cord injury and postinjury durations of years or more [] Moderate or severe grade depressivestatus is also associated with neurologic bowel dysfunction in SCI patients Of these risk factors our patienthad complete cord injury that had occurred more than years ago Also many bowel symptoms appear in patients with SCI eg constipation distension incontinence abdominal pain bowel accidents nausea diarrheastrainingautonomichyperreflexia headaches or sweat relieved by a bowelmovement [ ] However our patient showedno appetite loss a sufficient amount of food intake andnonsevere bowel symptoms Thus the presence of malnutrition despite adequate food intake and low levels oflipidsoluble vitamins that were unresponsive to supplementation led us to suspect SIBOrectal bleeding hemorrhoidsAlthough gastrointestinal symptoms are frequently observed in patients with SCI there are few reports ofSIBO in SCI patients Cheng report that of of SCI patients were diagnosed with SIBO basedon the glucose hydrogenmethane breath test [] However the prevalence of SIBO among SCI patients as confirmed by the consensus diagnostic criteria ofthepresence of more than CFUmL bacteria or anyamount of E coli in upper digestive tract content is unknown In patients with SCI absent central nervous system innervation of the digestive tract can change theinhabiting environment of bacteria Gungor reportdifferences in gut microbial patterns between SCI patients and control individuals as measured by bacterialgenome sequencing [] Specifically they found thatbutyrateproducing bacteria were specifically reduced inSCI patients Thus it is possible that SIBO is overlookedin patients with SCI In our caseit is unclear whenSIBO occurred relative to the time of SCI but we suspect that it arose due to gastrointestinal motility disorder caused by autonomic disturbancesDisturbances in fat absorption and deficiency in fatsoluble vitamins ie vitamins A K E and D3 areobserved in patients with SIBO [] Excess bacteria inthe small intestine promotes a change from conjugatedbile acid into deconjugated bile acid which decreasesthe micellar solubilization of dietary fat Bacterial fermented short chain fatty acid causes osmotic watermovement to the intestinal lumen which results in diarrhea and malabsorption [] Intestinal epithelial damagein SIBO also interferes with fat absorption Mucosaldamage is caused by metabolites of aerobic bacteria endotoxins of anaerobic bacteria and lithocholic acidwhich is a bacterial degradation product of unconjugatedbile acid [] Our patient however showed constipation rather than diarrhea in spite of SIBO Whether ornot diarrhea occurs in patients with SIBO is determinedby multiple factors Constipation frequently occurs inpatients with SCI due to decreased physical activity andautonomic dysfunction De Looze reported that therate of constipation in the patients with SCI is []A certain proportion of the patients with SCI show constipation in spite of the coexisting SIBO Cheng reported that in patients with both SCI and SIBO showed constipation [] We believe that the factorsleading to constipation in our patient were stronger thanthose leading to diarrhea Vitamin D deficiency in SIBOcauses osteomalacia Our patientshowed multiplefractures and osteoporosis with serum vitamin D3 levelsbelow thetosupplementationrefractorydetectionlimitandThere is no consensus on the choice dose or durationof antibiotics for treating SIBO [] In principle antibiotics should be chosen based on the results of an antimicrobial susceptibility test but this approach cannotaddress the great diversity in microbiota of the digestivetract [ ] Metronidazole is a firstline choice forSIBO [] with other choices being rifaximin ciprofloxacin norfloxacin amoxicillinclavulanate trimethoprimsulfamethoxazole cephalexin or their combination []However these antibodies are selected based on customrather than scientific evidence [] In our case we usedoral polymyxin B and amphotericin B in accordancewith SDD which was first reported as a method of preventing ventilationassociated pneumonia and microbialtranslocation of gramnegative rod bacteria and fungi incritically ill patients treated in the intensive care unit[] Polymyxin B administered to the digestive tractis nonabsorbent into the human body and has strongbactericidal power against gramnegative rod bacteriaexcept for naturally polymyxinresistant bacteria such asProteus Providencia Manella Burkholderia and Serratia [] Amphoteric B is an antifungal drug that isalso nonabsorbent into the human body when administered to the digestive tract In our case after startingSDD fatsoluble vitamins were increased and osteoporosis was improved No obvious adverse effects of SDD 0cKubota BMC Gastroenterology Page of such as antibioticassociated diarrhea were observed inour caseWhen and how to stop antibiotherapy for the treatment of patients with SIBO are difficult problemsFew reports are available for the method and timingfor making a decision to stop antibiotherapy in SIBOLauritano reported that the recurrence rate at months after stopping antibiotherapy in SIBO patientsis [] They also showed that an older age history of appendectomy and chronic use of PPIs areassociated with SIBO recurrence Bures reportedthat cyclical gastrointestinal selective antibiotics areneeded for SIBO treatment [] These reports indicatethat in many patients with SIBO it is actually impossible to stop antibiotherapy because of the underlyingconditions that lead to SIBO Similarly in our case itwas difficult to ameliorate the underlying condition ofdecreased motility of the digestive tract due to SCIWe were compelled to continue SDD for a long duration We did however succeed in gradually reducingthe dose of polymyxin B and end the use of amphotericin B without signs of SIBO recurrence Withcareful consideration it may be possible and feasibleto stop SDD completelyProbiotics are also a treatment approach for SIBO assome species of bacteria are thought to protect againsthigh numbers of E coli and fungi in the digestive tract[] However the role and effects of probiotics are stillunclear The digestive tract microbiome has both pathogenic potential and a protective role in maintaininghealth However metagenomic analysis reveals that of microanisms in the digestive tract cannot becultured under laboratory conditions [] The effects ofSDD and probiotics on the digestive tract microbiome inpatients with SIBO should be investigated to furtherunderstand the pathogenesis of the diseaseIn conclusion we treated a patient with a sacral pressure sore who also had SCI multiple fractures withosteoporosis and malabsorption especially of fatsolublevitamins Based on culture of upper digestive tract content we diagnosed the patient with SIBO and startedSDD using polymyxin B and amphotericin B which effectively ameliorated the absorbency disturbance andallowed healing of the pressure sore In light of severalcommon risk factors between pressure sores and SIBOsuch as decreased physical activity our case providesadditional information on the associations among pressure sores malnutrition and SIBOAbbreviationsCFU Colony forming units SCI Spinal cord injury SDD Selectivedecontamination of the digestive tract SIBO Small intestinal bacterialovergrowth 25OHVitD3 25hydroxy vitamin D3AcknowledgementsWe thank the many personnel involved in this interdisciplinary diagnosticworkup as their effective technical assistance enabled a comprehensiveapproach to this difficult diagnosisAuthors contributionsYK and TT treated the patient conceived of and wrote the manuscript KISK and TK treated the patient and collected the data SA and NMinterpreted the data HN analyzed the data and created the figures andtables All authors read and approved the final manuscriptFundingNo funding was receivedAvailability of data and materialsData on this case not reported in the manuscript are available from thecorresponding author upon reasonable requestEthics approval and consent to participateEthical approval was not necessary for the reported investigations as theywere performed in a routine clinical setting with therapeutic intentionConsent for publicationThe patient provided written consent for reporting her case in aninternational published medical journal including clinical details and imagesCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Plastic Surgery Chiba University Inohana ChuokuChibacity Chiba Japan 2Department of Molecular DiagnosisChiba University Inohana Chuoku Chibacity Chiba Japan3Department of Plastic Surgery Chiba Emergency Medical Center Isobe Mihamaku Chiba JapanReceived July Accepted August ReferencesStone JM NinoMurcia M Wolfe VA Perkash I Chronic gastrointestinalproblems in spinal cord injury patients a prospective analysis Am JGastroenterol Liu CW Huang CC Chen CH Yang YH Chen TW Huang MH Prediction ofsevere neurogenic bowel dysfunction in persons with spinal cord injurySpinal Cord Eglseer D Hodl M Lohrmann C Nutritional management of olderhospitalised patients with pressure injuries Int Wound J Bures J Cyrany J Kohoutova D Forstl M Rejchrt S Kvetina J Vorisek VKopacova M Small intestinal bacterial overgrowth syndrome World JGastroenterol Matsui Y Furue M Sanada H Tachibana T Nakayama T Sugama J Furuta KTachi M Tokunaga K Miyachi Y Development of the DESIGNR with anobservational study an absolute evaluation tool for monitoring pressureulcer wound healing Wound Repair Regen Sachdev AH Pimentel M Gastrointestinal bacterial overgrowth pathogenesisand clinical significance Ther Adv Chronic Dis Groah SL Schladen M Pineda CG Hsieh CH Prevention of pressure ulcersamong people with spinal cord injury a systematic review PM R Vantrappen G Janssens J Hellemans J Ghoos Y The interdigestive motorcomplex of normal subjects and patients with bacterial overgrowth of thesmall intestine J Clin Invest Zoetendal EG Raes J van den Bogert B Arumugam M Booijink CC TroostFJ Bork P Wels M de Vos WM Kleerebezem M The human small intestinalmicrobiota is driven by rapid uptake and conversion of simplecarbohydrates ISME J Miazga A Osinski M Cichy W Zaba R Current views on theetiopathogenesis clinical manifestation diagnostics treatment andcorrelation with other nosological entities of SIBO Adv Med Sci 0cKubota BMC Gastroenterology Page of Khoshini R Dai SC Lezcano S Pimentel M A systematic review ofdiagnostic tests for small intestinal bacterial overgrowth Dig Dis Sci Heintschel M Heuberger R The potential role of zinc supplementation onpressure injury healing in older adults a review of the literature WoundsPublishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations Han TR Kim JH Kwon BS Chronic gastrointestinal problems and boweldysfunction in patients with spinal cord injury Spinal Cord Ebert E Gastrointestinal involvement in spinal cord injury a clinicalperspective J Gastrointestin Liver Dis Gungor B Adiguzel E Gursel I Yilmaz B Gursel M Intestinal microbiota inpatients with spinal cord injury PLoS One 2016111e0145878 Harari D Sarkarati M Gurwitz JH McGlincheyBerroth G Minaker KLConstipationrelated symptoms and bowel program concerning individualswith spinal cord injury Spinal Cord Menter R Weitzenkamp D Cooper D Bingley J Charlifue S Whiteneck GBowel management outcomes in individuals with longterm spinal cordinjuries Spinal Cord De Looze D Van Laere M De Muynck M Beke R Elewaut A Constipationand other chronic gastrointestinal problems in spinal cord injury patientsSpinal Cord Lynch AC Wong C Anthony A Dobbs BR Frizelle FA Bowel dysfunctionfollowing spinal cord injury a description of bowel function in a spinalcordinjured population and comparison with age and gender matchedcontrols Spinal Cord Krogh K Nielsen J Djurhuus JC Mosdal C Sabroe S Laurberg S Colorectalfunction in patients with spinal cord lesions Dis Colon Rectum Chen CY Chuang TY Tsai YA Tai HC Lu CL Kang LJ Lu RH Chang FY LeeSD Loss of sympathetic coordination appears to delay gastrointestinaltransit in patients with spinal cord injury Dig Dis Sci Cheng X Zhang L Xie NC Xu HL Lian YJ Association between smallintestinal bacterial overgrowth and deep vein thrombosis in patients withspinal cord injuries J Thromb Haemost Kirsch M Bozdech J Gardner DA Hepatic portal venous gas an unusualpresentation of Crohn's disease Am J Gastroenterol Jones RM Neish AS Recognition of bacterial pathogens and mucosalimmunity Cell Microbiol Hoog CM Lindberg G Sjoqvist U Findings in patients with chronicintestinal dysmotility investigated by capsule endoscopy BMCGastroenterol Singh VV Toskes PP Small bowel bacterial overgrowth presentationdiagnosis and treatment Curr Treat Options Gastroenterol Quigley EM AbuShanab A Small intestinal bacterial overgrowth Infect DisClin N Am viiiix Melchior C Gourcerol G Bridoux V Ducrotte P Quinton JF Leroi AMEfficacy of antibiotherapy for treating flatus incontinence associated withsmall intestinal bacterial overgrowth a pilot randomized trial PLoS One2017128e0180835 VandenbrouckeGrauls CM Vandenbroucke JP Effect of selectivedecontamination of the digestive tract on respiratory tract infections andmortality in the intensive care unit Lancet Silvestri L van Saene HK Casarin A Berlot G Gullo A Impact of selectivedecontamination of the digestive tract on carriage and infection due togramnegative and grampositive bacteria a systematic review ofrandomised controlled trials Anaesth Intensive Care Camus C Salomon S Bouchigny C Gacouin A Lavoue S Donnio PYJavaudin L Chapplain JM Uhel F Le Tulzo Y Shortterm decline in allcause acquired infections with the routine use of a decontaminationregimen combining topical polymyxin tobramycin and amphotericin Bwith mupirocin and chlorhexidine in the ICU a singlecenter experienceCrit Care Med Olaitan AO Morand S Rolain JM Mechanisms of polymyxin resistanceacquired and intrinsic resistance in bacteria Front Microbiol Lauritano EC Gabrielli M Scarpellini E Lupascu A Novi | Thyroid_Cancer |
"clinical development of immune checkpoint inhibitors ICIs therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit Therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of ICIs and more indepth and comprehensive understanding has beencontinuously explored in recent years Predictive markers of ICIs efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers With thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers Comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs and discuss their future directions in achievingprecision immunooncologyKeywords Neoplasm Immune checkpoint inhibitor Predictive biomarker Tumor mutation burden Programmeddeath ligand1BackgroundImmune checkpoint inhibitors ICIs therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors Antiprogrammed cell death1programmed cell deathligand PD1PDL1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma RCC head and neck squamous cell carcinoma HNSCC and gastroesophageal Correspondence cuijwjlueducnCancer Center the First Hospital of Jilin University Xinmin StreetChangchun Jilin Chinacancer [ ] However despite the breakthrough in clinical treatment with ICIs most patients do not benefitPembrolizumab or nivolumab has an objective responserate ORR of in firstline melanoma and insecondline nonsmall cell lung cancer NSCLC []Therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of ICIs and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid 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 0cBai Biomarker Research Page of technologyimmunohistochemicalmade many new advances in the corresponding fieldsWith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers The development of predictive biomarkers contributes to revealing the therapeutic mechanisms of ICIs and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs It should be pointed out here that when reading and collating we try to read and include all therelevant s In the process of selecting s we include the authoritative s published in highleveljournals or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedAdvances of multiple predictive biomarkers toICIs efficacyi Tumor genome and neoantigen biomarkersTumor mutation burdenSignificant correlations between high tumor mutationburden TMB and response to ICIs have been reported inseveral cancer types [] including urothelial carcinoma[] small cell lung cancer SCLC [] NSCLC []melanoma [] and human papilloma virus HPVnegative HNSCC [] A metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log TMB [] The National ComprehensiveCancer Network NCCN guidelines have adopted TMBas the recommended test for patients with NSCLC receiving immunotherapy Although the results in some clinicalstudies of RCC [] HPVpositive HNSCC [] and melanoma receiving antiPD1 after recurrence [] showedthat TMB alone also did not clearly distinguish respondersand predict OS it is still exciting that multiple studies inthe American Society of Clinical Oncology ASCOmeeting have confirmed the predictive value of TMB inimmunization or combination therapy KEYNOTE061study [ ] CONDOR study [] EAGLE study []EPOC1704 study [] etc consolidating its status ofTMB as an independent predictor And in April theUS Food and Drug Administration FDA prioritized theapproval of TMB as a companion diagnostic biomarkerfor pembrolizumabNonetheless the cutoff values of TMB were defineddifferently across studies and assay platforms such asatezolizumab mtMb in urothelial cancer pembrolizumab mtMb in NSCLC and atezolizumab¥ ¥ or ¥ mtMb in NSCLC [] andnivolumab plus ipilimumab ¥ mtMb in NSCLC [] which needs further study to confirm the optimalcutoff value in different tumors Moreover the NGSpanels have approved by the FDA that can be used to estimate TMB include the MSKIMPACT and FoundationOne CDx panel the detection results of which arehighly consistent with whole exome sequencing WES[ ] and other solutions are under development Astudy detecting TMB cutoff value at mtMb in NSCLC patients with the FoundationOne platformcontaining a gene panel found that compared withTMBL patients overall survival OS and DCR was significantly improved in TMBH patients treated withantiPD1L1 drug [] Both WES and targeted NGS a422cancergene panel performed in patients withNSCLC treated with antiPD1L1 demonstrated thatTMBH population has a significantly better durableclinical benefitDCB and progressionfree survivalPFS [] These findings demonstrate the feasibility ofcomprehensive genomic profiling CGP but the designof optimal next generation sequencing NGS panel thatis more accurate comprehensive and costeffective isstill not clear In addition given that bTMB was identified as a predictor of PFS but failed to differentiate patients with OS benefits researchers consider the need toexplore other more precise factors eg allele frequencyAF A study that developed a new bTMB algorithm intwo independent cohorts POPLAR and OAK showedthat modified bTMB low AF bTMB LAFbTMB mutation counts with an AF was significantly associated with favorable HR 95CI p PFS HR 95CI p andORR p after immunotherapy but required tobe prospectively validated [] Finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions A recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment TMB was only associated with OS in untreated patients while early 4week ontreatment changein TMB ÎTMB was strongly associated with antiPD1response and OS in the entire cohort [] The detectionof ÎTMB is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cBai Biomarker Research Page of In addition epigenetic changes are associated withTMB The latest study investigated the association between TMB and DNA methylation DNAm to explorepotential complimentary biomarkers for NSCLC immunotherapies The results showed that high TMBNSCLCs had more DNAm aberrance and copy numbervariations CNVs showing certain value in predictingefficacy such as HOX gene methylation status and TMB[] Thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction Exploration in Chinese NSCLC patientsshowed that NSCLCs with high TMB had DNAm aberrance and CNVs Some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens [] In the pancancer analysis of cancer types evaluated in The Cancer Genome AtlasTCGA RCC had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnsSNVs[] Somatic copy number alterations SCNAs are another feature of the genomic landscape of tumors andpancancer TCGA analysis revealed an inverse correlation between SCNAs atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested [] and this result was subsequently replicated in a larger study of TCGA []single nucleotide variantsDNA damage response pathwaysGenetic variation involved in DNA mismatch repairMMR pathway can lead to microsatellite instabilityMSI a specific type of high TMB tumors and increased numbers of CD8 tumor infiltrating lymphocytesTILs PD1TILs and indoleamine 23dioxygenaseIDO tumor cells have been shown in MMR deficiencydMMR colorectal cancer [] Recently five clinical trials Keynote016 including multipletumor types have shown that patients with dMMRMSIH can achieve durable responses to pembrolizmabBased on this pembrolizumab is approved by the USFDA for the treatment of any advanced solid tumor withdMMRMSIH and nivolumab in combination with ipilimumab has also shown promising response in dMMRMSIH colorectal cancer [] In addition dMMR canalso cause mutations in the DNA polymerase gene epsilondelta POLEPOLD1increasing the mutationload and neoantigen load Analysis of POLEPOLD1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher TMB and OS Therefore it may be an infordependentInidentifying patients who benefitaddition pathways of base excision repairBERand prognostic markerfrom ICIs []risk factorhomologous recombination repair HRR MMR in theDNA damage response DDR signaling network contribute more significantly to TMB or neoantigens whichhave the highest levels when comutated [] It hadbeen identified that comutations in the DDR pathwaysof HRR and MMR or HRR and BER defined as comutare associated with increased levels of TMB neoantigenload and immune gene expression signatures Comutpatients showed a higher ORR and longer PFS or OS indicating that comut can be used as predictors of response to ICIs and provide a potentially convenientmethod for future clinical practice []Specific mutated gene pathways in tumor cellsIt is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy Patients with mutations in the interferon IFNγpathway genes IFNGR12 JAK12 and IRF1 are poorlyresponsive to ICIs treatment and confer resistance []A study found that in patients receiving immunotherapytumor cells can downregulate or alter IFNγ signalingpathways such as lossoffunction alleles of genes encoding for JAK12 and changes in STAT1 to escape the influence of IFNγ [] resulting in poor efficacy andresistance Recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling SWISNF complex and unique genes of the PBAFcomplex Pbrm1 Arid2 and Brd7 lead to sensitivitiesto ICIs [ ] Loss of function of the PBAF complexincreased chromatin accessibility to transcription regulator elements of IFNγinducible genes within tumorcells and subsequently increased production of CXCL9CXCL10 chemokines leading to more efficient recruitment of effector T cells into tumors [] In human cancers expression of Arid2 and Pbrm1 are related toexpression of T cell cytotoxicity genes which confirmedin Pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic T cells and responsive to immunotherapy [ ]In addition doublestranded RNAdsRNA editing enzyme adenosine deaminase acting onRNA ADAR1 protein can block the IFNγ signalingpathway and lead to poor ICIs efficacy and resistanceLoss of function of ADAR1 in tumor cells can reduce AtoI editing of interferoninducible RNA species and leadto dsRNA ligand sensing by PKR and melanomadifferentiationassociated protein MDA5 This resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy [] Finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding PDL1 and IFN signaling pathway genes sensitizingto anticytotoxic Tlymphocyteassociatedprotein4 CTLA4 therapy []it 0cBai Biomarker Research Page of In addition to the IFNγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy Epidermal growthfactor receptor EGFR and anaplastic lymphoma kinaseALK mutations have been shown to be associated withreduced response rates to ICIs and low TMB and therefore the FDA does not recommend firstline ICIstreatment in patients with EGRF or ALK positive tumors[ ] certain types of mutations in MDM2MDM4and ARID1A can predict nonresponse to ICIs in highTMB tumors [] NSCLC with KRAS and STK11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antiPD1 therapy [] and STK11 deficiency was an independent indicator of poor antiPD1response in NSCLC with KRAS mutant however at the American Association for Cancer Research AACRmeeting of patients in the Keynote042 studyNCT02220894 update data were tested for STK11 andKEAP1 and the results showed that patients could benefit from pembrolizumab regardless of STK11 and KEAP1status but patients with STK11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted NGSpanels suggested that duration of ICIstreatment was associated with certain BRAF and MET alterations butnot TMB status [] NOTCH signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells Recent breakthrough findings have distinguished deleterious NOTCH mutation showing that itcan be used as a potential predictor of favorable ICI response in NSCLC potentially via greater transcription ofgenes related to DNA damage response and immune activation [] Another tumorspecific inheritance thatmay influence ICIs efficacy is the aberrant expression ofendogenous retroviruses ERVs Pancancer analysisidentified a positive correlation of transcript expressionof ERVs with Tcell activity in various tumors [] andpatient prognosis [] Furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones For example the analysis of studydata of secondline PD1L1 inhibitor therapy found thatthe mPFS of patients with KRAS G12C or G12V was significantly better than that of patients with KRAS mutations at other sites []In addition several pancancer biomarkers are recentlyapproved by the FDA For example given the effectiveORR of and a disease control rate DCR of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent FDA approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor FGFR2 fusion or rearrangement and the comprehensive genomicassay FoundationOne CDxdeveloped by Foundation Medicine as a companiondiagnostic Also exciting is the recent FDA approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic NSCLC with MET exon skippingMETex14 mutations including firstline patients andpreviously treated patients also using FoundationOneCDx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofNeoantigen loadNeoantigen load the number of mutations actually targeted by T cells may be directly related to the responseto ICIs [] A retrospective study showed thatclonal neoantigen burden was associated with the longerOS in primary lung adenocarcinomas p []Traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexMHC binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall TMB inpredicting ICIs efficacy or survival [] In recent practice this neoantigen can be assessed by the difference inpredicted MHCI binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index DAI reflectingclinically relevanttumor peptide[] A high DAI value indicates that the mutant peptidesignificantly increases binding affinity to MHC compared to the wildtype sequence and can generate moreimmune responses Studies on previously published cohorts treated with three ICIs have shown that DAI outperforms TMB and the traditionally defined neoantigenload in predicting survival [ ] In additionlowneoantigen intratumour heterogeneity might also be important for ICIs response Analysis of the lung adenocarcinoma TCGA database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with OSand longer lasting clinical benefit than either variablealone [] Anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the Immune Epitope Database IEDB [] butit does not account for all possible human leukocyteantigen HLA contexts In addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes A study developed the Neopepseealgorithm using a machine learning approach incorporating 0cBai Biomarker Research Page of integration of nine immunogenicity features and gene mutation expression levels [] and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction Recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion [] indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii Tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score IPS Herbst [] showed that response toatezolizumab treatment was significantly associated withhigh levels of PDL1 expression on the surface of TILs before treatment but not with PDL1 expression on tumorcells p Finally other inhibitory immune pathways may affect the response to ICIs therapy including Tcelllymphocyte activationgene3 LAG3 and Vdomain Ig suppressor of Tcell activation VISTA which can be used as potential biomarkers for ICIs responseimmunoglobulin3 TIM3PDL1 expressionGiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between PDL1 expression and response to ICIs or OS even in firstlinecombination therapy [] pembrolizumab is currently approved by the FDA for use in patients with PDL1 PDL1 ¥ of tumor cells in firstline treatmentand ¥ in secondline treatment NSCLC and PDL1immunohistochemistry IHC as a companion diagnosticfor antiPD1 therapy in NSCLC patients [ ] However some studies have not detected a significant correlation between PDL1 expression and response to ICIs[ ] and PDL1 negative patients can still benefitclinically with treatment with ICI or combination treatment with ICIs [] with ORRs ranging from to Therefore PDL1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing FirstlyPDL1 assay and antibody are not standardized []Secondly PDL1 expression is temporally and spatiallyheterogeneous [] A study of metastatic NSCLCtreated with ICIs showed that PDL1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases Andthe predictive value of PDL1 at different biopsy sites forthe benefit of ICIs in NSCLC may vary higher PDL1 inlung or distant metastasis specimens was significantly associated with higher response rate PFS and OS whilePDL1 in lymph node metastasis biopsy was not associated with either response or survival [] Thirdly positive score and cutoff value of PDL1 expression is notstandardized [] At present PDL1 positive scoremainly focuses on the PDL1 expression level of tumorcells that is tumor proportion score TPS But PDL1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scoreCPS which is the proportion score of the sum of PDL1 expressed by tumor cells and tumorassociated immune cells In addition PDL1 expression on immuneresponseto ICIsimmunetreatmentBiomarkers of tumorinfiltrating immune cellsOverall immune status of tumor microenvironmentThe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert [] Immuneinflamed is characterizedby the presence of CD8 and CD4 T cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules [] indicating a potential antitumor[]immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] Analysis of pretreatment samples forantiPD1PDL1 revealed a relatively high abundance ofCD8T cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of CD8T cells into tumor parenchyma []while immunedesert phenotype is characterized by theabsence of abundant T cells in the parenchyma orstroma of tumors and poor response to ICItreatment[] Recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment TME classifying tumors aswell as predicting treatment response and prognosis[] which involves the density of two lymphocyte populations CD8 and memory [CD45RO] T cells in thecenter and invading margin of tumor [] Mlecnik et al[] evaluated immunoscore in specimens of stageIIV colorectal tumor and confirmed that it was significantly associated with PFS DFS and OS and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival The valueof immunoscore to predicting ICIs efficacy is being validated internationally in clinical trials of melanoma andNSCLC []A wider assessment of active immune responses withinTME by immune gene expression profiling might effectively predict clinical benefit to ICIs strategies Analysisof total RNA and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cBai Biomarker Research Page of expression of immunerelated genes in clinically active patientsincluding cytotoxic T cell markers egCD8A perforin granzyme B Th1 cytokines or chemokines MHCII and other immunerelated genes egNKG7 IDO1 [] Ascierto [] screened morethan immunerelated genes in patients with recurrent breast cancer years after treatment and thosewithout recurrence more than years later and foundthat five genes IGK GBP1 STAT1 IGLL5 and OCLNwere highly overexpressed in patients with recurrencefree survival In addition IFNγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with ICIs The study developed IFNγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding IFNγ granzymes AB perforin IDO1 and other immunerelated genesBoth gene scores showed significant associations withbest overall response rate and PFS Optimized cutoffvalues for IFNγ scores based on receiver operatingcharacteristic curve ROC curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders []Immune cells with specific phenotypes in TMEThe phenotype of TILs also influences the efficacy ofICIs The study used singlecell mRNA sequencingscRNAseq data analysis to identify two major CD8Tcell phenotypes within melanoma memorylike andexhausted [] the proportion of which is strongly correlated with response to ICIs The research furtherfound that the transcription factor TCF7 is selectivelyexpressed in memorylike T cells so the ratio ofCD8TCF7 to CD8TCF7TILs is strongly correlatedwith improved response and survival in melanoma patients treated with antiPD1 [] Balatoni []found that of immune cells in TME were positivelyassociated with OS after treatment including CD4 andCD8 T cells FOXP3 T cells CD20 B cells CD134and CD137 cells and NKp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes Researchers found that only asmall proportion of CD8 TILsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential CD39 expression was the key molecule thatdistinguished the two populations [] Analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of CD39 on CD8 TILs indicating thatCD39CD8TIL may be a promising predictive biomarker [] The fact of very low level of CD39 expression on CD8TILs in of EGFRmutant NSCLC isconsistent with their low response rate to antiPD1immunotherapyIn addition a study showed that Fc domain glycan ofthe drug and Fcγ receptor FcγR expressed by the hostbone marrow cells could determine the ability of PD1tumorassociated macrophages TAMs to capture antiPD1 drugs from the surface of T cells which leads toPD1 inhibitor resistance [] and the association ofTAMs and poor antiPD1 response was reported inmelanoma cohorts [] antiPD1 response was associated with an increase in CD8T cells and natural killercells NK cells and a decrease in macrophages [] andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mPFS after antiPDL1 therapy in RCC emphasizing the inhibitory role of myeloidcells in response to ICIs [] In conclusion immunecells in TME show a great promise in the developmentof predictive biomarkers for ICIsimmunerepertoireDiversity of immune repertoires in TMEEffective T cell responses involve the activation and expansion of specific antigenreactive T cell clones so diversity ofin intratumoral orperipheral may correlate with ICIs responses and can bequantified as richness and clonality [] However theresults seem to be complex with some studies finding apositive correlation between TIL clonality and the response to ICIs before [] or after [] treatment whileothers showing that only an increase in TIL clonalityduring treatment is associated with the response to antiPD1 [ ] others show that intratumoral T cellclonality is not associated with survival while peripheralT cell clonality is inversely associated with PFS and OS[] Tumeh [] further investigated whetherbaseline TILs have a narrow T cell receptor TCR repertoire focusing on tumorspecific immune responsesand whether this narrow TCR repertoire correlates withpembrolizumab responses They found that respondingpatient had more restricted usage of the TCR beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients Notablybaseline TCR clonality was not highly correlated withTIL density suggesting that some patients with restricted TCR clonality specific for tumor antigens maystill benefit from antiPD1 therapy even though TILdensity is low Recently researchers have proposed theimmune repertoire IRIndex the average frequency ofshared TCR clones in T clones in TILs and peripheralPD1CD8 T cells They found that neoantigenstimulated TCR agreed with IRIndex and patients withhigh IRindex had better immune activation and highergene expression profiles GEPs score subsequently they 0cBai Biomarker Research Page of confirmed the predictive value of IRindex to ICIs efficacy DCRPFS But considering that it is difficult tosort out PD1CD8 T cells in tumor tissue based ontwo separate patient cohorts a research confirmed thatTCR repertoire diversity and clonality of peripheral PD1CD8T cells may serve as noninvasive predictors ofclinical outcomes after ICIs in patients with NSCLC[] The viewpoints of T cell diversity and TCR clonality as markers of ICIs efficacy need to be further validated in a large patient populationiiiLiquid biopsy biomarkersPeripheral blood cell biomarkersPeripheral blood is a noninvasive source to explore potential biomarkers for ICIs and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies Analysis of melanoma treated with ipilimumabshowed that improved OS and PFS were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio NLR low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of FoxP3 Treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased FoxP3Treg concentratreatmenttions and increased lymphocyte and eosinophil counts[] Reports in patients with melanoma treated withpembrolizumab and in patients with NSCLC treatedwith nivolumab have shown that NLR is associated withworse tumor response [ ] Multivariate analysis inmelanoma patients treated with antiPD1 antibodiesshowed that NLR was the only factor associated withworse ORR and shorter PFS indicating that NLR is astrong predictor of worse outcome in patients treatedwith ICI [] Low baseline lactate dehydrogenase LDHlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedOS in antiPD1 and CTLA4 treated melanoma [] Given that previous studies have proposed the importance of baseline derived NLR dNLR and LDHlevels as prognostic markers a recent study proposed acomposite prognost | Thyroid_Cancer |
thyroid cancer diagnosis by Raman spectroscopyMarco Sbroscia15 Michael Di Gioacchino1 Paolo Ascenzi1 Pierfilippo Crucitti2 Alessandra di Masi1 Isabella Giovannoni3 Filippo Longo2 Davide Mariotti1 Anda Mihaela Naciu4 Andrea Palermo4 Chiara Taffon3 Martina Verri3 Armida Sodo1 Anna Crescenzi3 Maria Antonietta Ricci1Over the last years the incidence of human thyroid cancer disease has seen a significative increment This comes along with an even higher increment of surgery since according to the international guidelines patients are sometimes addressed to surgery also when the fine needle aspiration gives undetermined cytological diagnosis As a matter of fact only of the thyroid glands removed for diagnostic purpose have a post surgical histological report of malignancy this implies that about of the patients have suffered an unnecessary thyroid removal Here we show that Raman spectroscopy investigation of thyroid tissues provides reliable cancer diagnosis Healthy tissues are consistently distinguished from cancerous ones with an accuracy of ¼ and the three cancer typology with highest incidence are clearly identified More importantly Raman investigation has evidenced alterations suggesting an early stage of transition of adenoma tissues into cancerous ones These results suggest that Raman spectroscopy may overcome the limits of current diagnostic toolsDetection of nodular thyroid pathology is increasing worldwide paired with an increased number of thyroid cancer diagnosis1 Whether this epidemic spread is real or is due to an exaggerated use of imaging tools in clinical practice is object of a vivid debate within the scientific community Anyway management of patients with thyroid nodule causes a clinical problem and a social burden as well2 To avoid overdiagnosis and overtreatment of these patients physicians need additional information about the biology of each thyroid nodule In particular there is need for a tool able to distinguish between benign and malignant follicular lesions and between indolent and aggressive tumors3 The genomic profile of thyroid tumors has been extensively investigated4 and several immunocytochemical and molecular markers have been proposed for a more accurate classification of thyroid nodules5 However none of these markers reaches a reliable sensibility and specificity to be routinely used in clinical setting6 Moreover although the major genetic alterations of thyroid tumors are known only little information if any is available about changes in protein lipids and other cellular constituents due to thyroid oncogenesisTo correctly manage patients with thyroid nodules and to avoid unnecessary surgery we strongly need a method of analysis that overcomes the limits of current diagnostic tools by identifying specific tumoral markers possibly with high spatial resolution and low impact on the patient Cells and tissues are characterized by a specific biochemical composition Each pathology or cellular abnormality is accompanied by biochemical molecular changes Optical and spectroscopic techniques that correlate the biochemical composition molecular structure and their variations with the morphological alterations represent a powerful diagnostic and potentially clinical tool8Among the experimental techniques available for chemicalphysical studies of matter at the molecular level Raman spectroscopy RS is a good candidate for our needs This technique offers a wealth of molecular information while being not invasive and achieving spatial resolutions as high as a few μm in microRaman apparatuses RS probes the vibrational dynamics of the sample through a fast and not destructive analysis It does not require any sample preparation and can consequently be applied in a0vivo For these characteristics it has been recently applied on a variety of biological and medical samples to identify specific metabolic states of cells tissues and 1Dipartimento di Scienze Universit¡ degli Studi Roma Tre Rome Italy 2Unit of Thoracic Surgery Campus BioMedico University Rome Italy 3Pathology Unit Campus Biomedico University Hospital Rome Italy 4Unit of Endocrinology and Diabetes Campus BioMedico University Rome Italy 5Present address Dipartimento di Fisica Sapienza Universit¡ di Roma Rome Italy email armidasodouniroma3itScientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Raman spectra A Typical Raman spectra of the examined thyroid tissues labelled according to the histology report Stars label the Raman characteristic peaks of cytochrome c triangles those of carotenoids The black vertical dashed lines allow an easier comparison among the spectra B Centroids Raman spectra for the four identified clusters by Kmeans analysisbacteria and in particular to differentiate between healthy and cancer tissues9 Preliminary studies on thyroid tissues by RS suggest that this technique may have high impact in thyroid cancer diagnosis13In a RS experiment a laser beam is focalized on the sample and the scattered light is analyzed in frequency Typical spectra after background subtraction are shown in Fig a01A These are characterized by peaks and bands with frequency centered at the vibrational frequency of the intramolecular bonds characteristic of the main sample componentsWe have applied RS augmented by cluster analysis to investigate histological samples from human thyroid Assignement of the RS peaks to carotenoids or cytochrome c in different amounts has allowed to distinguish healthy tissues from cancerous ones and to discriminate for the first time among the three categories of carcinoma with larger incidence namely Papillary carcinoma PTC follicular variant of Papillary carcinoma FVPTC and Follicular carcinoma FC We have also analyzed samples labelled after histology as follicular adenoma FA We found that these samples are spread over four categories reported above by the cluster analysis Subsequent more specific analyses of protein expression have confirmed an anomalous level of tumoral or benign molecular markers in the adenoma samples labeled in the non healthybenign categories by RS All the samples analyzed by RS are reported in a table as Supplementary informationResultsFigure a01A shows from bottom to top the Raman fingerprint region cm of four samples with a clear histology diagnosis namely HealthyBenign average of the spectra of samples Hea49 and Hea64 PTC average of the spectra of samples TIR51 and TIR58 FVPTC sample TIR56 and FC average of the spectra of samples TIR47 and TIR55 carcinoma tissues Each spectrum has been normalized to its integrated intensity We notice that each tissue has its own characteristic spectral lineshape According to Rau et a0al15 and references therein this spectral range shows the bands ascribed to proteins lipids and aminoacids Here we will not report a detailed assignment of each band to a particular biomolecule as this is sometimes tricky and not relevant to our aim We will instead put the attention on the distinctive set of peaks which characterize each histological sample We notice that the spectrum of the healthy tissues is dominated by an intense broad and structured band centered around ¼ cm possibly due to the presence in the sample of amide I and lipids19 The sharpest structure of this band falls at cm and along with the bands at · and cm Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Agglomerative hierarchical clustering analysis Dendrogram of the Raman spectra of human thyroid tissues as extracted from the AHCA analysis Individual samples are represented by the label Hea for healthy or TIR for not healthy followed by the patient anonymous ID code Those plotted in Fig a01A are labelled healthy FC FVPTC and PTC respectively Dashed squares identify the four clusters namely healthybenign orange Follicular carcinoma or FC black follicular variant of papillary carcinoma or FVPTC magenta and papillary carcinoma or PTC blue The arrows indicate the adenoma samples All samples within the light blue shaded area are carcinomasrepresets the fingerprint of cytochrome c20 This is a protein localized in the mitochondrial intermembrane space under normal physiological conditions The spectrum of a classical papillary carcinoma labeled PTC in Fig a01A is totally different and unambigously shows the presence of carotenoids bands at and cm not present in healthy tissues as already reported in the literature10 In the follicular carcinoma spectrum labeled FC in Fig a01A we observe a marked enhancement of the cytochrome c bands As a matter of fact the broad band at ¼ cm that dominates the HealthyBenign spectra is barely visible in this case after spectral intensity normalization Interestingly the spectrum of the Papillary Follicular carcinoma labeled FVPTC in Fig a01A looks like a superposition of the PTC and FC spectra showing the fingerprints of carotenoids along with a clear enhancement of the bands ascribed to cytochrome c with respect to the spectra of HealthyBenign tissuesThe presence of carotenoids in both PTC and FVPTC carcinoma is peculiar and worthy of interest The cellular process of carotenoids uptake not directly synthetized by human anisms and their proper identifications is under biochemical investigationAs Raman spectroscopy is able to detect the differences between healthy and cancerous tissues it may be considered as a promising tool for in a0situ cancer diagnosis We have analyzed a total of histological samples of human thyroid tissues samples diagnosed as adenomas including one inadequate TIR59 for RS and not further considered for statistical analysis healthy ones and carcinomas ones namely PTC FVPTC FC and Hyalininzing Trabecular Tumor HTT All samples are labelled as TIR or Hea followed by the patient anonymous ID codeWithin each histology class spectra may differ for noise background or fluorescence contributions although keeping unaltered the characteristic fingerprints commented above This observation suggests that the experimental spectra can be analyzed on quantitative grounds by cluster analysis after backgrounds and fluorescence subtraction This aims at classifying each Raman spectrum into homogeneous groups based on its characteristic features in other words on the presence and relative intensity of specific bands The analysis has been performed following two distinct approaches see MethodsThe first approach called agglomerative hierarchical clustering analysis AHCA builds a dendrogram of the entire data set by iteratively selecting pairs of the closest spectra or groups of spectra as a function of their Euclidean distance The results of this analysis are reported in Fig a0 where each sample is represented by means of the label Hea or TIR depending on whether the tissue has received a healthy or not healthy histological diagnosis followed by a progressive number This figure tells us that our spectra are consistent with a classification of the dendrogram into clusters assuming a distance threshold of compared to a maximum distance of The four clusters have been labelled according to the diagnosis of the four spectra shown in Fig a01A a priori selected as the most representative of each histology report namely the less noisy with a clear and confirmed diagnosis HealthyBenign and PTC tissues are very well distinct each other and have the smallest dispersion in terms of Euclidean distance within each cluster Indeed the smaller is the dispersion of the Euclideian distances the better is the homogeneity of the group in the present case HealthyBenign and PTC tissues are the most homogeneous The other two clusters FC and FVPTC are more dispersed although equally distinguishable Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Kmeans analysis Samples distribution within the four KM clusters as a function of their Euclidean distance from the associated centroid The same labels and colours as in Fig a0 have been used Black triangles refer to adenomasCluster Cluster Cluster Cluster Cluster Cluster Cluster Cluster Table Euclidean distance between the cluster centersTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples healthy and carcinomas tissues This shows that RS has identified False negative diagnosis among the negative Medical diagnosis and different samples among the with a positive diagnosis which fall in a cluster different from those expected on the basis of the medical diagnosisfrom the others The FC cluster is the closest to the HealthyBenign one as both are characterized by the presence of cytochrome c and distinguished only on the basis of its Raman fingerprint relative intensity The FVPTC tissues fall between FC and PTC clusters as predictable from copresence of carotenoids and citochrome c peaks in their Raman spectraThe second unsupervised statistical approach considered is called Kmeans KM its results are reported in Fig a0 This confirms that four clusters properly describe our data set Clusters contain samples from the histology reports of PTC HealthyBenign FVPTC and FC respectively The agreement between the clustering results of the two approaches is very good as we notice only one difference concerning sample TIR60 which switches from a Raman diagnosis of PTC to that of FVPTC Fig a0 shows that healthybenign tissues spectra have on the average the shortest distance from their centroid and the smallest dipersion On the contrary the spectra of FVPTC tissues are the most dispersed possibly due to a different balance of concentration between carotenoids and cytochrome c in each sample Table a0 reports the distance matrix among the clusters centroids Cluster showing the largest centroid distance from the others is readily isolated its Raman spectrum is dominated by the carotenoid fingerprint while discrimination among the others requires consideration of more spectral detailsKM analysis returns also the Raman spectrum calculated for each cluster centroid these are reported in Fig a01B for direct comparison with the prototypical spectra of the four classes of tissues The similarity of the spectra reported in Fig a01AB is astonishing and proves that our clustering is solidBased on these results we have computed a confusion matrix providing the accuracy of our analysis in discriminating between healthybenign and pathological tissues regardless of the specific pathology As reported in Table a0 our accuracy is of ¼ Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples including adenomasThis shows that RS has identified false negative diagnosis among the negative medical diagnosis and different samples among the with a positive diagnosis which fall in a cluster different from those expected on the basis of the medical diagnosisFigure a0 Immunohistochemistry images Microscopic pictures of A TIR43 sample stained with immunohistochemistry for Galectin3 high power field Hematoxylin counterstained and B TIR64 sample stained with immunohistochemistry for HBME1 medium power field Hematoxylin counterstained Panel A shows a mosaic pattern with negative cells intermingled with cells showing positive reaction in cytoplasm as well as in nuclear matrix Panel B shows strong positive reaction due to a microfocus of papillary carcinoma within the follicular adenomaTotal of samplesMedical diagnosisRamanTrue negative False negative ClassificationFalse positive True positive AccuracyTable Confusion matrix based on samples including adenomas after immunohistochemistry revisionThis shows that RS has identified false negative diagnosis among the negative medical diagnosis and that the number of false positive diagnosis goes down to Finally we have applied the same analysis including the spectra of the samples classified as adenoma by the histological diagnosis Their position is highlighted by arrows in the AHCA dendrogram of Fig a0 and in the distributions of Fig a0 These figures show that the spectra of adenoma tissues spread over all four clusters defined by healthy and cancerous tissues This is a quite unexpected result as adenomas are classified as benign tissues and therefore should fall into the healthybenign cluster Accordingly the confusion matrix after inclusion of adenoma samples reported in Table a0 returns an accuracy of the statistical analysis of After the RS analysis four adenoma samples TIR43 TIR44 TIR64 TIR71 falling within the cancerous clusters have been in depth reanalyzed as far as their expression of benignity and cancerous markers are concerned by immunohistochemistry test The lowering of benignity markers CD56 or sometimes the onset of tumoral markers Galectin3 HBME1 are revealed in all these cases The immunohistochemistry images of TIR43 and TIR64 samples are shown in Fig a0 as representative of the all four cases These results could be symptomatic of early stages of progression of adenomas into a specific type of carcinoma Such type of transformation has never been demonstrated and our Raman observation s a new light about the malignant potential of thyroid FA and about the possibility to assess which of these lesions may constitute a clinical concern Interestingly the confusion matrix after this revision gives an accuracy of ¼ A few anomalies visible in Figs a0 and a0 deserve a comment Sample Hea56 and sample Hea63 apparently fall into a wrong cluster namely the FVPTC cluster but interestingly very close to their TIR counterpart FVPTC and TIR63respectively This may happen when the Raman spectrum of the healthy portion is collected from a tissue region too close to the pathologic one where normal and altered cells are almost intermingled giving the superposition of healthy and cancerous Raman spectra In particular the case of sample Hea72 falling within the FVPTC cluster may be due to the diffusion of carotenoids in normal parenchyma at the border Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Biochemical analysis Expression levels of cytochrome c have been assessed in thyroids of five patients for each patient the healthy Hea and pathological TIR slices have been analysed A Levels of cytochrome c have been normalized to actin Data represent the mean values ± SDs derived from three replicates normalized to healthy counterparts Students t test P a0 a0 P a0 a0 compared with control B Exemplificative images of filters blotted with cytochrome c and actin primary antibodies Images have been gathered at the same timeof the PTC nodule as already observed in previous Raman chemigram map of thyroid carcinomas15 This gives in the Raman spectrum the superposition of the fingerprint of carotenoids with fingerprint of healthy tissues containing cytochrome c thus explaining the wrong assignment obtained by Raman analysisAdditionally levels of cytochrome c have been further evaluated by biochemical analysis Fig a0 on a few cases Indeed only for patients and PTC and and FC a sufficient amount of thyroid tissue was available to perform immunoblot analyses As shown in Fig a0 cytochrome c levels significantly increase in TIR45 134fold induction compared to its healthy counterpart Hea45 P a0 a0 TIR46 27fold induction compared to Hea46 P a0 a0 and TIR47 456fold induction compared to Hea47 P a0 a0 In agreement with Raman results cytochrome c levels are similar in TIR and Hea slices of patients and In conclusion this study demonstrates the capability of Raman spectroscopy as diagnostic tool for thyroid cancer Indeed through a multivariate statistical analysis of the spectra it is possible to readily separate healthy from cancerous tissues and in the majority of the cases to discriminate among the most common cancer typologies see Supplementary information for details on the individual cases Last but not least our findings highlight the possible usefulness of Raman spectroscopy as a tool for in a0situ and early diagnosis of thyroid pathologyScientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0cMethodsStudy enrollment From January to January we enrolled consecutive subjects females and males with thyroid nodular pathology referred to the Endocrinology Unit of Campus BioMedico University Hospital Their age distribution is centered at a0years with a standard deviation of a0years All the study population had received a cytological diagnosis of indeterminate suspicious or malignant lesion with a formal indication to surgery total thyroidectomy according to the international guidelines24 After signing the informed consent these patients underwent total thyroidectomy at the Surgical Unit of the same InstitutionNeck ultrasound and clinical evaluation All subjects were submitted to thyroid US evaluation using a frequency range of MHz on a MyLab Esaote Genova Italy US scan of thyroid gland and neck area were performed by experienced endocrinologists at the Endocrinology Unit Nodules were classified according to ACR TIRADS risk stratification criteria25 without prior knowledge of the cytological results When there was a disagreement the endocrinologists conducted a separate session to reach an unified consensus We collected demographic data including age gender family history of thyroid cancer and using the medical records we evaluated the thyroid function and the presence of autoimmune thyroiditisThyroid tissues preparation At the time of surgery the removed specimens were immediately submitted unfixed to the Pathology Unit in an appropriately labelled container The pathologist valued the gross anatomy of the samples and a tissue slice of about cm wide cm length mm of thickness was cut including both healthy and neoplastic areas avoiding surgical margins The slice was snap frozen on a metallic coldplate inside a cryostat A μm section was stained with haematoxylineosin in order to confirm the presence of healthy and neoplastic tissues After this step four consecutive cryostatic sections were cut at μm of thickness collected on separate slides and stored at ¦C until the Raman evaluation Our Raman study was exclusively performed on these frozen unfixed samples The residual slices were defrosted formalin fixed and paraffin embedded with the paired surgical samples for definitive histology Final diagnosis was reported in agreement with current edition of WHO classification of endocrine tumours26 Immunohistochemical analysis for Galectin3 Gene Tex CD56 Agilent and HBME1 Agilent was performed in each case on paraffin sections from neoplastic areas using an automatized immunostainer Omnis AgilentNodules with diameter wider than cm were submitted for biochemical analysis In such cases at the time of gross dissection further slices of about a0cm were cut from the surgical specimen sampling both healthy and neoplastic tissue Slices were snap frozen and stored at ¦C until biochemical evaluationRaman spectra collection Raman spectra have been collected by means of a Renishaw InVia MicroRaman spectrometer In this setup unpolarised spectra are collected through a Leica DM2700 M confocal microscope equipped with a Leica LWD and an Olympus objective The required highcontrast rejection for the elastically scattered light is provided by an holographic edge filter A diffraction grating with a0groovesmm disperses the Raman inelastic scattering contribution providing a spectral resolution of about cm A Peltier cooled pixel CCD detector collects the dispersed light A solidstate diode laser source at nm with a nominal output power of nearly mW has been used as excitation source To prevent photodamage neutral density filters have been used for lowering the laser power at the sample The spot size has been shrink down to few microns when necessary to isolate the contribution of cells about ten microns in size from the surrounding cytoplasm thus reducing spectral interference Spectra have been collected in the extended scan mode covering the cm frequency shift range and accumulating scans with an equivalent integration time of nearly s per scan Measurements have been collected on points per sample Wire Renishaw software has been used to collect the raw spectra Wire and LabSpec software has been used to perform the preliminary data reduction eg background and fluorescence subtraction while Origin OriginLab Corporation software has been employed to perform the statistical analysis After background and fluorescence subtraction all the spectra have been normalised to their integral in order to avoid any bias due to fluctuations of the experimental conditionsCluster analysis of Raman spectra In order to gain better confidence on the clustering analysis we have selected two unsupervised multivariate statistical approaches to analyze the Raman spectra the agglomerative hierarchical clustering analysis AHCA and the Kmeans one KM Both methods are based on the Euclidean distance among spectra as a measure of their similaritydissimilarity In detail in these analyses spectra are represented by their distance with respect to an origin thus generating a dissimilarity matrix Among the possible choices for the spectral distance we have chosen the Euclidean one since it maximizes the similarities among the spectra The choice to use these two methods was made to exploit their complementarity of hierarchical and nonhierarchical approaches and consequently to give solidity to the obtained results13 The AHCA method aims at classifying observations each Raman spectrum into homogeneous groups the clusters based on the measured characteristics Euclidean distance associated to each Raman spectrum In particular we have used the completelinkage algorithm in order to enhance differences among clusters It works by pairing the most similar spectra those showing the lowest dissimilarity value in the matrix and then looking for the largest distance between such pair and the rest of the data By iteratively repeating such an approach a classification of spectra into wellseparated groups is obtained and visualised as a dendrogramThe KM method minimizes the variance within each cluster with respect to an a priori number of centroids randomly distributed This protocol starts by associating each data entry Euclidean distance associated to each Raman spectra to the cluster with the closest centroid The centroids are iteratively updated towards convergence The number of clusters k is an input of the routine In order to choose the proper value of k the most Scientific RepoRtS 101038s41598020701650Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Spread of the individual Kmeans clusters as a function of their number Data are reported by using the same colors as in Fig a0 for the individual clusters Notice that four clusters give the largest spread among the clustersused strategy is the elbow curve based on the explained variance within the entire dataset This analysis in our case gives only two centroids one for samples containing mainly carotenoids and one for all other cases Thus following the suggestion of the histology diagnosis we have set k a0 a0 In order to verify the reliability of this choice we have calculated the spread of each cluster with respect to its centroid as a function of k Intestingly we find that k a0 a0 is the highest kvalue with the lowest spread as shown in Fig a0We have tested the predictive accuracy of both AHCA and KM methods by generating a confusion matrix This reports the comparison between the medical and spectroscopic diagnosis The accuracy is defined asAccuracy True negative True positivenumber of total samplesBiochemical analysis Frozen thyroids have been weighted homogenized and sonicated in lysis buffer mM TrisHCl pH mM NaCl mM EDTA glycerol vv Triton X100 vv and protease inhibitors Twentyfive micrograms of protein extracts previously quantified with the Bradford assay BioRad Hercules CA have been resolved by SDSPAGE and transferred onto PVDF membranes BioRad After blocking with BSA wv dissolved in TBS buffer Tween20 vv membranes have been probed overnight at ¦C with anticytochrome c mouse mAb sc13560 Santa Cruz Biotechnology Dallas TX USA and antiactin mouse mAb sc47778 Santa Cruz Biotechnology primary antibodies Membranes have been then incubated for h at room temperature with antimouse HRPconjugated secondary antibody BioRad All the experiments have been repeated trice Blots have been acquired and processed using the ChemiDoc Imaging system BioRad Cytochrome c quantification has been performed using the Image Lab software version BioRad Laboratories Results are shown as mean ± standard deviation SD the statistical significance of differences has been tested using the Students t test GraphPad Prism version ethics statement The study was approved by the Ethical Committee of the University of Rome Campus Biomedico UCBM prot TS ComEt CBM and prot PAR ComEt CBM The informed consent was collected from patients before surgery Enrolled patients were recorded in a codified file with an anonymous ID code which was registered in the software database of the Pathology Unit of the UCBM All experiments were performed in full accordance with the principle of Good Clinical Practice GCP and the ethical principles contained in the current version of the Declaration of HelsinkiReceived March Accepted July References Vaccarella S et al Worldwide thyroidcancer epidemic The increasing impact of overdiagnosis N Engl J Med Udelsman R Zhang Y The epidemic of thyroid cancer in the United States the role of endocrinologists and ultrasounds Thyroid Rusinek D et al Current advances in thyroid cancer management Are we ready for the epidemic rise of diagnoses Int J Mol 101056NEJMp 101089thy20130257 Sci Scientific RepoRtS 101038s41598020701650Vol1234567890wwwnaturecomscientificreports 0c TCGAR Network Integrated genomic characterization of papillary thyroid carcinoma Cell 101016jcell201409050 Cantara S et al Molecular signature of indeterminate thyroid lesions current methods to improve fine needle aspiration cytology fnac diagnosis Int J Mol Sci 103390ijms1 Haugen B R et al American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association guidelines task force on thyroid nodules and differentiated thyroid cancer Thyroid 101089thy20150020 Paschke R et al European thyroid association guidelines regarding thyroid nodule molecular fineneedle aspiration cytology diagnostics Eur Thyroid J 10115900046 Krafft C Popp J Raman4clinics the prospects of Ramanbased methods for clinical application Anal Bioanal Chem Kallaway C et al Advances in the clinical application of Raman spectroscopy for cancer diagnostics Photodiagn Photodyn Ther 101007s0021 101016jpdpdt Li Z et al Surfaceenhanced Raman spectroscopy for differentiation between benign and malignant thyroid tissues Laser Phys Lett 1010881612201111404560 Krafft C Popp J The many facets of Raman spectroscopy for biomedical analysis Anal Bioanal Chem 101007s0021 Palermo A et al Raman spectroscopy applied to parathyroid tissues a new diagnostic tool to discriminate normal tissue from adenoma Anal Chem 101021acsanalc hem7b036 Harris A T et al Raman spectroscopy and advanced mathematical modelling in the discrimination of human thyroid cell lines Teixeira C S B et al Thyroid tissue analysis through Raman spectroscopy Analyst 101039B8225 Rau J V et al Raman spectroscopy imaging improves the diagnosis of papillary thyroid carcinoma Sci Rep Head Neck Oncol 78H 101038srep3 Rau J V et al Proofofconcept Raman spectroscopy study aimed to differentiate thyroid follicular patterned lesions Sci Rep 101038s4159 MedeirosNeto L et al In a0vivo Raman spect | Thyroid_Cancer |
"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai 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 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e | Thyroid_Cancer |
CytoskeletonAssociated Role ofPDLIM5Xiaolan Huang Rongmei Qu Jun Ouyang Shizhen Zhong and Jingxing DaiGuangdong Provincial Key Laboratory of Medical Biomechanics Department of Anatomy School of Basic MedicalSciences Southern Medical University Guangzhou ChinaRegenerative medicine represented by stem cell technology has become one of thepillar medical technologies for human disease treatment Cytoskeleton plays importantroles in maintaining cell morphology bearing externalforces and maintaining theeffectiveness of cellinternal structure among which cytoskeleton related proteins areinvolved in and play an indispensable role in the changes of cytoskeleton PDLIM5 isa cytoskeletonrelated protein that like other cytoskeletal proteins acts as a bindingprotein PDZ and LIM domain PDLIM5 also known as ENH Enigma homolog isa cytoplasmic protein with a molecular mass of about KDa that consists of a PDZdomain at the Nterminus and three LIM domains at the Cterminus PDLIM5 bindsto the cytoskeleton and membrane proteins through its PDZ domain and interactswith various signaling molecules including protein kinases and transcription factorsthrough its LIM domain As a cytoskeletonrelated protein PDLIM5 plays an importantrole in regulating cell proliferation differentiation and cellfate decision in multipletissues and cell types In this review we brieï¬y summarize the state of knowledge onthe PDLIM5 gene structural properties and molecular functional mechanisms of thePDLIM5 protein and its role in cells tissues and an systems and describe thepossible underlying molecular signaling pathways In the last part of this review wewill focus on discussing the limitations of existing research and the future prospects ofPDLIM5 research in turnKeywords PDZ and LIM domain microï¬lament actin cytoskeleton cytoskeletonassociated proteinINTRODUCTIONPDZ and LIM domain also known as ENH ENH1 L9 and LIM is a cytoskeletonrelated proteinthat was ï¬rst discovered by Kuroda using yeast twohybrid technology with proteinkinase C PKC as the bait protein PDLIM5 which consists of a PDZ domain and one or moreLIM domains is a PDZLIM family member whose sequence is highly conserved across speciesThe proteins of the PDZLIM family EnigmaLMP1 ENH ZASPCipher RIL ALP and CLP36have been suggested to act as linkers to direct LIM binding proteins to the cytoskeleton Vallenius PDZLIM protein can act as a signal modulator to aï¬ect actin dynamics regulate cellstructure and control gene transcription to promote the assembly of protein complexes The PDZLIM protein family which function as proteinprotein interaction modules act as scaï¬olds to bindto ï¬lamentous actinassociated proteins a range of cytoplasmic signaling molecules and nuclearEdited byMarina PajicGarvan Institute of Medical ResearchAustraliaReviewed byClment ThomasLuxembourg Institute of HealthLuxembourgHongqiang ChengZhejiang University ChinaCorrespondenceJun OuyangjouyangsmueducnShizhen ZhongzhszhsmueducnJingxing Daidaijxsmueducndaijx2013163comSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationHuang X Qu R Ouyang JZhong S and Dai J AnOverview of theCytoskeletonAssociated Roleof PDLIM5 Front Physiol 103389fphys202000975Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5proteins allowing this family to carry out diverse functionsduring development and adulthood Krcmery Several members of the PDZLIM proteins family play aregulatory role in the invasion and migration of cancer cellsDysfunction of the proteins of the PDZLIM family is knownto aï¬ect the maintenance of an function and weaken theinvasion ability and metastatic potential of cancer cells BagheriYarmand TanakaOkamoto Accordingto recent studies PDLIM5 may be involved in the progressionof multiple tumor types Eeckhoute Edlund Heiliger Li The important role ofPDLIM5 in various anizational systems have led to a deeperunderstanding of its physiological function This review aimsto summarize the state of knowledge and progress related toPDLIM5 from multidisciplinary perspectivesTHE PDLIM5 GENE AND ITSEXPRESSION AND THE STRUCTUREAND DISTRIBUTION OF PDLIM5The PDLIM5 GeneThe PDLIM5 gene is located on the human chromosome 4q223between markers W12900 and W13273 and spans exonsUeki Te Velthuis and Bagowski PDLIM5can be categorized as long isoforms with three LIM domainsand short isoforms without LIM domains according to thepresence or lack of three LIM domains and the long and shortisoforms each contain ¼ subtypes Cheng Ito Mouse PDLIM5 isoform I mENH1 encodes afulllength 591aminoacid protein containing a PDZ domainand three LIM domains two smaller transcripts called mousePDLIM5 isoform and mENH2 and mENH3 encode a aminoacid protein and a 239aminoacid protein respectivelyBoth mouse PDLIM5 isoforms and lack three LIM domainsNiederlander Zheng In humans fourPDLIM5 splice isoforms have been identiï¬ed one long isoformENH1 which contains three LIM domains at its Cterminaland is widely expressed in all tissues and three short isoformsENH24 which are mainly expressed in cardiac and skeletalmuscle Kuroda Ueki Analysis of humanPDLIM5 transcripts showed that three transcripts hENH12 were similar to those of mice while the fourth transcripthENH4 encodes a 215aminoacid protein lacking three LIMdomains Niederlander The Expression Structure andDistribution of PDLIM5is a memberThe PDLIM5 protein also known as ENHofthe Enigma family which consists of an NterminalPDZ domain and three Cterminal LIM domains The mainfunction of PDZ and LIM domains is to participate inproteinprotein interactions Table The PDZ domain oneofischaracterized by a highly conserved amino acid sequenceconsisting of six antiparallel strands and two αhelicesthe most common proteinprotein binding domainsTABLE Binding partners of PDLIM5 and their functionsProteinDomain FunctionsReferencesProtein kinaseA PKAαactininMyotilinLtype calciumchannelYAPProtein kinaseC PKCProtein kinaseD1 PKD1CREBID2PDZPDZPDZPDZPDZLIMLIMLIMLIMNtype Ca2channelsAMP activatedprotein kinaseKAE1Protein kinaseLin Sarcomere Zline proteinSarcomere Zline proteinCalcium channels inmyocytesTranscriptionalcoactivatorProtein kinaseProtein kinaseCAMP relatedtranscription factorsDifferentiation inhibitorNtype calcium channelsin nervous systemProtein kinaseNakagawa Ito Ito Maturana Elbediwy Kuroda Maturana Maturana Ito Lasorella and Iavarone Nakatani et alMaenoHikichi et alYan Liu Kidney anion exchanger Su Fanning and Anderson Sheng and Sala The PDZdomain provides a proteinbinding interface that facilitates theformation of multiprotein complexes with a variety of partnersincluding membraneassociated proteins cytoplasmic signalingproteins and cytoskeleton proteins Jelen Krcmery Zheng Ito The LIM domainis approximately amino acids long and is characterized byhighly conserved and spatially deï¬ned cysteine and histidineresidues that coordinate the binding of two zinc ions to formtwo zinc ï¬ngerlike structures LIM domains can exist in proteinsalone or in combination with other domains Dawid Bach Kadrmas and Beckerle Te Velthuisand Bagowski The LIM domains can combine highlydiverse partners ranging from signaling molecules and actincytoskeletal components to transcription factors and it alsosupport cellular functions Dawid In particularthe crosslinking with actin cytoskeleton such as LIM domainprotein can maintain the functional structure of cardiomyocytesby a mechanism involving its own binding and actin ï¬lamentcrosslinking which plays an important role in the developmentof heart disease Hoï¬mann In addition LIM domainproteins have also been reported to be involved in the invasionand metastasis of cancer as components and targets of thecytoskeleton Hoï¬mann exhibitThe PDLIM5 splicing isoformstissuespeciï¬cexpression the long isoforms are widely expressed in varioustissues while the short isoforms are only highly expressedin cardiac and skeletal muscle Yamazaki Thisdiï¬erential expression of PDLIM5 may be related to theirdiï¬erent roles in diï¬erent tissues and an systems Table Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5TABLE List of different disease involved in PDLIM5Disease and developmentReferencesRelated signalingpathwaysRASERKAMPKPKCMicroRNA17¼Papillary thyroid carcinomaProstate cancerWei Koutros Liu Li Gastric cancernonsmall cell carcinomaEdlund Cancer associated with steroid use Wang Heart developmentCardiomyocyte hypertrophyTGFSmadPulmonary hypertensionDilated CardiomyopathyBipolar disorderDepressive disorderSchizophreniaAlcohol dependence type diabetes and hypertensionNakagawa Yamazaki Bang Chen Cheng Cheng Zhao Liu Numata Owusu THE DIFFERENTIAL ROLES OF PDLIM5IN VARIOUS AN SYSTEMSThe Role of PDLIM5 in the NervousSystemPDZ and LIM domain is widely expressed in diï¬erent regionsof the brain such as the hippocampus thalamus hypothalamuscerebral cortex and amygdala MaenoHikichi Incentral neurons PDLIM5 is mainly localized in the membraneand cytoplasm where it regulates neuronal calcium signaling andcolocalizes with neurotransmitterprotruding vesicles Numata these observations indicate that PDLIM5 playsa role in brain development Some studies have shown thatthe expression of PDLIM5 is associated with multiple mentaldisorders such as bipolar disorder major depression andsusceptibility to schizophrenia Liu Zhao Herrick Wang In the nervous system PDLIM5 plays an important rolein the formation of nerve growth cones and promotes thediï¬erentiation of nerve cells Some studies have shownthat PDLIM5 expression is upregulated during neuraldiï¬erentiation and it has been shown that its ectopic expressionin neuroblastoma cells leads to the translocation of ID2 whichis one of the four members of the ID protein family called adiï¬erentiation inhibitor from the nucleus to the cytoplasmresulting in the inactivation of transcriptional and cellcyclepromoting functions ofthe latter Lasorella and Iavarone Furthermore PDLIM5 can form large complexes withPKC and Ntype Ca2 channels to promote the regulationof Ntype calcium channel activity MaenoHikichi Ren showed that PDLIM5 and PKCεcoexist in the nerve growth cone Through interaction withαactinin PDLIM5 may be involved in regulating microï¬lamentremodeling in neurons and the formation of the PDLIM5PKCεcomplex in the nerve growth cone which acts as a scaï¬old tomediate PKCε translocation to the membrane during PMAinduced growth cone collapse It is suggested that PDLIM5participates in a variety of functions of the nervous systemas well as in a signaling pathway involving the sequestrationof the transcription factor ID2 in the cytoplasm Howeverthe precise mechanisms by which PDLIM5 regulates thefunctions of the nervous system via ID2 blockade requiresfurther elucidationPDLIM5 in the Heart and Skeletal MusclePhysiological Roles of PDLIM5 in the HeartThe heart undergoes development and begins to function in theearly stages of embryonic development PDLIM5 is expressedat high levels in the skeletal muscle and myocardium and isconsidered to be a heart and skeletalmusclespeciï¬c scaï¬oldprotein to regulates mouse heart development Mu PDLIM5 is capable of binding to αactinin throughthe PDZ domain and colocalizing in the zdisk region ofcardiomyocytesindicating that this protein plays a role incardiac development Studies have shown that PDLIM5 mRNAsare mainly expressed in the heart and skeletal muscle of adultrats and that PDLIM5 acts as a scaï¬old protein to mediatethe transmission of PKC signals in cardiomyocytes playing animportant role in development of the muscle cell in an earlydevelopmental stage Nakagawa Zheng The PDZLIM protein family is highly expressed in the striatedmuscle PDLIM5 is similar to other PDZLIM members in thestriated muscle in which the PDZ domain binds to αactininwhile the LIM domain binds to several protein kinases C andprotein kinase D Kuroda Nakagawa For example PDLIM5 traditionally activates the PKC throughthe direct binding of its LIM domain Maturana and interacts to PKA Lin Transcription factorCREB which is one of the ï¬rst transcription factors activated byneurohumoral factors stimulation is a transcription factor cAMPresponse element binding protein is a known target of the PKCand protein kinase D1 PKD1 pathways Thonberg Ozgen The interaction between PDLIM5 isoform and CREB is necessary for the phosphorylation of CREB at theaminoacid residue Ser133 which promotes the transcriptionalactivation and nuclear localization of CREB the phosphorylatedCREB enters the cardiomyocyte nucleus to play the role oftranscription factor and promote the growth of cardiomyocytesIto Moreover in neonatal rat cardiomyocytesPDLIM5 interacts with PKD1 through its LIM domain and formscomplexes with PKD1 and cardiac Ltype voltagegated calciumchannelα1C subunits to regulate the activity of Ltype voltagegated calcium channels Maturana Although theformation of protein complexes such as PDLIM5PKCPKD1is well understood the downstream molecular events remainto be elucidatedThese results suggest that the localization of PDLIM5 at somesubcellular sites and its ability to interact with multiple functionalproteins play an important role in cellular and physiologicalfunctions Furthermore the role of PDLIM5 in the heart wasFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5studied using a heartspeciï¬c PDLIM5knockout mouse modelIt was found that the ablation of PDLIM5 disrupted the stabilityof the PDLIM5CiphersCalsarcin complex formed in the zdiskregion thus interfering with the connection between adjacentsarcomeres and extracellular matrix These eï¬ects were found toresult in the loss of optimal force transmission and a signiï¬cantdecrease in cardiac shortening fractionleading to dilatedcardiomyopathy Cheng Novel polymorphisms inthe PDLIM5 gene encoding the Zline protein have also beenshown to increase the risk of idiopathic dilated cardiomyopathyWang that underpinsPDZ and LIM domain is additionally involved in skeletalmuscle development Myogenesis is an important biologicalprocessskeletal muscle regeneration andpostnatal growth The silencing of PDLIM5 increases the nuclearaccumulation of diï¬erentiation inhibitor Id2 which inhibits theproliferation and diï¬erentiation of myoblasts Nakatani Qiu In addition the diï¬erentiation of andmorphological changes in skeletal muscle is regulated by a groupof transcription factors known as myogenic regulators PDLIM5isoform overexpression leads to the upregulation of MyoDand myogenin while PDLIM5 isoform knockout signiï¬cantlydecreases the expression of these two proteins these ï¬ndingsindicate that the main eï¬ect of PDLIM5 isoform on musclecells is to stimulate the transcription of MyoD andor myogeninencoding genes Ito Although the main role of PDLIM5 is as a speciï¬c scaï¬oldprotein which to bridge the connection between cytoskeletonand membrane proteins and promote the formation of proteincomplexes it is capable of generating numerous splicing isoformsENH24 that exert various eï¬ects on the development ofheart and skeletal muscle In vitro PDLIM5 isoform hasbeen shown to promote the expression of myogenic genes andmyotube formation while the short PDLIM5 isoform hasbeen found to abrogate myotubelike morphological changeswithout altering the expression of the myogenic transcriptionfactors MyoD and myogenin Ito Furthermorethe overexpression of PDLIM5 isoform prevented ventricularcardiomyocyte hypertrophy induced by vascular stress hormonesYamazaki Western blotting analysis of muscle tissueusing a nonisoformspeciï¬c antiPDLIM5 antibody showed thatPDLIM5 isoform is only expressed in skeletal muscle witha speciï¬c distribution of PDLIM5 members between skeletalmuscle and myocardium Niederlander These results suggest that PDLIM5 plays a key role in thedevelopment of the myocardium and skeletal muscle Howeverthe signal transduction mechanisms underlying the role ofPDLIM5 in heart and skeletal muscle remain to be furtherstudied For example the speciï¬c molecular mechanisms bywhich PDLIM5 regulates the development of myocardium andskeletal muscle through binding protein kinases are unknownFurthermore the mechanisms by which PDLIM5 sequestersnuclear protein Id2 in the cytoplasm remain to be elucidatedThe Role of PDLIM5 in Cardiovascular DiseasesPDZ and LIM domain is mainly distributed on the zline ofthe sarcomere of cardiomyocytes therefore the eï¬ect of PDLIM5on myocytes may be related to the contractile function of thesecells PDLIM5knockout mice exhibit dilated cardiomyopathywhich is characterized by thinning of the left ventricular wallenlargement of the left ventricular cavityimpaired cardiaccontraction and reduced ejection function Cheng PDLIM5 can regulates vascular remodeling which canas a new proatherosclerotic factor to be a therapeuticallytargeted Huang Cardiac remodeling which isindicative of progression in many cardiovascular diseases ischaracterized by cardiomyocyte hypertrophy and myocardialï¬brosis which lead to heartfailure Swynghedauw Barry and Townsend microRNA miR21 derivedfrom cardiac ï¬broblasts exosomes is a strong paracrine RNAmolecule that induces cardiomyocyte hypertrophy Thum Recentlyit has been reported that PDLIM5 is thedirect target of miR17¼ Bang Chen By acting on its target gene PDLIM5 miR participates in the interaction between cardiac ï¬broblastsand cardiomyocytesthus inducing myocardial pathologicalhypertrophy Bang PDLIM5 also plays a role invascular smooth muscle AMPactivated protein kinase is anintracellular energy receptor of AMPK which is activatedunder hypoxia ischemia glucose loss and stress Steinberg andKemp AMPK is generally considered to be an energysensor kinase and requires AMP for its activation Carling Nakano reported that AMPK controls microtubuledynamics by phosphorylating cytoplasmic connexin170 CLIPthus regulating cell migration Nakano In addition AMPK is involved in the regulation of actincytoskeleton dynamics and plasma membrane reanizationBae Kondratowicz Studies haveshown that AMPK activation plays an important role inneovascularization and metastasis HoyerHansen and Jaattela In vascular smooth muscle cells AMPK phosphorylatesPDLIM5 at Ser177inhibiting the downstream Rac1Arp23signaling pathway to mediate cell migration Yan In pulmonary artery smooth muscle cells PASMCsSMCspeciï¬c knockout of PDLIM5 enhances hypoxiamediatedvascular remodeling while overexpression of PDLIM5 inhibitsthe TGFSmad signal pathway and prevents hypoxiainducedpulmonary hypertension elevation in vivo Cheng In addition PDLIM5 silencing induces the activity of TGF3 TR1 and TGF and increases the overall expressionlevel of Smad2 The suppression of PDLIM5 has beenfound to enhance the nuclear staining of Samd23 Chen indicating that PDLIM5 participates in thedevelopment of cardiovascular disease by negatively regulatingthe TGF3Smad23 signal pathway Additionally demonstratedthat PDLIM5 plays an important role in the cardiovascularsystem through miRmediated regulation of the phenotype ofpulmonary artery smooth muscle cells miR17¼ regulatesthe diï¬erentiation of PASMCs through its target PDLIM5indicating that the miR17¼92PDLIM5TGFSmad pathwayis essential for vascular remodeling during the developmentof pulmonary hypertension PDLIM5 therefore represents apromising therapeutic target for future cardiovascular drugdiscovery eï¬ortsFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5The Role of PDLIM5 in TumorAs an actin adaptor protein PDLIM5 is not only involved incytoskeletal tissue cellular processes and an development butis also considered to play roles in tumorigenesis and developmentEeckhoute Edlund Heiliger Li PDLIM5 is expressed in many cancer celllines In a study of neurologic tumor it was found that thetranscription factor ID2 binds to the PDLIM5 LIM domains andin these cancer cells high levels of PDLIM5 sequester ID2 in thecytoplasm preventing neuronal diï¬erentiation and promotingcell proliferation Lasorella and Iavarone PDZ and LIM domain is additionally upregulated inpapillary thyroid carcinoma PTC tissues elevated PDLIM5expression promotes the migration invasion and proliferation ofPTC cells by activating the RASERK pathway Wei PDLIM5 may therefore serve as a therapeutic target in a varietyof cancers It can promote the invasion and metastasis of cancercells Genotyping chip detection experiments have shown thatPDLIM5 is overexpressed in prostate cancer tissue Koutros and some studies have proved that the utility of serumand urine PDLIM5 levels as indicators for auxiliary diagnosisof prostate cancer with potential value in predicting the risk ofprogression in advanced prostate cancer PCA Ma PDLIM5 plays a key role in regulating the proliferation invasionand migration of malignant tumor cells by binding to AMPKand regulating its activation and degradation Liu PDLIM5 may therefore act as an oncogene in the developmentand progression of PCAIn view of the important role of PDLIM5 in cancer somestudies have indicated that it is involved in the growth of gastriccancer cells and suggested that PDLIM5 silencing through theuse of small interfering RNAs siRNA may be a potentialstrategy for the treatment of gastric cancer Li In addition Edlund found that the increased expressionof PDLIM5 is related to high tumor proliferation rates innonsmall cell carcinoma Edlund Steroids suchas corticosteroid medications play an important role in thedevelopment of cancer it has been reported that singlenucleotide polymorphisms SNPs in PDLIM5 interact withsteroids thus aï¬ecting the occurrence and development of cancerWang The above ï¬ndings show that PDLIM5 plays an important rolein the occurrence and development of cancer and that PDLIM5represents a candidate oncogene in various cancersThe Role of PDLIM5 in Other DiseasesreportedIn addition to playing a role in the diseasesinvolved in the link between alcoholabove PDLIM5 isdependence and diabetes Owusu found thatPDLIM5 gene polymorphism is associated alcoholdependentAD type diabetes T2D and hypertension and Severalgenetic variants of the PDLIM5 gene can aï¬ect AD T2Dand hypertension indicating that PDLIM5 is a shared geneamong the three diseases Therefore elucidation oftheunderlying molecular mechanisms and identiï¬cation of hithertoundiscovered molecular functions of PDLIM5 are expectedto enable the development of eï¬ective clinicalfor these diseasestherapiesIn addition PDLIM5 plays a role in the formation of cellextensions Being a scaï¬old protein PDLIM5 is involved inpromoting the activity of microï¬lamentassociated proteinsMicroï¬lamentassociated proteins play a central role in theprocess of cell extension Lanier Some studieshave found that PDLIM5 recruitment to cell extensions and isnecessary to form these extensions and that PDLIM5 knockoutreduces the assembly of actin ï¬laments in cell extensionsYuda THE RELATIONSHIP BETWEEN PDLIM5AND INTEGRINS AND ITS POTENTIALROLE IN THE REGULATION OF STEMCELL DIFFERENTIATIONSome studies have shown that the PDZ domain of PDZLIMprotein binds to αactinin protein at the adhesion junctionwhich is the site of integrin localization Xia Pomies Vallenius Tamura The functional interaction with integrin indicates that PDZLIM protein can participate in the adhesion signal cascadewhich transmits extracellular signals via intracellular regulatorypathwaysthereby modifying the actin cytoskeleton Theseï¬ndings show that the PDZLIM protein plays an overall role incellcell and cellmatrix interaction and cell migration Krcmery The regulation of PDZLIM activity plays animportant role in preventing uncontrolled actin recombinationproliferation and cell migration For example PDLIM5 canalso bind to the integrin protein kinase ILK acting as ascaï¬old bridge between renal ion exchanger KAE1 and ILKproviding a bridge between KAE1 and potential microï¬lamentsSu It has been reported that PDLIM5 is recruited from thecytoplasm to the integrin adhesion and Factin stress ï¬bersand responds to stress by directly binding to the key stressï¬ber component αactinin Microï¬laments control the nuclearand cytoplasmic localization oftranscriptional coactivatorsYAP and TAZ to regulate gene expression and mediate thediï¬erentiation of MSCs Dupont Halder The eï¬ective domains of some proteins that are recruited intothe actin structure in a forcedependent manner through theLIM domain regulate actin signal transduction Smith The action of mechanical force on integrin results in therecruitment of PDLIM5 to activate the YAP pathway duringmechanical transduction Elbediwy PDLIM5 acomponents of the integrin adhesion complex mediates therelationship between integrin and the cytoskeleton Horton et al2016ab Proteomic analysis of integrinrelated complexes fromMSCs has demonstrated the formation of signiï¬cant amountsof a vinculinpositive adhesion complex on a hard substratecoated with ï¬bronectin FN in MSCs a subset of whichcolocalized with or closely to clusters of PDLIM1 or PDLIM5Ajeian Frontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5PDZ and LIM domain undergoes tensiondependentrelocalization in cells Both embryonic and induced pluripotentstem cells can diï¬erentiate into derivatives of the third germlayer as a result human pluripotent cells are important toolsin regenerative medicine Thomson Takahashiand Yamanaka Studies have reported that duringcardiogenesis embryonic stem cells exhibit dramatic changes inthe expression of metabolic enzymes and cytoskeleton proteinsKonze in particular Zdisk related proteins withPDZ and LIM domain proteins including PDLIM5 are upregulated during cardiogenesisFurthermorethe expression of NKX25 an importantmyocardial transcription factor results in the generation ofspeciï¬c PDLIM5 splicing variants during the early developmentof cardiomyocytes which in turn aï¬ectsthe myogenicdiï¬erentiation of myocardium Konze At presentthere are few studies on PDLIM5 in stem cells elucidation of itsmechanism in diï¬erent stem cell types is warranted to identify itsfunctions in these cellsPROBLEMS AND PERSPECTIVESIn this we brieï¬y reviewed the known functionsof the PDLIM5 protein the progress in elucidation ofitsroles in various cellular and physiological processes and thesignaling pathways in which it participates As a member ofthe PDZLIM protein family PDLIM5 is involved in actinbinding αactinin binding protein kinase binding acts asa scaï¬old bridge between connective proteins and plays anindispensable role in various cellular processes However so farthe speciï¬c molecular mechanisms underlying the functions ofPDLIM5 remain unclearFuture research directions in investigation of PDLIM5 shouldseek to answer the following questionsFirst research on PDLIM5 has mainly focused on its roles intumor the nervous system and the cardiovascular system As amicroï¬lamentassociated protein does PDLIM5 play the samerole in other physiological systems or cell lines Is the PDLIM5gene expressed in multiple systemsREFERENCESAjeian J N Horton E R Astudillo P Byron A Askari J A and MillonFrmillon A Proteomic analysis of integrinassociated complexes frommesenchymal stem cells Proteomics Clin Appl 101002prcaBach I The LIM domain regulation by association Mech Dev 101016S0925477399003147Bae H B Zmijewski J W Deshane J S Tadie J M Chaplin D D andTakashima S AMPactivated protein kinase enhances the phagocyticability of macrophages and neutrophils FASEB J fj11190587BagheriYarmand R Mazumdar A Sahin A A and Kumar R LIMkinase increases tumor metastasis of human breast cancer cellsvia regulationof the urokinasetype plasminogen activator system Int J Cancer 101002ijc21650Bang C Batkai S Dangwal S Gupta S K Foinquinos A and HolzmannA Cardiac ï¬broblastderived microRNA passenger strandenrichedSecond although the role of PDLIM5 in diseases hasbeen studied eg its expression is upregulated during tumordevelopment the speciï¬c mechanisms by which it exerts theseeï¬ects are unknown and further elucidation of the underlyingmechanisms and other functions is warrantedThird PDLIM5 has four splicing isoforms which performdiï¬erent functions what are the mechanisms by which they playdiï¬erent roles Are these diï¬erences in their roles attributable tothe presence or absence of LIM domains Additionally what isthe mechanism by which they play diï¬erent rolesFourth in regard to strategies used for the inhibition ofPDLIM5 expression only viral transfection has been reportedto date no pharmaceutical compounds that inhibit PDLIM5expression have been identiï¬ed Therefore additional research onPDLIM5 inhibitors is also criticalFinally although PDLIM5 plays a crucial role in binding actinand has attracted much attention as a connecting protein thereare few studies on its eï¬ects on the actin cytoskeleton or othercytoskeleton such as binding to cytoskeletonrelated proteinsbridging the connection with the cytoskeleton Does PDLIM5aï¬ect the shape and location of the cytoskeleton in the processof participating in the biological functions of cellTheeï¬ects of PDLIM5mediated modulation ofthecytoskeleton on cell diï¬erentiation proliferation and othercellular functions remain to be explored in detail in future studiesAUTHOR CONTRIBUTIONSAll authors participated in the conception and writing of themanuscript SZ JO and JD reviewed and suggested modiï¬cationsto the content JD designed the structure of the reviewFUNDINGThis work wassupported by the National Key RDProgram of China grant number 2017YFC1105000 andthe Sanming Project of Medicinein Shenzhen grantnumber SZSM201612019exosomes mediate cardiomyocyte hypertrophy J Clin Invest 101172JCI70577Barry S P and Townsend P A What causes a broken heartmolecularinsights into heart failure Int Rev Cell Mol Biol S1937644810840031Carling D Mayer F V Sanders M J and Gamblin S J AMPactivatedprotein kinase natures energy sensor Nat Chem Biol nchembio610Chen T HuangJ B DaiJ Zhou Q RajJ U and Zhou Gthe miR17¼ signaling that PAI1 is a novel component ofregulates pulmonary artery smooth muscle cell phenotypes Am J PhysiolLung Cell Mol Physiol L149L161 101152ajplung00137Chen T Zhou G Zhou Q Tang H Ibe J C and Cheng H Loss of microRNA17 approximately in smooth muscle cells attenuatesexperimental pulmonary hypertension via induction of PDZ and LIM domain Am J Respir Crit Care Med 101164rccm2014050941OCFrontiers in Physiology wwwfrontiersinAugust Volume 0cHuang et alAn Overview of PDLIM5Cheng H Chen T Tor M Park D Zhou Q and Huang J B A highthroughput screening platform targeting PDLIM5 for pulmonary hypertensionJ Biomol Screen Cheng H Kimura K Peter A K Cui L Ouyang K and Shen T Lossof enigma homolog protein results in dilated cardiomyopathy Circ Res 101161CIRCRESAHA110218735Dawid I B Breen J J and Toyama R LIM domains multiple roles asadapters and functional modiï¬ers in protein interactions Trends Genet 101016s0168952598014243Dupont S Morsut L Aragona M Enzo E Giulitti S and Cordenonsi M Role of YAPTAZ in mechanotransduction Nature 101038nature10137Edlund K Lindskog C Saito A Berglund A Pontn F and G¶ranssonKultima H CD99 is a novel prognostic stromal marker in nonsmallcell lung cancer Int J Cancer 101002ijc27518Eeckhoute J A celltypespeciï¬c transcriptional network required forestrogen regulation of cyclin D1 and cell cycle progression in breast cancerGene Dev 101101gad1446006Elbediwy A Vanyai H DiazdelaLoza M Frith D Snijders A P andThompson B J Enigma proteins regulate YAP mechanotransductionJ Cell Sci 131jcs221788 101242jcs221788Fanning A S and Anderson J M PDZ domains fundamental buildingblocks in the anization of protein compl | 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 |
"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjonThis 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 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partners occupation and economic statusPatients occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatients marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patients hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patients family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet 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 Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0c" | Thyroid_Cancer |
Adjunctive Therapy to Achieve Preoperative Euthyroidism in Graves Disease A Case Report Authors Contribution Study Design A Data Collection B Statistical Analysis C Data Interpretation D Manuscript Preparation E Literature Search F Funds Collection G ABDEF Noor Abdulghani AlghanimABDEF Shymaa M AlkahtaniAEF Fatimah S AssariAEF Sarah W AlnosaierAEF Reham M BaderAEF ABEF Mariam M HendazAEF Amal AlhefdhiIsra E Elmahi Corresponding Author Conflict of interest Noor Abdulghani Alghanim email nalghanimalfaisaleduNone declared College of Medicine Alfaisal University Riyadh Saudi Arabia Breast and Endocrine Surgery King Faisal Specialist Hospital and Research Center Riyadh Saudi ArabiaPatient Final Diagnosis Symptoms Medication Clinical Procedure Specialty Male 37yearoldGraves diseaseDifficulty breathing ¢ voice change ¢ weight gainTotal thyroidectomySurgery Objective Background Case Report Conclusions Unusual clinical courseGraves disease is an autoimmune disease of the thyroid gland and it is considered the most common cause of hyperthyroidism It is characterized by particular eye manifestations skin changes and pretibial myxedema in addition to the signs and symptoms of hyperthyroidism Graves disease can be diagnosed based on clinical presentation and low thyroid stimulating hormone TSH and elevated free T4 FT4 levels Presence of TSH receptor antibody TRAb in the serum confirms the diagnosis of Graves disease Imaging studies like radioactive iodine scan will show a high and diffuse uptake Graves disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcomeThis is a case of a 37yearold Saudi male known to have Graves disease for years who presented to the endocrine surgery clinic with neck swelling difficulty breathing and change in voice After multiple attempts to control his fluctuating thyroid levels the team eventually managed to achieve a euthyroid state in the patient with the addition of saturated solution of potassium iodide SSKI and thus rendering him eligible for urgent surgeryWe report this case to show that SSKI can be used as adjunctive therapy to achieve a preoperative euthyroid state in refractory Graves disease MeSH Keywords Graves Disease ¢ Hyperthyroidism ¢ Hypothyroidism Fulltext PDF wwwamjcaserepcomindexidArt923342 e9233421Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves disease¦ Am J Case Rep e923342Figure Typical eye manifestations of Graves disease proptosis and periorbital edemaFigure Ultrasound showing an enlarged and hypervascular left thyroid lobe with no suspicious nodulesof the examination was unremarkable except for right scrotal swelling and delayed deep tendon reflexesThe patient then underwent ultrasound US of the thyroid which showed an enlarged and hypervascular gland compatible with Graves disease with no suspicious nodules Figure A computed tomography CT scan was done which revealed homogeneous diffuse swelling of bilateral thyroid lobes with no retrosternal extension along with bilateral proptosis Figures Therefore the patient was admitted to achieve a euthyroid state before proceeding for a total thyroidectomy The patient was managed by a multidisciplinary team with the goal of clearing him for surgery During the patients 3week hospital stay the dosage of methimazole was continuously altered because serial thyroid function tests showed a change from hyperthyroid to hypothyroid status His TSH and FT4 levels ranged from mUL to mUL and pmolL to pmolL respectively A few days after administration of saturated solution of potassium iodide SSKI was initiated drops three times daily the patient achieved a euthyroid state with TSH mUL and FT4 pmolL so urgent surgery was performed Intraoperatively the patients thyroid gland was found to be enlarged and vascular with each lobe measuring approximately to cm The gland was excised bilaterally along with the pyramidal lobe because it was also enlarged The postoperative BackgroundGraves disease is an autoimmune disease affecting the thyroid gland [] It is characterized by presence of autoantibodies that target thyroid stimulating hormone TSH receptors causing stimulation of the thyroid gland [] Patients with Graves disease usually present with signs and symptoms of hyperthyroidism that include fatigue heat intolerance sweating weight loss palpitations and tremor along with particular eye manifestations and sometimes skin changes [] It is considered the most common cause of hyperthyroidism accounting for approximately to of cases [] The diagnosis of Graves disease can be straightforward in the presence of typical signs and symptoms along with low thyroid stimulating hormone TSH and elevated free T4 FT4 levels [] Measuring TSH receptor antibody TRAb is helpful for confirming the diagnosis as it is present in of patients If the cause of hyperthyroidism remains uncertain a radioactive iodine uptake scan should be considered The scan helps to distinguish Graves disease from thyroiditis and other causes of hyperthyroidism In Graves disease iodine uptake is increased and diffuse [] Treatments of choice for hyperthyroidism include antithyroid medications radioactive iodine and surgical approaches [] The success rate for antithyroid medications is almost compared to and with radioactive iodine and surgery respectively [] Whenever surgery is indicated careful preoperative management to achieve euthyroidism is needed to optimize the surgical outcome In most cases a euthyroid state is reached within a few weeks of conventional antithyroid medications however in certain conditions as in drug malabsorption and in cases of predominantly high T3 levels it cannot be easily achieved and adjunctive therapy should be considered []Case ReportA 37yearold Saudi male presented to the endocrine clinic with palpitation sweating and weight loss He was diagnosed with Graves disease and treated with methimazole mg orally twice daily When symptoms of hypothyroidism developed the dose was decreased to mg orally twice daily The patient was referred to the endocrine surgery clinic complaining of obstructive symptoms in the form of difficulty breathing and voice changes due to neck swelling weight gain of kg during the last month and easy fatigability along with the typical eye manifestations of proptosis and periorbital edema Figure He was otherwise healthy and the rest of his history was unremarkable On physical examination the patient had a hoarse voice fine tremor in both hands and his skin was warm with diaphoresis There was proptosis lid retraction and diplopia involving both eyes Neck examination showed a diffuse tender swelling with bilateral lumps measuring cm on the right and cm on the left along with positive Pemberton sign The rest e9233422Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves disease¦ Am J Case Rep e923342Figure CT scan showing diffuse enlargement of the thyroid with no retrosternal extension or invasion of surrounding structurespathology report showed diffuse hyperplasia consistent with Graves disease with no evidence of malignancy After the surgery the patient was moved to the Intensive Care Unit ICU where he was assessed and found to be stable with no signs and symptoms of thyrotoxicosis or hypocalcemia Three days later the patient was discharged with orders to take calcium carbonate mg orally three times daily for days acetaminophen mg orally as needed for days levothyroxine mcg orally daily for days and calcitriol mcg orally daily for daysWhen the patient presented to the clinic weeks later for followup he was found to be in good health with no active complaints He had lost weight and there were no voice changes His eye manifestations had decreased but not disappeared completely Laboratory results showed a euthyroid state with a normal calcium levelDiscussionFigure CT scan demonstrating that the distance from the anterior margin of the globe to the interzygomatic line exceeds mm indicating significant bilateral proptosisfor antithyroid medications is almost compared to with radioactive iodine therapy [] Surgical approaches are considered the most successful and definitive treatment with total thyroidectomy being the preferred choice [] A review of the literature done in showed that total thyroidectomy is times more successful than radioactive iodine therapy [] Another study concluded that the highest rates of longterm remission reaching up to are achieved with surgery [] Nonetheless there is no clear consensus on the best treatment modality for Graves disease and the choice should be individualized Choice of modality depends on several factors including age comorbidities size of the goiter and severity of thyrotoxicosis [] Surgery is recommended in certain conditions for example in patients with compression symptoms due to presence of a large goiter those with low radioactive iodine uptake suspected thyroid cancer moderate to severe Graves ophthalmopathy and patients who cannot tolerate antithyroid medications [] Whenever surgery is selected careful preoperative management is needed to optimize the surgical outcome Preparing a patient with antithyroid medications is recommended by the American Thyroid Association ATA to achieve a euthyroid state and thus lower risk of intraoperative complications []Graves disease can be managed with three different treatment modalities antithyroid medications radioactive iodine or surgical removal of the thyroid gland [] The success rate In most cases a euthyroid state is achieved within weeks of antithyroid treatment In certain conditions however that is difficult to achieve with conventional therapy and patients e9233423Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves disease¦ Am J Case Rep e923342Moreover the largest case series of patients with severe thyrotoxic Graves disease was published in The study involved patients who reached euthyroidism after days of an intensive treatment regimen The authors concluded that patients with severe hyperthyroid Graves disease can rapidly achieve preoperative euthyroidism with simultaneous administration of iopanoic acid dexamethasone betablocker and methimazole or propylthiouracil [] Another case of Graves disease resistant to antithyroid medications was reported in The patient was promptly managed preoperatively with both iopanoic acid and dexamethasone []Three scientific papers on resistant thyrotoxicosis due to Graves disease were published between to In all cases a euthyroid state could not be reached with the usual antithyroid medications and the patients received prednisolone andor lithium which resulted in complete normalization of thyroid function before surgery [] Furthermore several refractory cases of Graves disease unresponsive to usual preoperative management were reported in and The patients were successfully prepared for surgery with use of plasmapheresis []ConclusionsPreoperative management of Graves disease can sometimes be challenging There have been many attempts to achieve a euthyroid state with different approaches In the patient described here Graves disease was resistant to conventional antithyroid medication for establishment of preoperative euthyroidism Our experience demonstrates that SSKI can be used in a case like ours to not only decrease vascularity of the thyroid gland but also as adjunctive therapy to achieve preoperative euthyroidismshould be prepared for surgery using adjunctive therapy [] Resistance to conventional antithyroid medications is not commonly encountered in clinical practice however there are few reported cases addressing the use of adjunctive therapy to rapidly restore normal thyroid function [] SSKI has been used for many years in management of Graves disease [] ATA hyperthyroidism management guidelines recommend preoperative administration of potassium iodide solutions KI for thyroidectomy [] The main rationale of using KI preoperatively is to decrease vascularity and blood loss during the surgery however a few studies suggest that when combined with antithyroid medications it can decrease thyroid hormone levels [] In a retrospective study showed the effectiveness of adding KI as a rescue preoperative management in uncontrolled Graves disease In patients in the study use of KI was safe and effective as preoperative preparation for total thyroidectomy []Cholestyramine was first used to treat Graves disease in in Korea [] The patient in that study was a 22yearold female with severe refractory Graves disease who was initially managed with a maximal dose of methimazole and propranolol with no improvement She was admitted and treated with methimazole propranolol hydrocortisone and KI The next day cholestyramine was added which resulted in a rapid decline of FT4 Ten days after admission the patient underwent total thyroidectomy [] Several cases of refractory Graves disease were reported in the literature between to In all these cases the patients failed to achieve a preoperative euthyroid state with conventional antithyroid medications Within to weeks of administration of cholestyramine as adjunctive therapy they became euthyroid Two studies were published in and to evaluate the effectiveness of adding cholestyramine to the conventional treatment regimen in cases of resistant Graves disease The conclusion from these reports is that cholestyramine can be used to safely and rapidly achieve preoperative euthyroidism []References Pokhrel B Bhusal K Graves disease In StatPearls Treasure Island FL StatPearls Publishing Barbesino G Tomer Y Clinical review Clinical utility of TSH receptor antibodies J Clin Endocrinol Metab DeGroot LJ Graves disease and the manifestations of thyrotoxicosis In Feingold KR Anawalt B Boyce A eds Endotext South Dartmouth MA MDTextcom Inc Subekti I Pramono LA Current diagnosis and management of Graves disease Acta Med Indones Girgis CM Champion BL Wall JR Current concepts in Graves disease Ther Adv Endocrinol Metab Wiersinga WM Graves disease Can it be cured Endocrinol Metab Seoul Wong KK Shulkin BL Gross MD Avram AM Efficacy of radioactive iodine treatment of Graves hyperthyroidism using a single calculated I dose Clin Diabetes Endocrinol Piantanida E Preoperative management in patients with Graves disease Gland Surg Yang Y Hwang S Kim M Refractory Graves disease successfully cured by adjunctive cholestyramine and subsequent total thyroidectomy Endocrinol Metab Seoul Genovese BM Noureldine SI Gleeson EM What is the best definitive treatment for Graves disease A systematic review of the existing literature Ann Surg Oncol Bartalena L Diagnosis and management of Graves disease A global overview Nat Rev Endocrinol SebastianOchoa A QuesadaCharneco M FernandezGarcia D Dramatic response to cholestyramine in a patient with Graves disease resistant to conventional therapy Thyroid Calissendorff J Falhammar H Lugols solution and other iodide preparations Perspectives and research directions in Graves disease Endocrine e9233424Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0cAbdulghani Alghanim N Use of adjunctive therapy to achieve preoperative euthyroidism in Graves disease¦ Am J Case Rep e923342 Naafs MA Lugols solution in thyroid surgery A minireview Global Journal of Otolaryngology Muldoon BT Mai VQ Burch HB Management of Graves disease An overview and comparison of clinical practice guidelines with actual practice trends Endocrinol Metab Clin North Am Burch HB Cooper DS Management of Graves disease A review [published erratum appears in JAMA ] JAMA Calissendorff J Falhammar H Rescue preoperative treatment with Lugols solution in uncontrolled Graves disease Endocr Connect Chae SB Kim ES Lee YI Min BR A case of methimazoleresistant severe Graves disease Dramatic response to cholestyramine Int J Thyroidol Kadem SG Resistant hyperthyroidism responses dramatically to adjunctive oral cholestyramine Jourbnal of Diabetes and Endorinology Mercado M MendozaZubieta V BautistaOsorio R EspinozaDe Los Monteros AL Treatment of hyperthyroidism with a combination of methimazole and cholestyramine J Clin Endocrinol Metab Tsai WC Pei D Wang TF The effect of combination therapy with propylthiouracil and cholestyramine in the treatment of Graves hyperthyroidism Clin Endocrinol Oxf Panzer C Beazley R Braverman L Rapid preoperative preparation for severe hyperthyroid Graves disease J Clin Endocrinol Metab Pandey CK Raza M Dhiraaj S Rapid preparation of severe uncontrolled thyrotoxicosis due to Graves disease with Iopanoic acid a case report Can J Anaesth Saleem T Sheikh A Masood Q Resistant thyrotoxicosis in a patient with Graves disease A case report J Thyroid Res Nair GC C Babu MJ Menon R Jacob P Preoperative preparation of hyperthyroidism for thyroidectomy role of supersaturated iodine and lithium carbonate Indian J Endocrinol Metab Jude EB Dale J Kumar S Dodson PM Treatment of thyrotoxicosis resistant to carbimazole with corticosteroids Postgrad Med J Candoni A De Marchi F Vescini F Graves disease thyrotoxicosis and propylthiouracil related agranulocytosis successfully treated with therapeutic plasma exchange and GCSF followed by total thyroidectomy Mediterr J Hematol Infect Dis Ezer A Caliskan K Parlakgumus A Preoperative therapeutic plasma exchange in patients with thyrotoxicosis J Clin Apher e9233425Indexed in [PMC] [PubMed] [Emerging Sources Citation Index ESCI][Web of Science by Clarivate]This work is licensed under Creative Common AttributionNonCommercialNoDerivatives International CC BYNCND 0c' | 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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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 |
Signaling pathway analysis methods are commonly used to explainbiological behaviors of disease cells Effector genes typically decide functionalattributes associated with biological behaviors of disease cells by abnormal signalsthey received The signals that the effector genes receive can be quite different innormal vs disease conditions However most of current signaling pathway analysismethods do not take these signal variations into considerationMethods In this study we developed a novel signaling pathway analysis methodcalled signaling pathway functional attributes analysis SPFA method This methodanalyzes the signal variations that effector genes received between two conditionsnormal and disease in different signaling pathwaysResults We compared the SPFA method to seven other methods across GeneExpression Omnibus datasets using three measurements the median rank of targetpathways the median pvalue of target pathways and the percentages of significantpathways The results conï¬rmed that SPFA was the topranking method in termsof median rank of target pathways and the fourth best method in terms of medianpvalue of target pathways SPFAs percentage of significant pathways was modestindicating a good false positive rate and false negative rate Overall SPFA wascomparable to the other methods Our results also suggested that the signal variationscalculated by SPFA could help identify abnormal functional attributes and parts ofpathways The SPFA R code and functions can be accessed at githubcomZhenshenBaoSPFASubjects Bioinformatics Cell Biology Computational Biology Computational ScienceKeywords Signaling pathway analysis Functional attributes Cell behaviors SPFA Effector genesINTRODUCTIONRecently developed highthroughput functional genomics technologies have generatedlarge amounts of experimental disease data and detected new biological informationChallenge for biologists is understanding the biological behaviors of disease cells usingboth newly generated disease data and existing biological knowledge Signaling pathwayanalysis methods are used to better understand the biological behaviors of disease cellsHow to cite this Bao Z Zhang B Li L Ge Q Gu W Bai Y Identifying diseaseassociated signaling pathways through a noveleffector gene analysis PeerJ 8e9695 107717peerj9695Submitted February Accepted July Published August Corresponding authorYunfei Bai whitecfseueducnAcademic editorJun ChenAdditional Information andDeclarations can be found onpage 107717peerj9695Copyright Bao et alDistributed underCreative Commons CCBY 0cThe understanding of biological behaviors of disease cells beneï¬ts to understand thepathological scenery and treatment Overrepresentation analysis ORA based methodswere initially presented as signaling pathway analysis methods to help biologists identifyoverrepresented pathways from a list of relevant genes produced from experimentaldata ORAbased methods merely count the number of differentially expressed genes inspeciï¬c functional category gene sets such as the Gene Ontology GO Blake the Kyoto Encyclopedia of Genes and Genomes KEGG Kanehisa BioCarta Nishimura and Reactome Joshitope Then they determinesigniï¬cance of the overlaps via statistical tests such as Fishers exact test Many tools arebased on this method including OntoExpress Draghici Khatri Fisher Khatri Sirota Butte and the Gene Ontology Enrichment AnalysisSoftware Toolkit GOEAST Zheng Wang However ORAbased methods onlytake into account large changes in individual genes that significantly affect pathwaysand they do not account for smaller changes in sets of functionallyrelated genesie pathways capable of significant effects Functional class scoring FCS basedmethods have been used to avoid this limitation of ORAbased methods FCSbasedmethods take into account the coordinated gene expression changes in pathways such asgene set enrichment analysis GSEA Subramanian gene set analysis GSAEfron Tibshirani and meanrank gene set enrichment tests MRGSE Liu However ORAbased and FCSbased methods are both limited because theydo not take into account the complex interactions between genes or the complextopology of pathways To overcome this limitation pathwaytopologybased methodswere proposed Pathwaytopologybased methods integrate the complex interactionsbetween genes using pathway topology information speciï¬cally KEGG signalingpathway informationSignaling pathway impact analysis SPIA one of the most widelyused pathwaytopologybased methods considers both the number of differentially expressed genesDEGs in a given pathway and the topology information of that pathway Tarca Many improved methods based on SPIA have been proposed Li developed a method called subSPIA which used a minimum spanning tree way toprune signaling pathways and used the SPIA method to identify significant signalingsubpathways Li Bao developed two SPIAbased methodscalled PSPIA and MSPIA These two methods replaced or interaction strength inSPIA with the interaction strength of the Pearson correlation coefï¬cients and mutualinformation respectively Bao There are different pathwaytopology methodsthat make use of the topological information of signaling pathways For instance GeneGraph Enrichment Analysis GGEA uses prior knowledge derived from directed generegulatory networks Geistlinger Liu Xu Bao used a subgraphmethod to take advantage of pathway topological information Liu Xu Bao ROntoTools introduced a term of perturbation factor by considering the type ofinteractions to take the pathway topology into consideration Tarca Bao PeerJ 107717peerj9695 0cVoichita Donato Draghici SebastianLeon developed a method usingtopology to detect liner subpathways in a signaling pathway SebastianLeon These methods still have their disadvantages Pathwaytopologybased methods donot consider the importance of genes in pathways Geneweightbased methods havebeen proposed to overcome this limitation Pathway analysis with downweighting ofoverlapping genes PADOG uses the frequency of a present gene in the analyzedpathways to improve gene set analysis Tarca Functional link enrichment ofgene ontology or gene sets LEGO measures gene weights in a gene set according toits relative association with genes inside and outside the gene set in a functional associationnetwork Dong Fang proposed an improved SPIA method calledSPIAIS that measured and assigned the importance as the average output degree ofthe gene in the pathwayA signaling pathway is a cascade of molecular reactions that bring out the functionalattributes eg cell proliferation apoptosis associated with the biological behaviors ofdisease cells using effector genes Effector genes receive signals without outputting signalsto other genes in an individual signaling pathway SebastianLeon Diseasesare always related to the abnormal signal that the effector genes receive Thereforethe signal that the effector genes receive can be very different under disease andnormal conditions The limitation of the previously mentioned methods includinggeneweightbased methods is that they do not consider the signal variations betweendisease and normal conditionsAdditionally the functional attributes in the same signaling pathway may be verydifferent from one another and can sometimes be opposites For example there are twoopposite functional attributes on the axon guidance pathway axon repulsion and axonattraction see the hsa04360 pathway in the KEGG dataset We cannot determinewhich functional attributes contribute more to the disease using most current pathwayanalysis methods Furthermore some pathways consist of several parts each with verydifferent contributions For example the Wnt signaling pathway is significant acrossdifferent diseases and can be divided into three parts Most existing pathway analysismethods cannot determine which part of the Wnt signaling pathway most significantlycontributes to a speciï¬c diseaseWe propose a new method that considers the signal variations between normal anddisease conditions that effector genes received in pathways the signaling pathwayfunctional attributes analysis SPFA method SPFA calculates the gene expression changesin a given pathway using an ORA method and then combines the ORA method resultswith the signal variation results under two conditions normal vs disease The signalvariations can help identify functional attributes and abnormal pathways We tested thecapabilities of the proposed signaling pathway analysis method on a series of real datasetsusing three parameters We also showed that the two types of probabilities consideredin this method were indeed independent Ultimately we veriï¬ed the usefulness of thesignal variations the effector genes received under two different conditions using theSPFA methodBao PeerJ 107717peerj9695 0cMATERIALS AND METHODSData sources and preprocessingSignaling pathway analysis methods require two types of input a collection of pathwaysand a list of genes or gene products with accompanying expression values across differentsamples between the compared phenotypes We used the KEGG signaling pathway asit is the most common manuallycurated signaling pathway used for pathway analysisWe downloaded signaling pathways from the KEGG PATHWAY datasetWe acquired disease gene expression datasets from the KEGGdzPathwaysGEORpackage and KEGGandMetacoreDzPathwaysGEO Rpackages Table TarcaBhatti Romero Tarca Each disease gene expression dataset wasmatched with a corresponding disease KEGG pathway For example a colorectal cancerdataset was associated with the colorectal cancer pathway Tarca The corresponding disease KEGG pathways were called target pathways Three rules wereused to select the gene expression datasets The datasets DEGs were available If no DEGs were selected other comparable methodswould return null results The results of these datasets could be analyzed Pathway analysis result pvalues of could not be analyzed The target pathways of these datasets were KEGG pathways since we used KEGGpathways as examplesDEGs were selected if they contained more than genes with FDR adjustedpvalues Otherwise we selected more than genes with original pvalues and absolute log fold change If DEGs still less than genes we selected the top of genes ranked by pvalues as DEGs 0c 0cdse 0c 0cSPFA algorithm designTo assess the signal variations between two conditions normal vs disease that the effectorgenes received from upstream genes we calculated the sum of signal variations from allupstream genes to effector genes Given an effector gene ge and an upstream gene gsthe signal variation from the gene gs to the effector gene ge can be deï¬ned asese ¼ cordiseaseðgsgeà 00 cornormalðgsgeÃwhere cordiseaseðgsgeà and cornormalðgsgeà refer to the Pearson correlation coefï¬cientbetween the gene expression data of gene gs and gene ge in the disease and normal statesrespectively dse is the network distance between gene gs and gene ge The Pearsoncorrelation coefï¬cient is always used in gene coexpression networks to represent thestrength of interactions between two genes The Pearson correlation coefï¬cient can also beused to represent the strength of an interaction between two gene expression valuesStudies have shown that the genetic regulatory patterns in signaling pathways betweenBao PeerJ 107717peerj9695 0cTable Data sets used for assessing the proposed method and compared methodsIDTarget pathwayGEO IDReferencesColorectal cancerColorectal cancerColorectal cancerColorectal cancerColorectal cancerNonsmall cell lung cancerPancreatic cancerPancreatic cancerPancreatic cancerThyroid cancerThyroid cancerAlzheimers diseaseAlzheimers diseaseAlzheimers diseaseAlzheimers diseaseAlzheimers diseaseChronic myeloid leukemiaChronic myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaAcute myeloid leukemiaDilated cardiomyopathyDilated cardiomyopathyEndometrial cancerGliomaGliomaHuntingtons diseaseParkinsons diseaseParkinsons diseaseProstate cancerProstate cancerRenal cell carcinomaRenal cell carcinomaGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762Hong Galamb and Gyorffy SabatesBellver Hong Uddin SanchezPalencia Badea Pei Donahue He Liang Liang Liang Blalock Juan Affer Affer Le Dieu Le Dieu Stirewalt Barth Hever Liu Liu Runne Zhang Zheng Wallace Wallace Lenburg Wang genes are different under normal and disease conditions Jung If the geneticregulatory pattern between the two genes changes the signal transmitted between thetwo genes will be very different Thus we used the Pearson correlation coefï¬cient tocalculate the signal variations that the effector genes received from their upstream genesHowever if an upstream gene does not directly transmit a signal the signal may beattenuated Therefore we used the network distance dse between gene gs and gene ge as apenalty coefï¬cientBao PeerJ 107717peerj9695 0cFor each effector gene gi in a given pathway the accumulated signal variations betweeneijXsj¼1normal and disease conditions that the upstream genes received total s genes in theupstream of the gene gi were calculated using the formulaASVðgià ¼The accumulated signal variation ASVðgià of the effector gene gi in a pathway can helpus distinguish among the functional disease attributes Effector genes with high ASVðgiÃdemonstrate that these functional attributes significantly contribute to their correspondingdiseasesFor a given signaling pathway the total accumulated signal variation ASV can bedeï¬ned asASV ¼Xki¼1ASVðgiÃwhere k is the total number of effector genes in the given pathwayUltimately the probability Psd used to measure the signal variations between twoconditions normal vs disease that those effector genes received from genes upstream in agiven signaling pathway Px is based on the pathways ASVðPxà The same number of genesas the one observed on the given signaling pathway are randomly selected from allgenes random gene IDs and have any possible expression data in all samples in the rangeof the experimenter Therefore the observed signal variations were obtained by permutingthe gene IDs times ASVperðPxÃwas the total accumulated signal variation of thegiven pathway Px obtained in the perth time The probability PsdðPxà of the given pathwaywas calculated asPsdðPxà ¼IðASVperðPxà 15 ASVðPxÃÃPwhere I is a function that returns when the argument is true and when the argument isfalseThe probability Psd does not measure the gene differential expression in a givenpathway Thus we had to combine the probability Psd with the probability Pde which canmeasure the total gene differential expression in a given signaling pathway The probabilityPde of a given pathway Px can be calculated through the following hypergeometric test 13 12 13tr 12m 00 t 12 13n 00 rmnPdeðPxà ¼ 00where the whole genome contains a total of m genes n genes are the number of DEGs inthe m genes and the given pathway contains t genes and r DEGsBao PeerJ 107717peerj9695 0cThe probability Psd uses the Pearson correlation coefï¬cient of the two genes expressiondata but the probability Pde uses the number of DEGs in a pathway Thus the twoprobabilities are independent of each other The signiï¬cance of the given pathway wascalculated following the SPIA method which combines the probabilities Psd and Pde Tarca The formulas areP ¼ c 00 c 01 lnðcÃc ¼ Psd 02 Pdewhere c is a product of Pde and Psd P is the combined probability of the signalingpathwaySignificantly enriched pathway analysis using SPFAThe SPFA procedure identiï¬es significantly enriched pathways in two steps Fig The ï¬rst step measures the total gene differential expression in the signaling pathwaysDEGs need to ï¬rst be identiï¬ed from the gene expression datasets Then the DEGs aremapped onto the signaling pathways Finally the signaling pathway pvalues are calculatedusing a hypergeometric testThe second step is to measure the signal variations between the two conditions normalvs disease that effector genes received from upstream genes in the signaling pathwaysThis is completed by Finding all effector genes in each signaling pathway Ascertaining all paths from the upstream genes to the effector genes in each signalingpathway If a path exists between the upstream genes and effector genes an interactionmust exist between them The paths network distances are used to weight thecorresponding interactions Using the Pearson correlation coefï¬cient absolute difference values between the diseaseand normal samples to calculate the signal variations of the corresponding interactions Using the network distance of each interaction to decrease their signal variations Calculating the accumulation of the signal variations between the effector genes andupstream genes for each effector gene Calculating the sum of the accumulations of all effector genes in each signaling pathway Evaluating the statistical signiï¬cance of each pathway based on their scoreUltimately the results of the two steps are combined into one pvalue We used the FDRadjust method on the combined pvalue to determine the signiï¬cance of each signalingpathway The pathways with the adjusted combined pvalues smaller than a thresholdvalue were considered as significant pathwaysThe distribution of effector genes in the signaling pathwaysStudying the signal variations between two conditions normal vs disease that the effectorgenes received leads to a deeper understanding of the biological behaviors of disease cellsEffector genes are widely scattered throughout the signaling pathways If a gene has noBao PeerJ 107717peerj9695 0cFigure The workï¬ow of SPFA method The step by step to identify significant signaling pathways using SPFAFullsize \ue90d 107717peerj9695ï¬g1signal inputs in an individual signaling pathway the gene is not considered an effectorgene The distribution of effector genes in each signaling pathway can be seen in Fig One hundred and ninetyï¬ve of the signaling pathways contained effector genesComparison methods and measuresWe compared seven methods to SPFA including Fisher Khatri Sirota Butte GSA Efron Tibshirani GSEA Subramanian MRGSE Liu SPIA Ullah ROnoTools Tarca Voichita Donato Draghici and PADOG Tarca We selected these methods for their stability andprevalence they can be compared using the same R environment as SPFAThere is no universally accepted technique for the validation of the results of pathwayanalysis methods Current pathway analysis methods use the results of a very smallnumber of datasets based on searching corresponding published life literature Thisapproach has its limitations First the number of datasets used is small Second authorsoften search their own leading to biased results Third complex biological phenomenaalways directly or indirectly correspond to multiple signaling pathwaysTarca compiled an objective and reproducible approach based on multipledatasets Tarca This approach avoided a biased literature search and requiredtesting on a large number of different datasets at least In this work we followedBao PeerJ 107717peerj9695 0cseneg rotceffe fo rebmuNPathwaysFigure The distribution of the effector genes number in each signaling pathway A total of ofFullsize \ue90d 107717peerj9695ï¬g2 signaling pathways contain the effector genesTarca validation approach Two measurements were compared in thisvalidation approach The ï¬rst measurement was the median pvalue of the targetpathways of the disease datasets Smaller median pvalues meant higher sensitivityThe second measurement was the median rank of the disease target pathwaysThe higher ranked methods were more accurate To further validate the different pathwayanalysis method results we used a third measurement the ratio of significant pathwaysusing a signiï¬cance threshold of of the adjusted pvalue in the datasets Thismeasured the methods ability to control false positive and false negative ratesRESULTSThe independence between the two probabilitiesThe two probabilities Pde and Psd are theoretically independent under the null hypothesisWe veriï¬ed their independence by calculating the squared correlation coefï¬cient betweenthe two probabilities using the gene expression datasets Table Our results showed thatthe average squared correlation coefï¬cient of the datasets was R2 ¼ Only four ofthe squared correlation coefï¬cients were slightly higher than R2 ¼ These resultsindicated essentially no correlation between the two probabilitiesSPFA method performanceWe compared SPFA with the other seven methods using three measurements the medianpvalue of the target pathways the median rank of the target pathways and the ratioof significant pathways The signaling pathways with adjusted pvalues ¤ weresignificantWhen comparing the median rank of the target pathways SPFA ranked ï¬rst Fig When comparing the median pvalue of the target pathways SPFA ranked fourthBao PeerJ 107717peerj9695 0cTable The squared correlation coefï¬cients between the two probabilities using the geneexpression datasets The four squared correlation coefï¬cients which are slightly more than areshown in boldGEO IDGSE4107GSE4183GSE8671GSE9348GSE23878GSE18842GSE15471GSE16515GSE32676GSE3467GSE3678GSE5281_HIPGSE5281_ECGSE5281_VCXGSE1297GSE16759GSE24739_G0GSE24739_G1GSE14924_CD4GSE14924_CD8GSE9476GSE1145GSE3585GSE7305GSE19728GSE21354GSE8762GSE20291GSE20164GSE6956AAGSE6956CGSE781GSE14762AverageSquared correlation between the probabilities Pde and Psd661523E748134EFig Notably the methods with the highest ranking in one measurement did notnecessarily rank the highest in the others This is because different measurements analyzedifferent abilities For example MRGSE was ï¬rst in median pvalue but was sixth inmedian rank Fisher was second in median pvalue but ranked fourth in median rankTo better compare SPFAs performance against the other methods we added the ranks ofBao PeerJ 107717peerj9695 0csyawhtap tegrat fo sknaRSPFAPADOGSPIAFisherGSAMRGSEGSEA ROntoToolsFigure The distribution of the target pathways ranks of the eight methods using datasets SPFAperforms the 1st among eight methods in terms of the median ranks of the target pathwaysFullsize \ue90d 107717peerj9695ï¬g3the median pvalue and median rank values from each method together We found that thecombined value of SPFA and PADOG was the smallest Table To further assess the performance of the eight methods we collected the results fromother general pathways typically associated with cancer using the out of datasetswith a form of cancer in Table Apoptosis and Pathways in cancer When using theApoptosis pathway and Pathway in cancer pathway instead of target pathways SPFAsmedian ranks were both ï¬rst and the median pvalues of MRGSE were also both rankedï¬rst These results were in alignment with the target pathway results However when usingBao PeerJ 107717peerj9695 0ctsyawhap tegrat fo seuavplMRGSEFisherPADOGSPFASPIAGSEAGSA ROntoToolsFigure The distribution of the target pathways pvalues of the eight methods using datasetsSPFA performs the 4th among eight methods in terms of the median pvalues of detecting the tarFullsize \ue90d 107717peerj9695ï¬g4get pathwaysthe Apoptosis pathway and Pathway in cancer pathway instead of the target pathwaysPADOGs median pvalues were both ranked ï¬fth When using the Apoptosis pathwaySPFAs median pvalue ranked third When using the Pathway in cancer pathway SPFAsmedian pvalue ranked fourth All these results suggest that SPFA had the best accuracyand a good sensitivity when compared with the other seven methodsAdditionally our results showed that SPFAs ratio of significant pathways wasmoderate Fig compared to the others MRGSEs ratio of significant pathways wasalmost and it could be questioned whether a such number of pathways was realisticBao PeerJ 107717peerj9695 0cTable The combined rank values of the ranks in terms of the median pvalues and the medianranks of target pathways of eight methodsMethodsSPFAPADOGFisherMRGSESPIAGSAGSEAROnoToolsRanks of the median pvaluesRanks of the median ranksSumTable The results of other general pathways apoptosis and pathway in cancer typically associatedwith cancer using the out of datasets with a form of cancer For each pathway the values for thetype of methods with the smallest median pvalues and ranks strongest association with the phenotypeare shown in boldPathway statisticApoptosisSPFAFisherSPIAGSAGSEAMRGSERontoToolsPADOGpValues medianRanks medianPathway in cancerpValues median794E225E162E27ERanks medianGSAs ratio of significant pathways was lower than and it reï¬ected that the GSAmethod had a high false negative rate The methods had a modest ratio of significantpathways indicated that the method had a modest false positive rate and a modest falsenegative rate Thus the discriminative ability of SPFA was good when compared with theother seven methods In conclusion our results strongly supported that SPFA waswellsuited for signaling pathway analysis and conï¬rmed previously reported results inDong Sources of improvement for SPFAThe main source of improvement in SPFA is that it uses signal variations that effectorgenes received under normal and disease conditions SPFA is compared to the simplerORAbased method used to calculate the probability Pde without accounting for signalvariations Fig As shown in Fig the ORAbased method has a higher worse rankand pvalue than SPFA for the target pathwaysBao PeerJ 107717peerj9695 0csyawhtap tnacifings tion dna tnacfings fio oitar ehTsignificancenot significantsignificantSPFAFisherSPIAROntoToolsGSAMRGSEGSEAPADOGmethodsFigure Average percentage of the pathways detected as significant and not significant by eachmethod using the threshold of pvalues ¤ Fullsize \ue90d 107717peerj9695ï¬g5Validating the correlation between diseases and the signal variationsthat effector genes received under two different conditionsTo validate the correlation between diseases and the signal variations that effector genesreceived under two different conditions normal vs disease we analyzed a colorectalcancer dataset GSE4183 and an Alzheimers disease dataset GSE16759 The colorectalcancer microarray GSE4183 Affymetrix array HGU133 Plus20 included colorectalcancer samples and normal samples Galamb Gyorffy Bao PeerJ 107717peerj9695 0cABttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa ttt tttt tfff foooo sss seeekuuunaaaavvvRppplllttttssssyyyyaaaawwwwhhhhaaaappppeeeeggggrrrraaaa t tttt tttf fffoooo s sssekkkunnnaaaavRRRplSPFASPFASPFASPFAORAORAORAORASPFASPFASPFASPFAORAORAORAORAFigure Determining the contribution of signal variations received by effector genes between twodifferent conditions normal vs disease in SPFA performance The boxplots show the distribution ofFullsize \ue90d 107717peerj9695ï¬g6the target pathways ranks A and pvalues BThe Alzheimers disease dataset GSE16759 included four disease samples and four normalsamples Juan The Wnt signaling pathway was altered in of the colorectal cancer samplesGalamb We assessed the signal variations that effector genes received in theWnt signaling pathway using the GSE4183 dataset Fig The results of Galamb coincided with our signal variation results Galamb reported thatoverexpression of TNS1 could induce the activation of JNK ENTREZID and The signal variation that the effector gene ENTREZID received ranked ï¬rst inour results Galamb detected that RBMS1 is another overexpressed geneand modulator of cmyc ENTREZID cmyc can regulate cell cycles and cause cellsto transform pathways The signal variation that the effector gene ENTREZID received ranked second in our results Galamb also identiï¬ed that TCF4 is anoverexpressed gene that can participate in the transcriptional regulation of genesassociated with colon carcinogenesis These colon carcinogenesis associated genes includecmyc ENTREZID cyclin D1 ENTREZID PPARδ ENTREZID and MMP7 ENTREZID The signal variations that these effector genes receivedranked second fourth ï¬fth and sixth respectivelyBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoitairav liangSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695ï¬g7Many pathways can be studied in colorectal cancer datasets For example the PI3KAktsignaling pathway plays a critical role in the growth and progression of colorectal cancerJohnson The effector genes ENTREZID596 ENTREZID842 andENTREZID1027 have the highest signal variations and are linked to cell cycle progressionand cell survival Fig The GSE4183 dataset results further conï¬rmed the role of thispathway in colorectal cancer developmentThe Wnt signaling pathway is also closely related to the occurrence and development ofAlzheimers disease Inestrosa The signal variations that different effectorgenes received calculating based on the Alzheimers disease dataset GSE16759 in the Wntsignaling pathway were shown in Fig The signal variations that the effector genesENTREZID and received were considerably higher than the other effector genesin the Wnt signaling pathway This result validated evidence of crosstalk between theAlzheimers disease signaling pathway and the two effector genes upstream genes in theWnt signaling pathwayAll these results indicated the high correlation between diseases and the signalvariations calculated using the SPFA methodBao PeerJ 107717peerj9695 0cseneg rotceffe eht yb devecer snoiitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the PI3KAktsignaling pathway using colorectal cancer datasets GSE4183Fullsize \ue90d 107717peerj9695ï¬g8The other usages of the signal variations that effector genes receivedunder two different conditionsThe signal variations that effector genes received under two different conditions canshow the different contributions of different functional attributes contributed to theircorresponding diseases We can also identify which parts of the pathway contribute to theircorresponding diseases through the signal variations that effector genes receivedWhen looking at the Wnt signaling pathway results of GSE4183 Fig ï¬rst we knowthe functional attributes participating in the cell cycle have abnormal signal variationsbecause most effector genes with high signal variations participate in the pathway cell cycleincluding cmyc ENTREZID cyclin D1 ENTREZID and PPARδENTREZID and MMP7 ENTREZID Second we can know that theabnormal state of the ï¬rst and second parts of the Wnt signaling pathway may contributemore to colorectal cancer because that the effector genes with high signal variations areall in the two parts If we were only to observe DEG distribution in the Wnt signalingpathway using the GSE4183 dataset we would not know which abnormal part contributedto the disease Fig Through the result of the Wnt signaling pathway in GSE16759Fig on one hand according to this result we can know that the functional attributeslinked with the effector genes ENTREZID and which had the highest signalBao PeerJ 107717peerj9695 0cseneg rotceff eehi t ybdevecer snoitairav langSiEffector genesFigure The signal variations received by effector genes from the upstream genes in the Wntsignaling pathway using Alzheimers disease datasets GSE16759Fullsize \ue90d 107717peerj9695ï¬g9variations were abnormal in Alzheimers disease On the other hand this may dominatethat the ï¬rst part of the Wnt signaling pathway may be more related to t | Thyroid_Cancer |
EndocrinesyPostoperative vocal fold dysfunction in covid19 era are we still intime for a recoveryElena Bonati Elena Giovanna Bignami2 Paolo Del Rio1Received May Accepted July Springer ScienceBusiness Media LLC part of Springer Nature To the EditorThe novel coronavirus COVID19 is a highly contagious zoonosis produced by SARSCoV2 which arose inChina and spread all over the world transmitting from manto man through respiratory secretions In March itwas deï¬ned by the World Health anization WHO as apandemic to underline its spread and severityHealthcare professionals are one of the categories most atrisk of contracting the infection in particular when theiractivity involves the direct management of the patientsairways Among these categories we can count anesthetistshead and neck surgeons otolaryngologists maxillofacialsurgeons ophthalmologists and dentists For these reasonsthe latest evidencebased recommendations for otolaryngology and head and neck surgery practice suggest thathealthcare facilities should prioritize urgent and emergencyvisits and procedures until this condition stabilizes ceasingelective care []Nevertheless oncological surgical activity althoughslowed down did not stop in most hub hospitals Regardingthyroid cancer thyroid surgery is complex and the rate ofnerve damage is still considerable Immediate postoperativevocal fold rate is in our case study and decrease to after months Postoperative dysphonia can becaused by several factors other than nerve damage such astracheal intubation or scarring in the thyroid lodge It istherefore important to identify the cause of vocal corddysfunction and treat it correctly at the right time If anunilateral vocal fold paresisparalysis is diagnosedthetreatment consist in improving the speech while in case of Elena Bonatiebonati86gmailcom General Surgery Unit Department of Medicine and SurgeryParma University Hospital Parma Italy Unit of Anesthesiology Department of Medicine and SurgeryParma University Hospital Parma Italybilateral vocal fold paresisparalysis respiratory obstructionalso needs to be urgently treated Fortunately we havebroughtthe incidence of this last and most dangerouscomplication to at our Clinic since the introduction in of the routine use of intraoperative neuromonitoringduring thyroidectomyThe latest guidelines published by the Americanin March Association of Endocrine Surgeonsrecommend laryngeal examination in patients with knownor suspected new recurrent laryngeal nerve dysfunctionafter thyroidectomy for additional evaluation and possibletreatment with a speech pathologist According to theAmerican Academy of OtolaryngologyHead and NeckSurgery they assert that early referral weeks postsurgery to a laryngologistin combination with earlyintervention results in superior voice outcomes since theideal time for vocal fold augmentation is months afterthyroidectomy []A metaanalysis about therapy for vocal fold paresisparalysis after thyroidectomy concluded that the timingof therapy for unilateral vocal fold paralysis after thyroidectomy has a significant impact on the effect sizebeing significantly greater if therapy is performed within months This may be explained by progressive atrofolds and disappearance of nervephy offunction so that vocalfold movements cannot berecovered []the vocalPatients who underwent thyroid surgery from February and who had experienced a vocal fold disfunctionVFD were unable to undergo a laryngoscopy nor muchless a speech therapy according to health measuresnecessary to contain the spread of the virus This unfortunately causes a progressively reduced possibility ofrecovery increasing the speciï¬c morbidity related to surgery for thyroid cancer in this period The only indicationthat we can give to patients is the rest of the voice to avoidthe establishment of compensation mechanisms worseningthe clinical picture waiting to be able to resume the correcttreatment 0cTherapeutic diagnostic pathways in the COVID19 erahave become difï¬cult and dangerous logarithms that mustconsider the need for patient care and the possibility oftreatments delay in safety but also the risk of contagion ofthe patientsleast protection ofhealthcare personnel The hospital setup has been significantly changed and much of the economic structuraland human health resources have been dedicated to themanagement of the COVID pandemicthemselves and notIn parallel with the COVID19 emergency we areexperiencing another health emergencythe one thatinvolves the management of nonCOVID19 patients Evenin the second phase of the pandemic only urgent healthservices are provided A reanizing effort within theindividual healthcare companies is required to guaranteetreatment even for nonCOVID19 patientsMoreoverThe COVID19 pandemic highlighted the limits andweaknesses of our health system and now that the correctprotocols for the protection of healthcare personnel havebeen described allthe all healthcare companies shouldequip their staff with the appropriate materials such as N95masks hair cover protective coverall gown gloves faceshields goggles and shoe covers In the face of higherexpenses this would allow the resumption of activitiesminimizing the risk of an increase in the rate of infectionroutine health practices must be reconsidered preferring less invasive techniques in order toscreen patients who need secondlevel examination Evenif not used yet in our hospital transcutaneous laryngealultrasonography is a valid noninvasive and painlessalternative method in the assessment of vocal cords It hasbeen demonstrated in a recent prospective multicentricstudy that it has concordance with laryngoscopy in themajority of cases and so it can be a valid alternative asï¬rstline exam for vocalfold examination pre andpostoperativelyEndocrineFinally the growing use of virtual platforms for the needof social distancing could encourage their application evenin healthcare services that can be performed by teleconference such as speech therapyWe can assess that COVID19 pandemic is causingdirect morbidity and mortality and even a related one dueto missed or delayed treatment of multiple nonCOVID19diseases The delivery of the health service should beimproved and the health system itself must be modernizedto adapt to new needsCompliance with ethical standardsConï¬ict of interest The authors declare that they have no conï¬ict ofinterestPublishers note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional afï¬liationsReferences LP Kowalski A Sanabria JA Ridge WT Ng R de BreeA Rinaldo RP Takes A A Mkitie AL Carvalho CR Bradford V Paleri DM Hartl V Vander Poorten IJ Nixon C PiazzaPD Lacy JP Rodrigo O GuntinasLichius WM MendenhallA DCruz AWM Lee A Ferlito COVID19 pandemic effectsand evidencebased recommendations for otolaryngology and headand neck surgery practice Head Neck 101002hed26164 KN Patel L Yip CC Lubitz EG Grubbs BS Miller W ShenP Angelos H Chen GM Doherty TJ Fahey 3rd E KebebewVA Livolsi ND Perrier JA Sipos JA Sosa D StewardRP Tufano CR McHenry SE Carty The American Associationof Endocrine Surgeons Guidelines for the deï¬nitive surgical management of thyroid disease in adults Ann Surg e21e93 X Chen P Wan Y Yu M Li Y Xu P Huang Z Huang Typesand timing of therapy for vocal fold paresisparalysis after thyroidectomy a systematic review and metaanalysis J Voice 0c' | Thyroid_Cancer |
"Despite recent interest in the use of ketogenic diets KDs for cancer evidence of beneficial effects islacking This study examined the impact of a randomly assigned KD on quality of life physical activity andbiomarkers in patients with breast cancerMethod A total of patients with locally advanced or metastatic breast cancer and without a history of renaldisease or diabetes were randomly assigned to either a KD or a control group for this 12week trial Concurrentwith the first third and fifth chemotherapy sessions quality of life physical activity and biomarkers thyroidfunction tests electrolytes albumin ammonia ALP lactate and serum ketones were assessed Dietary intake wasalso recorded on admission and the end of the treatmentResults No significant differences were seen in quality of life or physical activity scores between the twogroups after weeks however the KD group showed higher global quality of life and physical activityscores compared to the control group at weeks P P Also serum lactate and ALP levelsdecreased significantly in the KD group compared to the control group at the end of the intervention ± vs ± ± vs ± P and P respectively A significant inverse associationwas observed between total carbohydrate intake and serum betahydroxybutyrate at weeks r P No significant differences between groups were observed in thyroid hormones electrolytes albuminLDH or ammonia Compliance among KD subjects ranged from to as assessed by dietary intakeand serum ketones levels of Continued on next page Correspondence khodabakhshiadelehyahoocom6Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranFull 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 0cKhodabakhshi Nutrition Journal Page of Continued from previous pageConclusion According to our results besides a higher global quality of life and physical activity scores compared tothe control group at weeks KD diet combined to chemotherapy in patients with breast cancer does not bringadditional benefit about quality of life and physical activity at weeks However decreases seen in levels of lactateand ALP in the KD group suggest that a KD may benefit patients with breast cancerTrial registration This trial has been registered on Iranian Registry of Clinical Trials IRCT under the identification codeIRCT20171105037259N2 wwwirctirtrial30755Keywords Ketogenic diet Breast cancer quality of life Physical activity Lactate Alkaline phosphatase chemotherapythere are stillIntroductionKetogenic diets KDs are high in fat and very low incarbohydrate They have been used as a dietary treatment in epilepsy for nearly a century [] RecentlyKDs have gained the attention of cancer researchersdue to their potential impact on cancer cell metabolism [] Despite the growing evidence of possibleantitumor benefitssome concernsabout potential adverse effects of KDs in cancer patientsincluding micronutrient deficiencies appetitereduction nausea constipation [] fatigue [] hyperlipidemia and especially unintended weight loss [ ]KDs are perceived as restrictive in nature which mayadd to the burden of cancer patients who already suffer from considerable physical emotional and financialstress all of which are known to negativelyimpact quality of life QoL In addition alterations inphysical and cognitive function during cancer treatment are pervasive It is estimated that of patients undergoingfromcancerrelated fatigue [] Prior studies have foundthat KD may improve physical and mental wellbeing[] Less fatigue has been reported in healthy overweight and obese adults following lowglycemic compared to highglycemic diets [] Results ofthreestudies using the validated European anization forResearch and Treatment core QoL questionnaire tofindings [] Aassess fatigue lacked consistentin advanced cancer patients showed imsmall trialprovementin sleep and emotionalfunction after athreemonth KD intervention [] Other studies havesuggested enhanced cognitive function [ ]cancertreatmentsufferTo date only four studies have assessed QoL in adultpatients with cancer [ ] Hunger is a reported sideeffect of restricted KDs however previous research hasfound that perceived hunger is reduced in low carbohydrate diets compared to low fat diets [] A recent systematic review has highlighted the need for additionallarger investigations on the impact of ketogenic diets onQoL [] The goal of this present trial was to assesswhether a KD had beneficial effects on QoL dietary intake physical activity and specific biomarkersinindividuals with breast cancer while also evaluating compliance to KD guidelines in these patientsThe protocol used in this trial [] and part of the resultsfrom this trial have been previously published [ ]MethodsThe study protocol was approved by the National Nutrition and Food Technology Research InstituteNNFTRI Shahid Beheshti University of MedicalSciencesIRSBMUNNFTRIREC1396187 All participants provided writteninformed consent prior to participating in the studySBMU TehranIranThis trial was a randomized controlled labelclinical trial to breast cancer patients with locally advanced or metastatic disease who were receiving chemotherapy for atleast weeks The studythe medical oncology clinic atwas conducted atShohadaeTajrish hospital Cancer Research CenterTehran Iran from July to October of Participation was to patients to years of ageExclusion criteria screened forsignificant cardiacrenal or neurologic comorbidities symptoms of malnutrition diabetes pregnancy and Karnofsky indexless than Using a block balanced randomizationmethod patients were assigned to the interventionn or controln groups Randomizationwas computergenerated by a statistician who was nota member of the medical team Blinding the participants or study personnel was not deemed feasible inthis dietintervention The project coordinator enrolled the participants and assigned them to their interventions Both the KD and the control diet werecalculated to be eucaloric using the MifflinSt Jeorformula The KD consisted of of calories fromCHO from protein from mediumchain triglyceride MCT oil and from fat A dietitianprovided specific nutritional counseling to each participantfacetoface meetings Patientsengaged in ongoing weekly counseling sessions viaphone WhatsApp or Telegram and were assessed forcompliance and possible adverse effects To furtherenhance compliance dietary recommendations werein individual 0cKhodabakhshi Nutrition Journal Page of individualized and appropriate recipes were providedto patients in the KD group were asked to refrainfrom eating any grains grain products starchy vegetables fruit or sugar Dietary carbohydrates were limited to nonstarchy vegetables and dietary proteinswere obtained primarily from egg meat poultry andfish Small amounts of lower carbohydrate berries andnuts were allowed as long as they did not exceed thecarbohydrate limit in the diet prescription Subjectswere encouraged to increase their fat intake and toselect from a variety of sourcesincluding olive oilbutter and cream cheese Patients were asked tochoose only the foods specified in the diet plan provided to them Patients were also encouraged to usemediumchain triglyceride MCT oil MCT oil anodorless and tasteless saturated fat does not requirebile or pancreatic enzymes for digestion It is easilyconverted to ketones in the liver thereby enhancingketosis Every weeks ml of MCT oilfromNutricia Erlangen Germany was provided to eachsubject in the KD group For better tolerance initialdosage of MCT was kept low and increased daily overa 6day period until maximum tolerable dosage wasachieved Dosage was reduced in a similar steppedprocessThe patients in the control group were instructed tofollow a standard diet consisting of CHO protein and fat Dietary compliance was checked byassessing blood betahydroxybutyrate levels every weeks and dietary intake at baseline and end of thestudyQoL assessmentQoL was assessed using the EORTC QLQC30 version and IORTC QLQBR23 questionnaires developed bythe European anization for Research and Treatmentof Cancer The validity and reliability of the questionnaires has previously been evaluated in Iran [ ]The questionnaires were completed at enrollment at weeks and at the end of the interventionDietary intake assessmentHospital dietitians used a 24h dietary recall 24HR toobtain a total of days intake one weekend day andtwo workdays through telephone and facetoface interviews both at the beginning and end of the study Theamount of each food consumed was estimated usingcommon household containers bowls cups and glassesand standard measuring cups and spoons as referencesThe mean quantity of total energy carbohydrate proteinand fat were estimated from the 24HRDietary intake wasanalyzed by Nutritionist IV software Version USPhysical activity assessmentPhysical activity was measured using the IPAC International Physical Activity questionnaire at baseline at weeks and at the end of the studyBiomarker assessmentFasting blood sampling for serum Na K Ca P lactate Mg LDH albumin ammonia and ALP were performed at baseline midway through the intervention weeks and at weeks T3 T4 and TSH were measuredat baseline and the end of the interventionStatistical analysisConsidering the power and α the sample sizewas calculated as individuals per group Assuming a dropout during the weeks of the study the finalnumber of participants was calculated as patients ineach groupStatistical analysis was carried out according to theintentiontotreat protocol Continuous variables weretested for normal distribution by the KolmogorovSmirnov test and then reported as mean ± standard deviation or median as appropriate Student ttest or MannWhitney U test was used to compare the continuousvariables between the two groups Paired sample ttestor Wilcoxon was used to compare the continuous variables within the two groups The ANCOVA test wasused to eliminate the effect of confounding factorsPearson correlation analyses were used to estimate associations between total carbohydrate intake and serumbetahydroxybutyrateData were analyzed using the SPSS version software Chicago IL USA and Stata version P was considered as statistically significantResultsDetailed patient demographics and a flow diagram werereported previously [] A total of women withbreast cancer were enrolled and randomly assigned to either the intervention n or control n groupsThree patients in the control group withdrew before beginning their assigned diet while10 patients in the KDgroup and patients in the control group withdrewfrom the study after beginning their assigned diet Ultimately patients in each group completed the studyand were included in the analysis No significant differences were seen between the two groups with regard toage cancer type metastasis and marriage or educationstatus P The intervention group included patients with locally advanced disease and patients withmetastatic disease liver bone lung liver andbone while the control group consisted of patientswith locally advanced disease and patients with 0cKhodabakhshi Nutrition Journal Page of metastatic disease bone liver lung liver andbone at other sites P Table Data related to quality of life are shown in Tables and No significant differences were seen in QoL betweenthe two groups after weeks however the KD groupshowed better global QoL compared to the controlgroup at week P Also at week diarrhea increased in the control groupcompared to the intervention P Data on week not shown Using the QoL questionnaire there was awithingroup decrease in reported hunger from baselineto weeks in the KD group P A withingroupdecrease was seen in physical performance measuresfrom baseline to weeks in both groups which was significant only in the KD group P In addition rolefunctioning and socialfunctioning scores significantlydecreased in the control group compared to the baselinebut not in the KD group P P Table Mean dietary intake is shown in Table and Fig The mean caloric and carbohydrate intake decreasedsignificantly at the end of the study compared to control P and P respectively while fatintake increased significantly in the KD group compared to the control group P After adjustingfor total energy intake this difference remained significant When data from both groups was combineda significant inverse association was observed betweentotalserum betaandr P hydroxybutyratealthough this effect was not seen when the KD groupwas analyzed separatelyintakeat weekscarbohydrateWithingroup analysis showed significant decreasesin energy carbohydrate and protein intake in bothgroups compared to the baseline Fat intake increasedsignificantly compared to the baseline in the KDgroup P and decreased significantly in thecontrol group P During the intervention of the subjects in the KDarm limited carbohydrates to g and of subjects consumed of calories from carbohydratesAt weeks of patients in the KD group hadserum ketones mmolL at 6weeks had ketone levels of mmolL As previously reportedserum ketone concentrations increased significantly inthe KD group ± to ± mmollP []At weeks physical activity improved in the KD groupcompared to the control group adjusted for cancer typeand baseline value P but after weeks physicalactivity did not show any significant differences in a between or withingroup analysis Fig No significant difference was observed in a betweenor withingroup analysis of thyroid hormones electrolytes albumin Ammonia and LDH Howeverlactateand ALP decreased significantly after intervention in theKD group compared to the control group P andP respectively ALP is adjusted for baseline valueand cancer type Table Data on thyroid hormones notshownDiscussionThe effect of KD on QoL physical activity dietary intake and biomarkers in patients with locally advancedand metastatic breast cancers was evaluated in thisstudy Based on our findings in the KD group globalQoL was higher at weeks perhaps in part because diarrhea was more frequent in the control group than theKD group No significant differences were seen in theQoL physical activity and biomarkers between the twogroups after the week intervention Lactate and ALPwere lower in the KD group compared to the controlEffect of diet on QoLIn our study in the KD group global QoL was higher at weeks No adverse effects were observed in thoseTable Baseline characteristics in breast cancer patients before interventionScale categoriesCancer TypeLocally AdvancedIntervention Ketogenic dietn Control Ordinaryn ERPRHER2Metastaticpositivenegativepositivenegativepositivenegative ER Estrogen receptor PR Progesterone receptorHER2 Human epidermal growth factor receptor aCalculated by chi square testbCategorical data shown as No p value008a057a043a079a 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before andafter intervention in KD group and control group as measuredby the EORTC QLQC30aFunctioningaPhysical functioningMD CIControlKDpvalueTable Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQC30SymptomsaFatiguepvalueControlKD Week Week pvalue33aNausea and vomitingWeek Week pvalueRole functioningWeek Week pvalue ± 11a ± ± ± Cognitive functioningWeek Week pvalue ± ± Emotional functioningWeek Week pvalueSocial functioningWeek Week pvalue ± ± ± ± Global quality of lifeWeek Week pvalue ± ± ± ± ± ± ± ± ± ± ± ± ± ± After adjusting for baseline value and chemotherapy status no significantdifferences were observedStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the two groupsData shown as mean and SDaThe higher values indicate higher level of functioning and quality of lifeparticipants assigned to the KD compared to the controlgroup after weeks Withingroup analysis showed decreased hunger and physical function in the KD groupcompared to the baseline In the control group role andsocial functioning decreased significantly compared tobaselineResults of a systematic review and metaanalysis haveshown that KDs suppress appetite [] Decrease in hunger or appetite in our study may be due to the high fatcontent of the KD as it decreases the ghrelin releasewhich in turn may reduce appetite High fat intake alsoslows digestion which could also impact the perceptionof hunger Previously we have shown that the KD resultsin weight loss [] As a clinical benefit KDinduced Week Week pvaluePainWeek Week pvalueReduction in appetiteWeek Week pvalueSleep difficultiesWeek Week pvalueDyspneaWeek Week pvalueConstipationWeek Week pvalueDiarrheaWeek Week pvalueFinancial concernsWeek Week pvalue MannWhitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate a higher grade of symptoms Data shown asmedian and quartile 0cKhodabakhshi Nutrition Journal Page of Table Quality of life in breast cancer patients before and afterintervention in KD group and control group as measured by theEORTC QLQBR23aKDControlpvalue aFunctioningFuture perspectiveWeek Week pvaluebSymptomsArmWeek Week pvalueBreastWeek Week pvalue Week Systemic therapy side effects Week pvalueConcerns over hair lossWeek Week pvalue Table Comparison of mean ± SD macronutrient intake atbaseline and 12weeksVariableMD CIpvalueKDMean ± SDControlMean ± SDEnergy KcaldayBefore ± ± ± AfterpvalueCarbohydrate grBefore ± ± ± ± AfterpvalueProtein grBeforeAfterpvalueFat gr ± ± ± ± ± 0001a 041aBefore ± ± ± ± AfterpvalueStudent ttest was used to compare the continuous variables between the twogroups Paired sample ttest was used to compare the continuous variableswithin the groupsMD Mean differenceCI Confidence intervalaAncova Adjusted for baseline value and energy0001aMannWhitney U test was used to compare the continuous variables betweenthe two groups Wilcoxon was used to compare the continuous variableswithin the two groupsaThe higher values indicate higher level of functioning and quality of lifebThe higher values indicate a higher grade of symptomsData shown as median and quartile decreases in appetite weight and body fat may result infavorable changes in breast cancer patients notably inoverweight or obese women [ ]In contrast with our findings Cohen found that aKD significantly enhanced physical function scores inwomen with ovarian or endometrial cancer comparedto the control group but appetite did not change atthe end of the study compared to the baseline []Part ofstudy andCohens trial may be explained by the design of thestudy While only of the participants in the Cohen study were undergoing chemotherapy all of ourpatients were receiving treatmentthe inconsistency between ourAlso timing of the administration of the questionnaires and whether the participants were in positive ornegative energy balance may have influenced ourfindingsNo significant difference was reported in QoL at theend of study compared to the baseline by TanShalaby [] However a slight decrease in physical androle functioning as well as temporary constipation andfatigue were reported in the KD group in one study []In our study constipation was noted by participants inthe KD arm during the early days which was managedby dietary changesAlso after weeks in the KD group physical activityscores was higher compared to the control group but at weeks differencessignificantbetween the two groupsin scores were notDietary intake and adherenceOur study data showed a significant decrease in carbohydrate intake and a significant increase in fat intake inthe KD group compared to the control Protein intakewas not significantly different between the two groupsbut decreased overall in both groups when compared tobaseline Total daily carbohydrate intake was similar toresults in the Cohen study [] We also assessed serumbetahydroxybutyrateIn the KD group ofpatients at weeks and at 6weekshad serum ketones and patients at weeks and weeks 0cKhodabakhshi Nutrition Journal Page of Fig Mean caloric intake and distribution of macronutrients as percentage of total kilocalories before and after week intervention in breastcancer patients in two groupsFig Comparison of trend changes in physical activity in breast cancer patients in two groups 0cKhodabakhshi Nutrition Journal Page of LBsvskwleuavpitnopdmisvskwleuavpLBsviitnopdmeuavlpinorrefnoBnodesabldetauclacerewseuavlpllaerusaemdetaepeRepytlsisyanAlavretnIecnedifnoCICecnereffiDnaeMDMpuwolloftsalroskeewskeewkeewropuwolloftsitnopdMiepytrecnacdnaeuavlenilesabrofdetsudAjavocnAsnosirapmoclepitlumrofnoitcerroc±±±±skeew±±itnopdMi±±enilesaB±±±±±±smArlairTDKICDMlortnoClortnoCDKlebairaVetatcaLluHDLstneitaprecnactsaerbdetaertDKdnalortnocnislevelrekramoBielbaTICDM±±±±lortnoCDKldgcmianommAICDMICDM±±±±±±lortnoCDK±±±±±±lortnoCDKICDMICDM±±±±±±±±±±±±ICDMlortnoCDKlortnoCDK±±±±±±±±±±lortnoCDKICDM±±ICDMlortnoCDKICDM±±±±±±lortnoCDKLdgmgMldginmubAllqemKlDgmPPLAldgmaClqemaNenilesaBLB 0cKhodabakhshi Nutrition Journal Page of had serum ketones mmoll Cohen reported that of patients had betahydroxybutyrate concentrations mmollA recent systematic study of KDs in adult cancerpatients reported a range of to with a adherence rate overall reported by [] According toour data the level of adherence to the KD interventionsuggests that the diet is a feasible option for women withbreast cancer who are receiving chemotherapyDespite the lack of any restriction in calorie intakein the study design and consistent with findings ofCohen [] the KD group showed a significant reduction in calorie intake compared to the control groupThe decrease in calorie intake may be due to reductions in appetite associated with ketosis as the subjects in the KD arm did not consume all of the fatcalculated for their diet This may also be due in partto customary practices surrounding meal preparationA decrease in appetite and subsequentinadvertentcalorie restriction most often results in weight loss inthe absence of malnutrition or cachexiathis mayhave antiinflammatory and proapoptotic propertieswhich in turn may exert a positive effect on thesehallmarks of cancer Ketosis may also enhance theeffectiveness of chemotherapy while reducing the sideeffects of treatment [ ]Effect of diet on biomarkersConsistent with the outcomes of the previous studiesour results revealed that the KD had no adverse effecton thyroid hormones electrolytes LDH urea and albumin Significant decreases were seen in serum levels oflactate KDs reduce glycolytic activity which in turn mayslow metastases by reducing the acidity of the tumormicroenvironment and lowering the availability of lactate as a substrate for biomass synthesis [] Decreaseswere also seen in ALP High levels of ALP in breast cancer patients is a negative prognostic marker often indicating progression of metastatic disease [] Moreresearch is needed to assess whether lower ALP and lactate as seen in this study contributes to slower rates ofdisease progressionTo our knowledge this is the first randomized controlled trial examining the effects of a KD on QoL inbreast cancer patientsThe primary limitation of this study was the heterogeneous nature of the sample in regards to cancer stageA secondary limitation was the small sample sizeConclusionAccording to our results besides a higher global QoLand physical activity scores compared to the controlgroup at weeks KD diet combined to chemotherapy inpatients with breast cancer does not bring additionalbenefit about QoL and physical activity at weeksWhile many blood biomarkers did not differ significantlybetween the two groups ketosis may still offer benefit tosome patients with breast cancer in part by decreasinglactate and ALPSupplementary informationSupplementary information accompanies this paper at doi101186s1293702000596yAdditional file figure Flow diagram of the patient treatmentprocessAdditional file figure Median confidence interval tyroidhormones in baseline and 12week by two trial arms in breast cancerpatientsAcknowledgmentsWe would like to thank all the patients at our clinic who took part in thisstudyAuthors contributionsKhodabakhshi carried out the conception Methodology performed theexperiments design of the diet and wrote the Davoodi and Seyfriedcollaborated in the design of the study Davoodi supervise on the thesisKalamian and Beheshti collaborated in the design of the diet Kalamian gavecritical review of the manuscript All authors have read and approved thefinal manuscriptFundingNot applicableAvailability of data and materialsData described in the manuscript code book and analytic code will bemade available upon request pendingEthics approval and consent to participateAll participants provided written informed consent prior to participating inthe studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nutrition School of Public Health Kerman University ofMedical Sciences Kerman Iran 2Physiology Research Center KermanUniversity of Medical Sciences Kerman Iran 3Biology Department BostonCollege Chestnut Hill MA USA 4Dietary Therapies LLC Hamilton MT USA5Department of Nutrition and Dietetics Mofid childrens hospital ShahidBeheshti University of Medical Sciences Tehran Iran 6Cancer ResearchCenter Shahid Beheshti University of Medical Sciences Tehran Iran7Department of Cellular and Molecular Nutrition Faculty of Nutrition Scienceand Food Technology Shahid Beheshti University of Medical SciencesTehran IranReceived March Accepted July ReferencesNeal EG Chaffe H Schwartz RH Lawson MS Edwards N Fitzsimmons G The ketogenic diet for the treatment of childhood epilepsy arandomised controlled trial Lancet Neurol Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab 0cKhodabakhshi Nutrition Journal Page of adverse effects on blood lipids a randomized controlled trial Nutr CancerFine EJ SegalIsaacson CJ Feinman RD Herszkopf S Romano MC TomutaN Targeting insulin inhibition as a metabolic therapy in advancedcancer a pilot safety and feasibility dietary trial in patients Nutrition Epub Zhou W Mukherjee P Kiebish MA Markis WT Mantis JG Seyfried TN Thecalorically restricted ketogenic diet an effective alternative therapy formalignant brain cancer Nutr Metab Mukherjee P Mulrooney TJ Marsh J Blair D Chiles TC Seyfried TNDifferential effects of energy stress on AMPK phosphorylation and apoptosisin experimental brain tumor and normal brain Mol Cancer Gatenby RA Gawlinski ET Gmitro AF Kaylor B Gillies RJ Acidmediatedtumor invasion a multidisciplinary study Cancer Res Singh A Pandey A Tewari M Kumar R Sharma A Singh K Advancedstage of breast cancer hoist alkaline phosphatase activity risk factor forfemales in India Biotech Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsHuebner J Marienfeld S Abbenhardt C Ulrich C Muenstedt K Micke O Counseling patients on cancer diets a review of the literature andrecommendations for clinical practice Anticancer Res Champ CE Palmer JD Volek JS WernerWasik M Andrews DW Evans JJ Targeting metabolism with a ketogenic diet during the treatment ofglioblastoma multiforme J NeuroOncol Zuccoli G Marcello N Pisanello A Servadei F Vaccaro S Mukherjee P et alMetabolic management of glioblastoma multiforme using standard therapytogether with a restricted ketogenic diet case report Nutr Metab Servaes P Verhagen C Bleijenberg G Fatigue in cancer patients during andafter treatment prevalence correlates and interventions Eur J Cancer Cohen C Fontaine K Arend R Soleymani T Gower B Favorable effects of aKetogenic diet on physical function perceived energy and food cravings inwomen with ovarian or endometrial Cancer a randomized Controlled TrialNutrients Breymeyer KL Lampe JW McGregor BA Neuhouser ML Subjectivemood and energy levels of healthy weight and overweightobesehealthy adults on highand lowglycemic load experimental dietsAppetite TanShalaby JL Carrick J Edinger K Genovese D Liman AD Passero VA Modified Atkins diet in advanced malignanciesfinal results of a safetyand feasibility trial within the veterans affairs Pittsburgh healthcare systemNutr Metab Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Klement RJ Sweeney RA Impact of a ketogenic diet intervention duringradiotherapy on body composition I Initial clinical experience with sixprospectively studied patients BMC Res Notes Schmidt M Pfetzer N Schwab M Strauss I Kämmerer U Effects of aketogenic diet on the quality of life in patients with advanced cancer apilot trial Nutr Metab Tóth C Clemens Z Halted progression of soft palate Cancer in a patienttreated with the Paleolithic Ketogenic diet alone a 20months followupAm J Med Case Rep Gibson AA Seimon RV Lee CM Ayre J Franklin J Markovic T Doketogenic diets really suppress appetite A systematic review and metaanalysis Obes Rev Sremanakova J Sowerbutts A Burden S A systematic review of the use ofketogenic diets in adult patients with cancer J Hum Nutr Diet Khodabakhshi A Akbari ME Mirzaei HR Kazemian E Kalantari K Kalamian M Effects of ketogenic diet for breast cancer treatment A protocol forrandomized controlled clinical trial J Biochem Technol Khodabakhshi A Akbari ME Mirzaei HR MehradMajd H Kalamian MDavoodi SH Feasibility safety and beneficial effects of MCTbasedKetogenic diet for breast Cancer treatment a randomized controlled trialstudy Nutr Cancer Khodabakhshi A Akbari ME Mirzaei HR Seyfried TN Kalamian M DavoodiSH Effects of Ketogenic metabolic therapy on patients with breast Cancer arandomized controlled clinical trial Clin Nutr in press Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe European anization for Research and Treatment of Cancer quality oflife questionnaire EORTC QLQC30 translation and validation study of theIranian version Support Care Cancer Montazeri A Harirchi I Vahdani M Khaleghi F Jarvandi S Ebrahimi M et alThe EORTC breast cancerspecific quality of life questionnaire EORTC QLQBR23 translation and validation study of the Iranian version Qual Life Res Champ CE Volek JS Siglin J Jin L Simone NL Weight gain metabolicsyndrome and breast cancer recurrence are dietary recommendationssupported by the data Int J Breast Ca | Thyroid_Cancer |
Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earths species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleeps quotidian normalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brains clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patients desired sleep time or augmenting factors that entrainthe bodys clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon siesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period Catchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances morning larks latephase delays night owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airways soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisordersspecifically sleep walkingalso occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women in males and in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economos encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while asleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in the loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleepor its encephalopathic analogmay affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i | Thyroid_Cancer |
distribution and reproduction in any medium provided the original work isproperly citedLacrimal gland neoplasms comprise up to of all orbital masses clinically and histologically Much of our current coreknowledge regarding lacrimal gland tumors stems from prior study of their more common counterparts the salivary glandsThe prognosis for each lacrimal gland tumor is contingent upon proper clinical evaluation and ultimately the histopathologicdiagnosis We describe a case of an invasive carcinoma expleomorphic adenoma CaexPA with a cystadenocarcinomacomponent arising from the lacrimal gland in the absence of any previously diagnosed pleomorphic adenoma benign mixedtumor or prior incisional surgery This case illustrates the importance ofincludingimmunohistochemistry and genetic testing to narrow a diï¬erential diagnosis and potentially aid or guide therapy in the futureOur ï¬nding suggests that carcinoma of the lacrimal gland may be derived from previously undiagnosed and perhaps evensubclinical pleomorphic adenomathe histopathologic assessment IntroductionMalignant mixed tumor of the lacrimal gland also knownas carcinoma expleomorphic adenoma CaexPA is thethird most frequent epithelial lacrimal gland tumor afterpleomorphic adenoma PA and adenoid cystic carcinomaACA [] Clinically aï¬ected patients tend to be to years older than those with PA and often present with aninsidiously progressive mass of a lacrimal fossa that suddenly becomes symptomatic Other reported clinical settings are patients without any previously known lacrimalgland tumor who develop a rapidly growing mass associated with pain and patients with one or more recurrencesof PA who undergo malignant transformation [] Diagnostic imaging is critical for the clinical diagnosis of a lacrimalgland neoplasm Computed tomography CT and magnetic resonance imaging MRI are ideal but MRI remainsthe preferred method to visualize surrounding tissue anddetect radiographic features concerning aggressive malignancy [] The incidence of a malignant transformation ofPA increases with the duration of the tumor Prior studiesreport that approximately less than of PA undergoesmalignant change within years after the ï¬rst treatmentand by the end of years [] CaexPA can arisein patients without a known preexisting lacrimal glandtumor The malignant component is most often adenocarcinoma not otherwise speciï¬ed however other histologicsubtypes have been described such as adenoid cystic carcinoma ductal carcinoma mucoepidermoid carcinoma andsquamous cell carcinoma 0cCase Reports in PathologyHerein we report a case of an invasive CaexPA with anepithelial component consistent with a cystadenocarcinomain the absence of any previously diagnosed PA and supportedby immunohistochemical and molecular studies Case PresentationA 64yearold male patient presented to the emergencydepartment due to left lateral canthal pain tearing and ipsilateral hearing loss over four months The initial examinationrevealed a visual acuity of bilaterally The pupils werereactive without an aï¬erent pupillary defect Intraocularpressures were and mmHg respectively Confrontational visual ï¬elds and color plates were unremarkable Therewas a complete abduction restriction of the left eye Theexternal examination revealed edematous upper and lowereyelids predominantly overlying the lateral orbital rim associated with temporal sloping and a nontender palpable andï¬xed mass of the temporal fossa a There wasptosis of the left upper eyelid with a reduced marginal reï¬exdistance of two and a half millimeters mm Exophthalmometry measured mm and mm with a base measurement of mm The anterior segment was otherwisenormal The fundus exam revealed symmetrically sharp andpink disc margins without pallor or edemaMaxillofacial CT scan with contrast showed a lytic lesion ofthe left orbital wall with associated heterogeneous soft tissuemass measuring cm medially displacing the left lateralrectus muscle b Magnetic resonance imaging of thebrain and orbits revealed an enhancing ltrating mass of theleft lateral orbital wall extending into the left supra zygomaticmasticator space c A core guided needle biopsywas performed and the hematoxylineosin HE stainedslide revealed a moderately diï¬erentiated adenocarcinomainvolving ï¬brous connective tissue and demonstrating a cribriform architectural pattern with moderate cytologic atypia andindividual cell necrosis Figures 2a and 2bPositron emission tomography and CT of the chest abdomen and pelvis did not reveal any underlying malignancy orevidence of metastases Subsequently the patient underwentleft orbital exenteration with eyelid sparing Grossly the specimen included orbital contents frontal bone portions of thefrontal sinus and zygomatic bone Serial sectioning revealeda cm multilocular cystic mass involving the lacrimalgland fossa abutting the globe superotemporallyHistopathologically the HEstained sections disclosedpredominantly neoplastic cystic structures in the proximityof the lacrimal gland acini measuring to mm in diameterltrating ï¬brous connective tissue and bone cA small focus of pleomorphic adenoma was identiï¬ed associated with a lowgrade ductal carcinoma in situ The invasivecystic component revealed intraluminal papillary architecture and cribriform arch formations of the lining epitheliumd The neoplastic epithelium was composed ofmedium to largesized cuboidal cells with intercalated ductcell appearance eosinophilic cytoplasm and apocrine features Small foci of invasive solid components were observeddemonstrating cribriform architecture moderate to severenuclear pleomorphism and up to mitotic ï¬gures per highpower ï¬eld Columnar cells with pseudostratiï¬ed nucleiwere also present with moderate nuclear atypia Foci of ruptured cysts with hemorrhage granulation tissue lymphocyticltrate and dystrophic calciï¬cation were also seen No lymphovascular or perineural invasion was identiï¬edImmunohistochemical studies using monoclonal antibodies were performed with appropriate positive controls encompassing Gata3 mouse monoclonal antibody predilute Cell Marque Rocklin CA gross cystic disease ï¬uidprotein GCDFP15 mouse monoclonal antibody dilute Thermo Fisher Scientiï¬c Fremont CA androgen receptor AR rabbit monoclonal antibody dilute Cell Marque Rocklin CA SOX10 rabbit polyclonalantibody dilute Cell Marque Rocklin CAprostatic speciï¬c antigen PSA mouse monoclonal antibody predilute Cell Marque Rocklin CAthyroid transcription factor TTF1 mouse monoclonalantibody predilute at micrograms Ventana Medical Services Tucson AZ p63 mouse monoclonal antibody predilute Biocare Medical Concord CA p53mouse monoclonal antibody predilute at microgramsVentana Medical Services Tucson AZ high molecularweight cytokeratin HMWK903 mouse monoclonalantibody predilute Cell Marque RocklinCA CAM mouse monoclonal antibody predilute Ventana Medical Services Tucson AZ estrogen receptor ERmouse monoclonal antibody diluted VectorLaboratories Burlingame CA progesterone receptor PRmouse monoclonal antibody prediluted Leica BiosystemsNewcastle UK Her2neu rabbit monoclonal antibody predilute Ventana Medical Services Tucson AZ and Ki67rabbit monoclonal antibody predilute Ventana MedicalServices Tucson AZ Immunohistochemical studies wereperformed on an automated stainer Leica BiosystemsInc BOND III System Buï¬alo Grove IL All antibodiesand testing were performed in a CLIAcertiï¬ed laboratoryThe invasive component of the tumor was positive fata3 AR HMWK903 and CAM52 and focally positivefor GCDFP15 while negative for p63 ER PR SOX10 PSAand TTF1 stains Figures 2e2h p53 was positive in lessthan of the tumor cells The Ki67 proliferative index ofthe tumor cells was low p63 and SOX10 stainshighlighted the PA and the in situ component of the tumorDetection of HER2 status by immunohistochemistry wasequivocal and reï¬ex testing using ï¬uorescence in situhybridization FISH was negative for HER2neu geneampliï¬cation based on the updated guidelines of theAmerican Society of Clinical OncologyCollege of AmericanPathologists criteria for HER2neu testing in breast cancerMolecular proï¬ling using a nextgeneration sequencingNGS based assay Foundation One was performed onthe lacrimal gland tumor paraï¬nembedded tissue to identify genomic alterations within cancerrelated genes Thefollowing genomic alterations were detected HRAS G13RPIK3CA E542K and BCORL1 H215fs Microsatellitestatus MDstable and tumor mutation burden TMB low1MutsMbThe combined histopathologic immunohistochemicaland molecular ï¬ndings supported the diagnosis of invasive 0cCase Reports in PathologyaabcbcFigure a Clinical photo of the patient at time of presentation direct view b Computed tomography maxillofacial axial cut Illustrating a cm mass of the lateral orbit eroding the lateral orbital wall with a soft tissue component extending into the orbit causing proptosis andnonaxial displacement of the globe c MRI brain and orbit T1 fatsuppressed image with gadolinium showing a lobulated mass withmixed cystic and solid components and inhomogeneous enhancement involving the left lateral orbital wall and suprazygomatic leftmasticator space Additionally the mass demonstrates edema and enhancement suggesting ltration by the massCaexPA disclosing a carcinomatous component with apredominant cystic growth pattern and focal solid areasreminiscent of a cystadenocarcinoma The surgical marginswere negative In addition the microscopic examination ofthe left eye globe revealed pseudoexfoliation syndrome andFuchs endothelial dystrophyThe patient underwent postoperative adjuvant chemoradiation followed by excision of the eyelids with no residualtumor Followup examination showed no evidence of recurrence or metastatic disease nine months after completingadjuvant therapy DiscussionMuch remains unknown about both the natural histologicprogression and malignant transformation of CaexPAWe know that lacrimal gland neoplasms comprise oforbital masses clinically [ ] and of orbital masseshistologically [ ] and nearly half of epithelial tumorsare malignant [] Although uncommon malignant transformation of PA can occur with incomplete excision InvasiveCaexPA can represent malignant transformation of PAmany of which develop recurrence or distant metastasis tothe lung bone abdomen and CNS [] This casehighlights the importance of an immunohistochemical andgenetic evaluation in complex lacrimal tumorsThe malignant epithelial component of CaexPA hasmorphological varieties including adenocarcinoma adenoidcystic carcinoma squamous cell carcinoma mucoepidermoid carcinoma and ductal carcinoma It could be likelya mixture of subtypes [] Rarely the only evidence ofpleomorphic adenoma is the presence of large areas of hyalinized stroma composed of myoepithelial cells and a fewductal structuresIn the current case the carcinomatous component of thetumor discloses predominantly an ltrative cystic growthpattern reminiscent of a cystadenocarcinoma previouslydescribed in neoplasms of the salivary and lacrimal glandsThe term cystadenocarcinoma of the salivary gland hasevolved It encompasses a variety of tumors depicting a cysticpattern of growth to a subset of papillary and cystic malignant neoplasms that have indolent behavior This is alsoobserved in other lowgrade salivary gland carcinomashowever it is important to note that they can demonstrateltrative growthlocal recurrence and metastasize toregional lymph nodes at the time of diagnosisPreviously Foss [] in a review of cases ofcystadenocarcinoma of the salivary gland used the followingdiagnostic criteria occurrence within a salivary gland invasive growth a predominantly cystic pattern ofgrowth with or without a papillary component and theabsence of acinar or mucoepidermoid diï¬erentiation or evidence of origin in a PA In the same review the predominantcell type varies among tumors and includes small cuboidalintercalated ductlike cellslarge cuboidal cells and tallcolumnar cells The subgroups of the large cuboidal cells 0cCase Reports in PathologyababcHMWCK903Androgen receptordedp53ep63fcfghghFigure a Moderately diï¬erentiated adenocarcinoma involving cores of ï¬brous connective tissue HE 2x b High power view of thetumor revealed a cribriform pattern moderate nuclear pleomorphism and scattered mitoses HE 40x c Neoplastic cystic structures inclose proximity to lacrimal gland tissue star in a hyalinized stroma and lymphoid proliferation HE 2x d Tumor that arose from afocus of pleomorphic adenoma star depicting cystic structures with small papillae HE 4x Inset papillary projections lined by largecuboidal cells with moderate nuclear atypia and apocrine features HE 20x e Tumor cells were strongly positive for HMWK903 4xf Nuclear expression of androgen receptor was present in the tumor 20x g p53 positive in less than of tumor cells 4x h p63was negative within the invasive component of the tumor 4xhave central nuclei abundant eosinophilic cytoplasm largenucleoli and apocrine features which are similar to thoseobserved in this case The fourth group has a combinationof cell types Furthermore Foss suggest that tumorswith predominantly columnar cells are associated withincreased metastatic potentialThe cystic growth pattern characteristic of this tumor isoften associated with an ammatory response due toruptures of the dilated structures as noted in this case Cystformation in neoplasias of the salivary and lacrimal glandscan behave as a mimicker of an ammatory process Pakdel report a case of a spontaneous rupture of a PAmasquerading as orbital cellulitis [] Histologically aruptured cystic space of a PA surrounded by a monocyticltrate and foreign bodytype granulomas is described Inthis paper the authors consider the spontaneous rupture ofthe cystic space as an underlying mechanism for the acutepresentation of this tumorCystadenocarcinoma can have a broad diï¬erentialdiagnosis The principal considerations include papillary cystic acinic cell adenocarcinoma secretory carcinoma mucoepidermoid carcinoma MEC and ductal carcinoma Aciniccell carcinoma and secretory carcinoma are typically indolent monotypic tumors that can disclose a papillary cysticarchitecture Histologicallysecretory carcinoma sharesnearly identical growth patterns to acinic cell carcinoma 0cCase Reports in Pathologybut instead shows a multivacuolated eosinophilic cytoplasmoften with luminal and intracytoplasmic mucin and no truezymogen granules Immunohistochemically secretory carcinoma is S100 and mammaglobinpositive and typicallynegative for DOG1 while acinic cell carcinoma shows theopposite staining proï¬le Additionally secretory carcinomaharbors t1215p13q25 resulting in an ETV6NTRK3 genefusion [] MEC is usually composed of a mixture ofpredominantly epidermoid squamoid cells abundant intermediate cells ranging from small basal cells with basophiliccytoplasm to larger cells with eosinophilic cytoplasm andmucous cells Welldiï¬erentiated MEC is a circumscribedtumor that can disclose glandular and cystic structures linedby a single layer of mucussecreting cells however the intermediate and highgrade tumors show solid nests or sheets ofcells composed of primarily epidermoid cells with a scant cystic component and obvious invasion severe pleomorphismnecrosis and increased mitosesOn the other hand primary ductal adenocarcinoma ofthe salivary gland SDC originates from the excretoryportion of the salivary duct is a rare aggressive malignantepithelial tumor and accounts for only of epithelial lacrimal gland tumors Histologically the tumor is highly ltrative and usually solid with occasional cystic areas disclosingbreastlike ductal carcinoma features with central necrosisOccasionally cystadenocarcinoma with large cuboidal cellseosinophilic cytoplasm and highgrade nuclear atypia bearssome similaritiesto ductal adenocarcinoma Howeverpapillarycystic invasive growth is not usually seen in ductaladenocarcinoma [] The invasive component features trabeculae ducts and sheets of neoplastic cells in a desmoplasticstroma with perineural and vascular invasion The latter isnot commonly observed in cystadenocarcinoma Immunohistochemically the tumor cells are positive for a low molecular weight cytokeratin CAM52 CK7 CEA EMA andGCDFP15 Other than epithelial markersthis tumorexpresses AR in up to of invasive cases HER2 positivityin of cases and p53 overexpression in all reportedcases The Ki67 proliferative index is over The tumoris ER and PR negativeFurthermore in this case the tumor also demonstratescytomorphologicalfeatures similar to that of a low tointermediategrade ductal carcinoma in situ of the breast Thelatter ï¬ndings might correlate with the previously describedlowgrade cribriform cystadenocarcinoma of the salivary glandalso known as lowgrade salivary ductal carcinoma and salivaryductal carcinoma in situ currently categorized as intraductalcarcinoma low grade and high grade respectively These typesof tumors show a variety of growth patterns both solid and cystic ranging from cribriform to solid to micropapillary and arereminiscent of lowgrade ductal carcinoma in situ of the breastFocal ltration may be noted []The exact pathogenesis of CaexPA remains controversial Prior studies have demonstrated that PA and CaexPA of the salivary and lacrimal glands share similar genomicproï¬les and frequently overexpress the PLAG1 oncoprotein[ ] Harrison [] in their review indicate that thedevelopment of CaexPA follows a multistep model of carcinogenesis with the progressive loss of heterozygosity at 8qthen 12q and ï¬nally 17p Alterations including ampliï¬cationgene fusion and translocations in 12q genes such as HMGICHMGA2 and MDM2 may play a role in the malignant transformation In the same study the authors also mention thatloss of 17p is usually common in CaexPA indicating tumorsuppressor gene p53 loss as this tumor evolves Additionallyit appears that CaexPA and other malignant epithelialtumors other than ACC do not harbor MYB gene rearrangements or fusions It is important to note that there does notappear to be a correlation between rearrangement statusand clinical outcome []Additional mutational analysis of the lacrimal gland carcinomas has been also evaluated [] demonstrating that RASKRAS NRAS PIK3CA and MET mutations are frequent indiverse epithelial neoplasms of the lacrimal gland with thehighest proportion of mutations found in adenoid cystic carcinoma PIK3CA and MET mutations can coexist with RASmutationsPIK3CA and HRAS mutations are detected in this casewhich correlates with alterations already described in lacrimal gland carcinomas however alterations in MDM2HMGA2 NTRK3 p53 PLAG1 and ETV6 among others werenot observedIn summary this case demonstrates an invasive CaexPA with a malignant epithelial component that resemblesthe cystadenocarcinoma mixed cell type described by Foss [] Immunohistochemically the tumor was ARpositive while negative for ER PR and Her2 with expression of p53 in less than of the tumor cells Theprevalence of AR varies between the diï¬erent subtypes ofsalivary gland carcinomas SGC In recent medical literature data AR expression has been detected in as many as of SDC [] Dalin [] in the same study alsoindicate that adenocarcinoma and acinic cell carcinoma ofthe salivary gland express AR in and of the casesrespectivelyOur ï¬ndings emphasize the importance ofThe current classiï¬cation of the salivary gland tumors[] and the combined histopathologic and immunohistochemical features suggest that this tumor could representthe results of the natural course of a lowgrade cystadenocarcinoma with focal transformation into an intermediatehighgrade invasive ductal carcinoma expleomorphic adenomafurtherexploration of CaexPA pathogenesis especially the extentof disease and the histologic subtype In additiontheimmunohistochemical and genetic testing provides important support for the diagnosis as well as potentially guidesfuture therapy and prognostic evaluation Correctly identifying the type of malignant epithelial component is a signiï¬cant factor in the survival of aï¬ected patients To ourknowledge this patient represents the ï¬rst case in whichCaexPA of the lacrimal gland reveals an indolent epithelial malignancy with a low proliferative index arising froman undiagnosed PA after many likely consecutive molecular alterationsLastly due to the rarity of these tumors arising from thelacrimal gland further studies are necessary to evaluate theirbiologic behavior and determine any correlation betweenCaexPA and pseudoexfoliation syndrome 0cCase Reports in PathologyEnd Results International Journal of Ophthalmology vol no pp [] F A Jakobiec J R Bily and R L Font Orbit in OphthalmicPathology An Atlas and Textbook W H Spender Edpp WB Saunders Co Philadelphia 4th edition[] M H Devoto and J O Croxatto Primary cystadenocarcinoma of the lacrimal gland Ophthalmology vol no pp [] R H Simpson Salivary duct carcinoma new developmentsmorphological variants including pure in situ high gradelesions proposed molecular classiï¬cation Head and NeckPathology vol Supplement pp [] R D Foss G L Ellis and P L Auclair Salivary gland cystadenocarcinomas a clinicopathologic study of cases TheAmerican Journal of Surgical Pathology vol no pp [] L Barnes J W Eveson P Reichart and D Sidransky Pathology and Genetics of Head and Neck Tumours IARC [] J Antony V Gopalan R A Smith and A K Y Lam Carcinoma ex pleomorphic adenoma a comprehensive review ofclinical pathological and molecular data Head and NeckPathology vol no pp [] F Pakdel N Pirmarzdashti S Soltani Z Nozarian F AAmoli and A Kassaee Spontaneous rupture of lacrimalgland pleomorphic adenoma Ophthalmic Plastic and Reconstructive Surgery vol no pp e41e43 [] R R Seethala Head and neck pathology Surgical PathologyClinics vol no [] S L von Holstein A Fehr M Persson Lacrimal GlandPleomorphic Adenoma and Carcinoma ex PleomorphicAdenoma Genomic Proï¬les Gene Fusions and Clinical Characteristics Ophthalmology vol no pp [] T Y Chen M G Keeney A V Chintakuntlawar et alAdenoid cystic carcinoma of the lacrimal gland is frequentlycharacterized by MYB rearrangement Eye vol no pp [] M Dalin P Watson A Ho and L Morris Androgen receptor signaling in salivary gland cancer Cancers vol no p Conflicts of InterestNone of the authors declare any competing interests Currently Dr Del Valle Estopinal is the Director of OphthalmicPathology and Assistant Clinical Professor of the Departments of Ophthalmology and Pathology at University ofCalifornia Irvine The City Dr S Orange CA Authors ContributionsDr Maria Del Valle Estopinal was the ocular pathologistthat performed the histopathologic immunohistochemicaland genetic evaluation Dr Bryant Carruth wastheoculoplasticstrained ophthalmologist who performed thecore biopsy The other authors participated in the clinicalcare and preparation of this manuscript All authors readand approved the ï¬nal paperAcknowledgmentsThe authors acknowledge the cooperation of the patient Theyacknowledge the help of the SUNY Upstate Departmentof Pathology and Eye Plastic and Reconstructive Surgeonsof Central New York in the contribution to evaluationand treatment of this patientReferences[] C L Shields J A Shields R C Eagle and J P Rathmelllacrimal glandClinicopathologic review of cases oflesions Ophthalmology vol no pp [] R L Font J O Croxatto and N A Rao Tumors of the Eye andOcular Adnexa American Registry of Pathology WashingtonDC [] W Harrison P Pittman and T Cummings Pleomorphicadenoma of the lacrimal gland a review with updates ontransformation and molecular genetics SaudimalignantJournal of Ophthalmology vol no pp [] SO Baek YJ Lee SH Moon YJ Kim and YJ JunPrimary adenocarcinoma of the lacrimal gland Archives ofPlastic Surgery vol no pp [] D Bell M C Sniegowski K Wani V Prieto and B EsmaeliMutationallacrimal gland carcinomas andimplications for treatment Head Neck vol Supplement pp E724E729 landscape of[] J A Shields C L Shields J A Epstein R Scartozzi and R CEagle Jr Primary epithelial malignancies of the lacrimalgland the Ramon L Font lecture Ophthalmic Plastic Reconstructive Surgery vol no pp [] Lacrimal Gland Tumor Study Group An epidemiologicalsurvey of lacrimal fossa lesions in Japan number of patientsand their sex ratio by pathological diagnosis Japanese Journalof Ophthalmology vol no pp [] S L von Holstein M H TherkildsenJ U PrauseG Stenman V D Siersma and S Heegaard Lacrimal glandlesions in Denmark between and Acta Ophthalmologica vol no pp [] W M Hassan M S Bakry H M Hassan and A S AlfaarIncidence of orbital conjunctival and lacrimal gland malignant tumors in USA from Surveillance Epidemiology and 0c' | 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 |
"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 |
"Lactation has a negative effect on female sexual function Hormonal changes during lactation causechanges which might lead to dyspareunia lack of libido and anasmia There are various pharmacological andnonpharmacological approaches to treat sexual dysfunction While pharmacological treatment has multipleunwanted side effects nonpharmacological therapies such as complementary medicine are a potential saferalternative The aim of this study is to evaluate the effect of ear acupressure on sexual function of lactating womenMethodsdesign This is a randomized clinical trial with a parallel sham control group In this study lactatingwomen between months and year after childbirth were referred to health care centers in Qazvin City andwould be invited to participate Participants will be divided into intervention n and control n groupsusing simple block randomization Both intervention and sham control groups will be visited over sessionswithin a 4day interval At each visit the adhesives containing Vaccaria seed will be adhered for the interventiongroup while nonlatexbased adhesives with no Vaccaria seeds will be placed on the same ear acupoints for thesham control group Selected ear acupoints include genitalia two ear points pelvic point master shoulder andposterior pituitary gland The women will be asked to hold the seeds on their ears for days and press each earpoint three times a day for s After days they will be asked to remove the seeds from their ears and rest for day Sexual function as primary outcome in both groups will be assessed using the Female Sexual Function Indexbefore and immediately after and months after the intervention Also Sexual Quality of Life as secondaryoutcome will be assessed using Sexual Quality of LifeFemale SQOLF before and months after intervention Datawill be analyzed using repeated measure ANOVA at the significant level of Discussion This study is expected to support the impact of ear channel ear acupressure on sexual function inlactating womenTrial registration Iranian Clinical Trial Registration Center IRCT20190626044028N1 Registered on August Keywords Ear acupressure Sexual function Lactation Correspondence nbahramiqumsacir2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin IranFull 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 0cBarghamadi Trials Page of BackgroundThe postchildbearing period is a crucial stage in thewomans life that involves physical hormonal mentalsocial and cultural changes [] Fatigue insomnia somestressors such as child care and changes in the bodyimage along with hormonal changes can reduce interestin sexual life and decrease the number of sexual intercourses during this period [] Endocrine function of lactation has a negative effect on sexual function becauseprolactin decreases sexual hormones such as androgenand estrogen This mechanism reduces sexual functionbecause of vaginal dryness vaginal epithelium atrophyand dyspareunia [] About two thirds of women duringlactating period experience at least one sexual problemsuch as decreased libido lack of sexual pleasure dyspareunia and vaginal dryness which usually are resolvedwithin year after childbirth [ ] The prevalence ofsexual dysfunction increases from to in the prepartum period [] to to in the postpartum period[] The results of various Iranian studies confirmed thehigh prevalence to of sexual dysfunctionduring lactation [ ] Therefore sexual dysfunction is acommon problem during lactation that needs moreattention from health care providersIn the postchildbearing period most women try to return to their sexual life within to weeks after childbirth Howeverit may take up to year to resumesexual relationships with the same quality of beforepregnancy [] Psychotherapy and pharmacotherapy aretwo main methods for treating female sexual dysfunctionFSD [] However the US Food and Drug Administration FDA does not recommend any foods or medicines for the treatment of FSD [] Therefore nonpharmacological therapies such as complementary medicine are a potentially safer alternative to pharmacologicaltherapy Acupressure is one of the noninvasive complementary methods oftraditional Chinese medicineTCM based on the principles of acupuncture []Acupuncture stimulates medical points and meridiansthat are distributed throughout the body to regulatephysiologic reactions [] Acupressurelike acupuncture modulates a persons vital energy by stimulatingacupoints and meridians that are distributed throughoutthe body []The external ear is one of the several somatotopicmicrosystems that can be used in acupressure Ear acupressure also known as auriculotherapy or auricularacupressure [ ] was first presented in ancient Chinese medicine to years BC but Dr Paul Nogierwas the first Western physician who put forward auriculotherapy in a scientific way [ ] Accordingto this theory each part of the body is associated with aspecific part of the ear that reflects the physiological orpathological state of the body [] In this method theouter surface of the ear auricle can be stimulated to reduce pathological conditions in other parts of the body[] Ear stimulation can be performed in various wayssuch as manual finger pressure electrical stimulation lasers differenttypes of needles magnetic seeds andseeds to strengthen neural connections [] The WorldHealth anization WHO has recognized ear acupuncture as a promising therapeutic approach becauseof its efficacy in managing health disorders [] Ear acupuncture has been recognized as a form of acupuncturethat can affect all body ans [] Ear acupressure isan easy noninvasive and safe therapy [ ] It is easyto use for mothers who need to take care of their childin the postchildbearing period []There are several studies supporting the effects of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] However the effect of ear acupressure on sexualfunction has not been studiedResearch aimThis study is designed to investigate the effect of earacupressure on the sexual function of lactating womenMethodsDesign and settingThis is a randomized clinical trial with a parallel shamcontrolled group The study is designed in accordancewith the CONSORT standards Lactating women referred to health care centers in Qazvin City Iran will beinvited to participate Figure shows the CONSORTflow diagram The current protocol is anized basedon the SPIRITTCM extension []ParticipantsIn this study breastfeeding women between monthsand year after childbirth will be referred to health carecenters in Qazvin City Iran and they will be invited toparticipate in the study Inclusion criteria will be willingness to participate in the study being literate beingprimiparous breastfeeding fullterm singleton deliverylesionfree auricle no ulcer and pain in the ear and ability to present for all intervention sessions Exclusion criteria will be being away from their spouse for more than month having complications during pregnancy orhaving postpartum depression in the postchildbearingperiod These criteria were diagnosed using the Edinburgh Postpartum Depression Scale substance abuse inthe individual or the spouse and history of illnessesaffecting sexual function in a woman or her spouse egpremature ejaculation cardiovascular mental health disorders thyroid diseases any form of cancer or injuries 0cBarghamadi Trials Page of Fig The CONSORT flow diagramof the genital area The use of drugs affecting sexualfunction such as psychotropic cardiovascular neurological and hormonal drugs will also be excluded Afterthe initial screening the Demographic and ObstetricQuestionnaire and Female Sexual Function Index FSFIwill be completed by the participants before anyinterventionRole and qualification of practitionersThe research team consists of one specialist in auriculotherapy TO two specialists in reproductive health ZAand NB and one midwifery postgraduate student Theprotocol of intervention was designed based on the literature review and expert opinion of TO who is an expert inauriculotherapy and is a journal editor for several international journals Other team members have been qualified in this technique before designing present researchParticipant screening and care providing will be done bySB and ZA and NB will monitor her during the first sessions to ensure the fidelity of providing interventionSample size calculationSample size is estimated according to the study ofBokaie with the mean and standard deviation of 0cBarghamadi Trials Page of total sexual function score has been reported as ± and ± in the intervention telephonecounseling group and control groups respectively []type error α Therefore considering the first confidence the second type error β power and error d and the possibility of loss tofollowup individuals for each group are requiredSo the total sample size will be peopleInterventionEar acupressure groupThe researcher will clean the auricle using alcoholand then place Vaccaria seeds using nonlatexbased adhesives on ear acupoints for genitalia pelvis shoulderand the posterior pituitary gland Figure illustrates theintended ear acupoints Intervention is designed for sessions within a 4day interval During these sessionsthe seeds are placed on the designated ear acupressurepoints The women will be instructed to keep the seedson their ears for days during which each point shouldbe compressed three times a day for s The compression should be performed with moderate stimulationthrough pressing steadily and slightly tighter until feelinga slight tingling and discomfort After days they willbe asked to remove the seeds from their ears and restfor a day [] The women will be reminded daily bysending the text and will be reminded by phone for thenext interventional session This procedure will be performed for ten sessions for each participant It should benoted that if a participant has problems during the useof the method for any reason displacement of seedsdiscomfort etc she can remove the previous seed andask the researcher to reattach new seedsSham control groupThe control group will have sessions that will be thesame as the intervention group except that no Vaccariaseeds will be placed on the ear acupointsMeasuresIn this studycollectionfour measures will be used for dataDemographic and Obstetric QuestionnaireThe demographic section includes questions such asage education level occupation place of residence economic status age at marriage and length of marriageThe obstetric section includes questions regarding thenumber of pregnancies type of contraceptive methoddelivery date type of delivery perinealinstrumental delivery history of painful intercourse in prenatalperiod infant gender birth weight first lactation number of intercourse per month before pregnancy onset offirst intercourse after delivery and average number ofintercourses per week during lactation Validity of thisquestionnaire will be confirmed by some of the facultymembers ofthe midwifery department of QazvinUniversity of Medical Sciences IraninjuryFig The selected ear acupoints 0cBarghamadi Trials Page of Female Sexual Function Index FSFIIt has been designed by Rosen to evaluate sexualfunction in women over the past weeks [] It consistsof items subdivided into six subcategories of sex desire items arousal items lubrication items asm items satisfaction items and pain itemsThese subcategories have response ranges from or to with higher scores indicating better sexual performance [] The questionnaire has been used in manystudies abroad and has shown to have a high degree ofinternal consistency reliability and validity [] Thefindings of Fakhri and Mohammadis study showed thatthe Farsi version of FSFI FSFIIV is a reliable and validinstrument with appropriate psychometric properties toevaluate womens sexual function [ ] Reliability ofthe scale has been calculated through stability analysisor calculation of the internal consistency coefficient TheCronbachs alpha coefficient was and was higher forall domains and for the whole scale [] In addition reliability was confirmed by the testretest method and thecorrelation coefficient was reported high indicating thatFSFIIV is reusable within a 4week interval [ ]The maximum score for each domain is and for thewhole scale is Also any score less than will beused to indicate sexual dysfunction []The Edinburgh Postpartum Depression Rating ScaleThis 10item scale has been designed to detect depression from weeks after delivery The Edinburgh Scalescores vary between and with cutoff point andabove to detect postpartum depression [] Psychometric evaluation of the Farsi version of this scale is carriedout in [] The Cronbachs alpha for the Edinburgh Scale has been reported as Validity of the Edinburgh Beck Scale has been reported as Thefindings of the study confirmed the high validity of theFarsi version of the Edinburgh Scale for the diagnosis ofpostpartum depression []The Sexual Quality of LifeFemale SQOLFIt is a short tool that specifically assesses the relationshipbetween sexual function and womens quality of life Ithas been developed by Symonds [] This 18itemquestionnaire focuses on sexual selfesteem emotionalaffairs and relationships Each item is responded with aLikert scale of strongly agree score to strongly disagree score Higher scores indicate better quality ofsexual life for women [] Validity and reliability of theFarsi version ofthis scale have been confirmed byMaasoumi []Primary outcomePrimary outcome of this study is to investigate changesof sexual function using the FSFISecondary outcomeThe secondary endpoint is investigating the quality ofwomens sexuallife which will be assessed using theSexual Quality of Life Questionnaire Any harm or sideeffects will be also reportedStudy procedureAfter obtaining the necessary permissions from the Ethics Committee of Qazvin University of Medical Sciencesregistration at the Iranian Clinical Trial RegistrationCenter IRCT and obtaining the permission from Qazvin Nursing and Midwifery School the researcher willattend for recruitment in Qazvin Health Centers Thelactating women of each center will be invited for participation The process and purpose of the research willbe explained to those who meet the inclusion criteriaAlso they will be ensured that their information will bekept confidential and that the questionnaires will be anonymous and coded in accordance with the researchethics They will enter into the study after signing thewritten consent form The researcher will ensure thatthey can leave the study at any time After enrolling eligible individuals a simple block randomization will beused Participants will be randomly assigned to the intervention and control groupsThe questionnaires will be filled out by the participants before the intervention Then the interventionacupressure will be performed as mentioned aboveEvaluation of sexual function in both the interventionand control groups will be performed using the FSFIquestionnaire immediately and months after theintervention Figure provides the timeline of recruitment allocation and assessmentStatistical analysisData will be analyzed using the chisquare test Fisherexact test and independent t test via the SPSS v24 software The KolmogorovSmirnov and Shapiro tests willalso be used to check for the normal distribution of sexual function scores Descriptive statistics number meanand standard deviation will be used to describe thedemographic characteristics of the participants The repeated measure ANOVA will be used to compare themean sexual function of women before and after theintervention The significance level of all tests is set asp Methods to protect against biasRandomization and allocationSampling will be made using a twostep samplingmethod In the first step Qazvin City will be consideredas five geographical districts Next two health centerswill be randomly selected in each of these five districtsof Qazvin City Secondly women referred to selected 0cBarghamadi Trials Page of STUDY PERIODEnrolment AllocationPostallocationCloseoutTIMEPOINT1t1t2t3t4t5t6t7t8t9t10t11t12t13tXENROLLMENTEligibility screenInformed consentRandomized by independent personAllocationINTERVENTIONSGroup A Auricular acupressureGroup B ControlASSESSMENTSFSFISQOLXXXXXXX X XXFig Schedule of enrolment interventions and assessmentshealth centers will be assessed for eligibility and in caseof willingness to participate in the study will be recruited They will be randomly assigned to two groupsas follows One letter is assigned to each group A intervention group B control group and all possible conditions for the 4block will be written and numbered asfollows 1AABB 2ABAB 3BBAA 4BABA 5ABBA6BAAB In a simple block randomization method using thetable of random numbers numbers of blocks will be selected until the specified sample size is achieved Therandom allocation sequence is specified For example ifthe numbers given are and respectively theallocation sequence will be as follows AABB ABABABAB BBAA As a result each participant will have aunique code n Allocation concealmentFor this purpose after preparing the allocation sequencethe sequence is annotated on paper and placed inopaque sealed envelopes which will be numbered consecutively The questionnaires will be coded in the samemanner A questionnaire with the same code will becompleted by the person who receives code intervention The assignment sequence and its concealment willbe performed by someone outside the research teamBlindingParticipants outcome assessor and statistical analyzerwill be blind to which group a participant was placedThe individual entering the data from the participantquestionnaires on demographic information postpartumdepression female sexual functioning and female qualityof life will be blind to which participant was assigned tothe auricular acupressure seed group or the sham control groupTreatment fidelityOne of the researchers SB is responsible to performintervention She has participated specific course on auriculotherapy and is supervised by acupuncturist MHAabout how to perform the intervention Approximately of intervention sessions will be run under supervision of the acupuncturist to ensure that all acupoints arechosen correctlyData managementOne of the researchers SB will be responsible for collecting data at each study stage and will assign anotherindividual for the task of data entry into the SPSS software This process will be performed under supervisionof NB and ZA 0cBarghamadi Trials Page of Ethical and safety issuesThe research protocol will be reviewed and approved bythe Ethics and Human Research Committee of QazvinUniversity of Medical Sciences decree code IRQUMSREC1398056 It is registered on the Iranian ClinicalTrial Registration Center underofIRCT20190626044028N1 In addition the following ethical considerations will be considered throughout this research obtaining written informed consent the volunteernature of participation in the study ability to withdrawfrom the study at any time and confidentiality of information even during the publication of findings All ethical issues related to clinical trials will be considered Informedconsent during recruitment phase will be obtained by SBdecreecodeDiscussionTo the best of our knowledge this study is the first randomized clinical trial investigating the effect of ear acupressure on the sexual function of lactating women Thestrength of this study is the randomized design with parallel treatment procedures and a large sample size butdue to the importance of the treatment practitionerknowing whether they are placing an adhesive patchwith a Vaccaria seed or without one a true double blindstudy is not possible Acupressure has no known side effects is noninvasive and is easy to use [ ] Therefore the therapist and clients can develop a sense ofclosenesstrust and confidence [] Several studieshave shown promising results about the effect of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] In the study of Oakley acupuncture wasaffective in premenopausal women with sexual function[] In line with previous literature this study wouldprovide insights about the impact of ear acupressure onsexual function in lactating womenAbbreviationsCONSORT Consolidated Standards of Reporting Trials SQOLF Sexual Qualityof LifeFemaleAcknowledgementsNot any in this stageTrial statusThe recruitment has not yet begun and necessary permissions are acquiredthe estimated date of recruitment is November The expected timefor completing the recruitment is March Protocol version registered on August Authors contributionsAll authors SB ZA NB and TO contributed to the design of the study SBNB and ZA drafted the preliminary manuscript TO revised the manuscriptand prepared the final version of the manuscript All authors revised themanuscript agreed to be fully accountable for ensuring the integrity andaccuracy of the study and read and approved the final version of themanuscript to be published All the authors met the criteria for authorshipand listed as coauthors on the title pageFundingThe research deputy of Qazvin University of Medical Sciences will providefinancial support Study funders have no role in the study design thecollection management analysis and interpretation of data the writing ofthe report and the decision to submit the report for publicationAvailability of data and materialsAnalyzed data and materials will be deidentified and publishedEthics approval and consent to participateResearch protocol is approved by the Ethics and Human ResearchCommittee of Qazvin University of Medical Sciences decree codeIRQUMSREC1398056 Permissions from responsible authority will beobtained before recruitment Informed consent will be acquired beforeparticipationConsent for publicationNot applicableCompeting interestsNone to declareAuthor details1Student Research Committee Qazvin University of Medical Sciences QazvinIran 2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin Iran 3Emperors College of TraditionalOriental Medicine Santa Monica CA USAReceived November Accepted August ReferencesAbdelhakm EM Said AR Elsayed DMS Effect of PLISSIT model sexualcounseling program on sexual quality of life for postpartum women Am JNurs Sci Avery MD Duckett L Frantzich CR The experience of sexuality duringbreastfeeding among primiparous women J Midwifery Womens HealthAcele Eà Karaçam Z Sexual problems in women during the firstpostpartum year and related conditions J Clin Nurs Mohammed YF Hassan HM AlDinary AM Rashed NS Sexual function afterchild birth according to the mode of delivery AAMJ Si H Kumamoto Y Sato Y Masumori N Horita H Kato R Prevalenceof female sexual dysfunction symptoms and its relationship to quality of lifea Japanese female cohort study Urology Williams A HerronMarx S Carolyn H The prevalence of enduring postnatalperineal morbidity and its relationship to perineal trauma Midwifery Ahmad Shirvani M Bagheri NM Sexual dysfunction and related factorsamong breast feeding women Iran J Obstet Gynecol Infertility Tork Zahrani S Banaei M Ozgoli G Azad M Investigation of the postpartumfemale sexual dysfunction in breastfeeding women referring to healthcarecenters of Bandar Abbas Iran J Obstet Gynecol Infertility Yörük F Karaçam Z The effectiveness of the PLISSIT model in solvingpostpartum sexual problems experienced by women Athens J Health Davis SR Van Der Mooren M van Lunsen RH Lopes P Ribot J Rees M et alEfficacy and safety of a testosterone patch for the treatment of hypoactivesexual desire disorder in surgically menopausal women a randomizedplacebocontrolled trial Menopause Belkin ZR Krapf JM Goldstein AT Drugs in early clinical development forthe treatment of female sexual dysfunction Expert Opin Investig Drugs Ozgoli G Mobarakabadi SS Heshmat R Majd HA Sheikhan Z Effect of LI4and BL32 acupressure on labor pain and delivery outcome in the first stage 0cBarghamadi Trials Page of of labor in primiparous women a randomized controlled trial ComplementTher Med Oleson T Auriculotherapy manual Chinese and Western systems of ear Mohammadi KH Heydari M Faghihzadeh S The Female Sexual FunctionIndex FSFI validation of the Iranian version Health Monit J Iran Inst HealthSci Res Burri A Cherkas L Spector T Replication of psychometric properties of theFSFI and validation of a modified version FSFILL assessing lifelong sexualfunction in an unselected sample of females J Sex Med Sidi H Abdullah N Puteh SEW Midin M The female sexual function indexFSFI validation of the Malay version J Sex Med Sun X Li C Jin L Fan Y Wang D Development and validation of Chineseversion of female sexual function index in a Chinese populationa pilotstudy J Sex Med Ter Kuile MM Brauer M Laan E The female sexual function index FSFI andthe female sexual distress scale FSDS psychometric properties within aDutch population J Sex Marital Ther Cox JL Holden JM Sagovsky R Detection of postnatal depressiondevelopment of the 10item Edinburgh Postnatal Depression Scale Br JPsychiatry Ahmadi kani Golzar A GoliZadeh Z Validation of Edinburgh PostpartumDepression Scale EPDS for screening postpartum depression in Iran J NursEduc Symonds T Boolell M Quirk F Development of a questionnaire on sexualquality of life in women J Sex Marital Ther Maasoumi R Lamyian M Montazeri A Azin SA AguilarVafaie ME HajizadehE The sexual quality of lifefemale SQOLF questionnaire translation andpsychometric properties of the Iranian version Reprod Health Oakley SH WaltherLiu J Crisp C Pauls R Acupuncture in premenopausalwomen with hypoactive sexual desire disorder a prospective cohort pilotstudy Sexual medicine 201643e176e81Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsacupuncture Yeh ML Chang YC Huang YY Lee TY A randomized controlled trial ofauricular acupressure in heart rate variability and quality of life forhypertension Complement Ther Med Tan JY Molassiotis A Wang T Suen LK Current evidence on auriculartherapy for chemotherapyinduced nausea and vomiting in cancer patientsa systematic review of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chien LC Chiang YC Lin SW Huang CK Ren D Reduction innausea and vomiting in children undergoing cancer chemotherapy byeither appropriate or sham auricular acupuncture points with standard careJ Altern Complement Med Abbate S Chinese auricular acupuncture Florida Routledge Yeh TL Chen HH Pai TP Liu SJ Wu SL Sun FJ The effect ofauricular acupoint stimulation in overweight and obese adults a systematicreview and metaanalysis of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chiang YC Hoffman SL Liang Z Klem ML Tam WW Efficacyof auricular therapy for pain management a systematic review and metaanalysis Evid Based Complement Alternat Med Kim JY Ryu HS Nam SH Park KS Effects of auricular acupressure therapy onnocturia and insomnia in the elderly Korean J Rehabil Nurs Wang YJ Hsu CC Yeh ML Lin JG Auricular acupressure to improvemenstrual pain and menstrual distress and heart rate variability for primarydysmenorrhea in youth with stress Evid Based Complement Alternat MedShergis JL Ni X Jackson ML Zhang AL Guo X Li Y A systematicreview of acupuncture for sleep quality in people with insomniaComplement Ther Med Dantas KKdL Auriculoterapia chinesa com o uso de sementes de colza nadismenorreia primaria relato de caso Universidade Federal do Rio Grandedo Norte Portman DJ Bachmann GA Simon JA Group OS Ospemifene a novelselective estrogen receptor modulator for treating dyspareunia associatedwith postmenopausal vulvar and vaginal atrophy Menopause Oleson T Flocco W Randomized controlled study of premenstrualsymptoms treated with ear hand and foot reflexology Obstet GynecolKwon SJ Park JS Effects of auricular acupressure on chemotherapyinducednausea vomiting and serum serotonin level Korean J Adult Nurs Di YM May BH Zhang AL Zhou IW Worsnop C Xue CC A metaanalysis ofearacupuncture earacupressure and auriculotherapy for cigarette smokingcessation Drug Alcohol Depend Huang ETY Di PhD YM Acupuncture therapies for chronic obstructivepulmonary disease a systematic review of randomized controlled trialsAltern Ther Health Med Dai L Cheng CW Tian R Zhong LL Li YP Lyu AP Standard ProtocolItems for Clinical Trials with Traditional Chinese Medicine recommendations explanation and elaboration SPIRITTCM extension Chin J Integr Med Bokaie M Hajimaghsoudi S Dehghani A Hosseini F The effect of sexualhealth counselling on the sexual function and satisfaction of breastfeedingwomen in the form of group consultation and telephone consultancyJ Adv Pharm Educ Res 20199S2191 Rosen CB Heiman J Leiblum S Meston C Shabsigh R Ferguson DD'Agostino R The Female Sexual Function Index FSFI a multidimensionalselfreport instrument for the assessment of female sexual function J SexMarital Ther Fakhri A Pakpour AH Burri A Morshedi H Zeidi IM The Female SexualFunction Index translation and validation of an Iranian version J Sex Med Wiegel M Meston C Rosen R The female sexual function index FSFI crossvalidation and development of clinical cutoff scores J Sex Marital Ther 0c" | Thyroid_Cancer |
coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from to scale range to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors knowledge is the first cancerspecific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of OtolaryngologyHead and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of OtolaryngologyHead and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of OtolaryngologyHead and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of OtolaryngologyHead and Neck Surgery McMaster University Hamilton Ontario Canada Department of OtolaryngologyHead and Neck Surgery Western University London Ontario Canada Department of OtolaryngologyHead and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and NeckEndocrine Oncology Moffitt Cancer Center Tampa Florida Department of OtolaryngologyHead and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of OtolaryngologyHead and Neck Surgery Stanford University Palo Alto California Department of OtolaryngologyHead and Neck Surgery University of Michigan Ann Arbor Michigan Department of OtolaryngologyHead and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of OtolaryngologyHead and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of OtolaryngologyHead and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of OtolaryngologyHead and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from to and lowpriority indications were assigned scores ranging from to Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a noncancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of and all other indications were assigned a weight of The total scale score ranged from to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLi gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEsrotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaOrdinal scale refers to the scale of to used to rate priority of surgery and Per Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition | 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 |
"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 |
"evaluate the clinicopathologic characteristics of Lymph Node metastasisbetween investing layer of Cervical fascia and deep fascia of infrahyoid strap Muscles LNCM in papillary thyroidcarcinoma PTCMethods Retrospective review of patients with PTC who underwent thyroidectomy and central compartment neckdissection CND from January to January was performed in two tertiary referral academic medicalcenters A total of consecutive patients with PTC who underwent thyroidectomy and CND were included inthe retrospective review The LNCM was resected as a separate specimen by the surgeon and the clinicopathologiccharacteristics of the patients were recorded Multivariate logistic regression analysis was performed to identify riskfactors for LNCM metastasisResults Of PTC patients patients had lymph nodes in the LNCM Among them caseswere confirmed to be positive in the LNCM In total the metastasis rate of LNCM in PTC patients was Univariate analysis revealed that the metastasis of LNCM were more likely to have a primary site in theinferior pole extrathyroidal extension ETE central cervical metastasis level III and level IV metastasis Multivariateanalysis further showed tumor location in the inferior pole ETE level III and level IV metastasis conferred asignificantly increased odds ratio for LNCM metastasisConclusion Attention should be paid to the lymph tissue in the LNCM for PTC patients especially in presence of aprimary site in the inferior pole ETE level III and level IV metastasisKeywords Thyroid carcinoma Surgery Central compartment neck dissection Recurrence Suprasternal spaceIntroductionPatients with papillary thyroid carcinoma PTC have afavorable prognosis with central neck locoregional recurrence varying from to [] The goal of a prophylacticor therapeutic central compartment neck dissection pCNDor tCND is to decrease the incidence of local recurrenceby removing all lymphatic tissue within the levels VI and Correspondence wugaosongwhueducn1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei Peoples Republic of ChinaFull list of author information is available at the end of the VII compartments which are generally the first and themost commonly involved with metastasis [] For patientswithout evidence of lymph node metastasis on preoperativeevaluation the additive value of a pCND at the time ofthyroidectomy is controversial Some authors advocatepCND considering high rate of occult metastaticnodal disease in cN0 PTC [] while other authors considerthat there is no highlevel evidence in favor of pCND []The performance of pCND is dependent on the weightgiven to the risks and benefits of pCND [] Consideringthe oncologic benefits of CND and the risks of a repeat 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 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of neck operation performing pCND is recommended toevery patient in China [ ]Although American Thyroid Association ATA guideline has defined the boundary of central neck compartment there is also significant variability in terms of theextent of CND In routine clinical practice CND canrange from sampling a few nodes in the paratrachealregion to a complete clearance from left carotid artery toright carotid artery and down to and including the uppermediastinum [] Owing to the variant extent of CNDsome central compartments are easily to be neglectedFor thyroid carcinoma patients with specific clinicopathologic characteristics incomplete lymph node dissectionmay result in increased recurrence reoperation andreoperationassociated complications [] Lymph Nodebetween investing layer of Cervical fascia and deep fasciainfrahyoid strap Muscles LNCM has not beenofreported The LNCM compartment is defined as followssuperiorly by the hyoid bonelaterally by the carotidarteries anteriorly by the investing layer of cervicalfascia and posteriorly by the deep fascia of infrahyoidstrap muscles LNCM space includes suprasternal spaceand intrainfrahyoid strap muscle spaceAnatomically LNCM is located anterior to the strapmuscles We consider that what is special about the concept of the LNCM is that it is belong to level VI but is aneasily overlooked anatomical area by a strap musculatureinvolving the sternohyoid and sternothyroid musclesduring selective or modified neck dissection Although themetastasis in LNCM was seldom it did occur in somePTC patients with regional recurrence As part of LCNMsuprasternal space metastasis for thyroid cancer wereinvestigated in three studies [] Thus we routinelydetected the suprasternal space and intrainfrahyoid strapmuscle space Fig This study was performed to identify the clinicopathologic characteristics and indication forlymph node metastasis dissection in the LNCMMaterials and methodsPatientsA retrospective review from the clinical and histopathologydatabase of two tertiary referral academic medical centersTongji Hospital of Huazhong University of Science andTechnology and Zhongnan Hospital of Wuhan Universityfrom January to January were conducted In theinstitutions preoperative examinations consisted of athorough physical examination neck ultrasound a clinicalevaluation of thyroid nodules and neck lymph nodes Fineneedle aspiration cytology FNAC were performed in patients who were suspected thyroid nodules or lymph nodeWith a pathological confirmation of PTC all the patientsreceived a thyroidectomy with CND Accordingly a pCNDwas performed for cN0 patients and a therapeutic CNDwas performed for cN1 patients The inclusion criteria forthe patients were as follows the clinical history completely recorded the LNCM was resected as a separatespecimen by the surgeon PTC patients who underwentthyroidectomy plus CND with or without lateral neckdissection A total of consecutive PTC patients wereenrolled The medical ethic committee of ZhongnanHospital of Wuhan University approved the procedure andinformed written consent was obtained from all patientsSurgical approachAll the operations were performed by the same seniorsurgeon Gaosong Wu with the patients under generalFig Four subdivisions Level VI 1st Level VI 2nd Level VI 3rd and Level VI 4th of central neck compartment are divided by deep fascia ofinfrahyoid muscles pretracheal visceral fascial and right recurrent laryngeal nerve 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of anesthesia Thyroidectomy was performed with a standard technique of fine capsular en bloc dissection andresection from inferior pole to superior pole []Intraoperative neuromonitoring was employed for all ofthe thyroidectomies [] Superior parathyroid glandswere identified and preserved in situ inferior parathyroidglands were protected in situ or autotransplanted in thesternocleidomastoid muscle according to three certaintypes based on their blood supply and location [ ]After the incision of the investing layer of cervicalfascia the interval between sternohyoid and sternothyroid muscles and the space anterior to the sternohyoidmuscle above the clavicle and the sternum weredetected If there was fibrofatty tissue Instead of the enbloc removal of the entire centrallymph nodes theLNCM was resected as a separate specimen if occurredThe presence or absence of lymph node metastasis wasdefined according to postoperative pathological reportsWhile dissecting paratracheal lymph nodes intraoperative neuromonitoring was employed to detect RLN fromdistally to proximally minimizing morbidity from injuryto RLN during compartment nodal dissection LNCMand other compartment lymphatic tissue were processedfor routine hematoxylin and eosine HE separatelyThe pathologic results were independently determinedby two qualified pathologists without any prior knowledge of the patients clinical dataData collection and statistics analysisTo determine the relation between LNCM metastasis andclinicopathologic factors such as age sex primary tumorsite lateral cervical lymph node metastasis level VI metastasis the chisquare test and Fishers exact test were usedas appropriate Multivariate logistic regression analysiswas performed to identify risk factors for LNCM metastasis of PTC P was considered statistically significantAll calculations were performed using SPSS for windows Postthyroidectomy hypocalcemia lasting for morethan months was considered as permanent VCP All patients were followed up every months postoperativelyResultsPatients detected with LNCMAfter reviewing patients who underwent thyroidectomy plus CND with or without lateral neck dissectionfrom January to January patients were detected with LNCM and patients wereabsent of LNCM The average tumor size of LNCM was cm and the mean number of lymph nodes sampledfrom LNCM was ranging from to Table showsthe comparison of clinicopathologic characteristics between the present LNCM group and the absent groupIn univariate analysis Hashimotos disease p multifocality leisions p the tumor located ininferior portion p extrathyroidal extensionETE p central cervical metastasis p level III and level IV metastasis p were significantly associated with high prevalence of LNCMPatients with metastatic LNCMAmong a total of patients with LNCM metastaticLNCM was found in patients Table compares the clinicopathologic characteristics between themetastatic LNCM group and the nonmetastatic LNCMgroup Three hundred eightythree patients were confirmed free of LNCM metastasis of themwith clinically negative node performed pCND and of them with clinically positive performed tCND All thepatients in the metastatic LNCM group performedtCND Lateral neck dissection was performed in cases in the metastatic LNCM group and cases in the nonmetastatic group all lateral neckdissection wastherapeutically performed Univariateanalysis was performed for the patients with and patients without metastatic LNCM Age at diagnosisgender and tumor size coexistent thyroid disease tumorfocality and level II metastasis were not correlated withLNCM metastasis Univariate analysis identified tumorlocated in the inferior pole central cervical metastasisETE level III and level IV metastasis as significant predictors of LNCM metastasis in our study population Multivariate analysis further showed that tumor location ETElevel III and level IV metastasis conferred a significantlyincreased odds ratio for LNCM metastasis Table ComplicationsThe median followup time was months range of patients had voice changes all of whomrecovered within months Temporary vocal cord paralysis was confirmed in patients by laryngoscope andthirteen permanent hypocalcemia was observed aftersurgeryDiscussionIn order to achieve the best chance of cure and effectivedisease control thoroughness of dissection has to betaken into account We prospectively performed comprehensive CND for PTC patients who underwent thyroidectomy and CND In addition data were analyzed for PTC patients to investigate the clinicopathologic characteristics for LNCM metastasis The occurrence rate ofLNCM was and of the patients harbored metastatic LNCM In total the positiverate of the LNCM was In this studymultivariate analysis revealed that a primary site in the inferior pole ETE level III and level IV metastasis were ofhigher LNCM metastasis rate which was consistent withthe findings by the previous report oflymph node 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for patients who had lymph nodes in LNCM compared tothose who did notVariablesAge mean ± SDAbsent n Present n P value¤ GenderFemale MaleTumor size cm ¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimotos diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes No LNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid Musclesmetastasis between sternocleidomastoid and sternohyoid muscle []Several studies have emphasized the importance of similar compartment in neck dissection for thyroid carcinomaSun pioneered the confirmation of the significantinvolvement of lymph node metastasis between sternocleidomastoid and sternohyoid muscle LNSS in lateralneck dissection [] which anatomically classified as part ofthe space of Burns They concluded that the positive rate ofLNSS was in clinically nodepositive cN PTCwhich was correlated with a primary site in the inferiorpole the lateral nodal metastasis level III and level IV nodalmetastasis [] Then Homma [] reported two casesof PTC patients with level III and IV lymph node metastases as well as metastasis in the suprasternal space Yu et al[] investigated the clinical significance of the suprasternalspace lymph node SSLN in pathological nodepositivepN PTC patients They concluded that metastasis rate ofSSLN was and the high SSLN metastasis of PTC wascorrelated with primary cancer site in the inferior thyroidpole strap muscle invasion level IV metastasis and LNSSmetastasis In our experience LNCM was rarely occurredin the central neck compartment and the positiveLNCM in PTC patients was infrequent as well Notably among the patients with pN PTC themetastasis rate of LNCM was which was muchlower than the metastasis incidence of SSLN described by Yu [] According to their results onefifth patients with pN PTC were performed incompleteCND and remained metastatic lymph nodes 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for positive LNCMVariablesAge mean ± SDMetastasis n Nonmetastasis n ¤ GenderFemale MaleTumor size cm ¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimotos diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes NoLNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid MusclesP value The total number of lymph nodes in the central neckcan range from to [] There is no consensus on thenumber of nodes removed or examined that would constitute an adequate dissection Aimed to allow surgeons tomore accurately report the extent of lymphadenectomyTable Multivariate analysis of predictors for LNCM positivityVariablesTumor locationp valueCentral cervical metastasisExtrathyroidal extensionLevel III metastasisLevel IV metastasisOR CI LNCM Lymph Node between superficial layer of deep Cervical fascia and deepfascia of infrahyoid Musclesvisceralwe divide the central neck compartment into four subdivisions by deep fascia of infrahyoid strap muscles pretrachealfascial and right RLN Fig Theproposed LNCM compartment is bounded superiorly bythe hyoid bone laterally by the carotid arteries anteriorlyby the investing layer of the cervical fascia and posteriorlyby the deep fascia of infrahyoid strap muscles which is defined as Level VI 1st In the current study suprasternalspace composed part of the LNCM Fig Compared toSSLN reported by Yu [] LNCM encompasseslymph nodes in the suprasternal space and lymph nodesbetween sternohyoid and sternothyroid muscles Figs and LNCM can fall under the normal subdivisions ofthe central compartment Subdivisions can actually recordthe extent of the CND which is able to provide detailedinformation for the possible second operation 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Fig LNCM coverage area in vivo Lymph nodes between sternohyoid and sternothyroid muscles a and lymph nodes in the suprasternal space bIncluding LNCM as an anatomical part of the centralneck allows for removal of previously unrecognized micrometastatic disease in of PTC patients with the inferiorportion lesions ETElevel III and level IV metastasisDissection of the LNCM space is less invasive and easy toachieve and is not timeconsuming It is at the entrance ofcentral neck compartment which is easy to expose and haslow risk of damaging RLN or parathyroid With the application ofintraoperative neuromonitoring and in situpreservation or autotransplantation of parathyroid theoccurrence of vocal cord paralysis and permanenthypoparathyroidism in the current study were lowerin this study [ ] Therefore in cases where LNCMspace metastasis is suspected or preoperative ultrasoundand CT suggests LNCM metastasis greater attentionshould be paid to the nodal tissue in the LNCM space inthyroid carcinoma patients These patients might benefitfrom a reduced risk of regional recurrence central neckreoperative morbidity and improved decision making inrelation to the use of radioiodine ablationThere are severallimitations in the present studyThe retrospective design is a limitation of the studyAnd this was two tertiary referral centers retrospective review and routine prophylactic nodal surgerywas offered in China however it is not standard elsewhere in the world which is a major limitation Aprospective randomized trial with a long time followup period may help to further evaluate the clinicalsignificance of LNCM in PTC patientsConclusionsIn summary additional dissection of nodes in theLNCM were accessible and might not increase morbidity Therefore attention should be paid to the lymphtissue between investing layer of cervical fascia and deepfascia ofinfrahyoid strap muscles for PTC patientsespecially in presence of inferior portion lesions ETElevel III and level IV metastasisAcknowledgementsThe authors thank the studied patients for their willingness to cooperatewith our studyAuthors contributionsGaosong Wu Study concepts and design Qianqian Yuan Study designmanuscript preparation and editing Jinxuan Hou Data analysis andmanuscript editing Yiqin Liao Data acquisition Lewei Zheng Manuscriptpreparation Fang Lu Data acquisition Kun Wang Quality control of dataand algorithms The authors read and approved the final manuscriptFundingThe authors have no support or funding to reportAvailability of data and materialsNot applicableEthics approval and consent to participateThis research was comprised of human participants and was approved byMedical Ethics Committee of Wuhan University Zhongnan Hospital Informedconsent was obtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei Peoples Republic of China 2Department of Thyroid and Breast Surgery Tongji Hospital of TongjiMedical College of Huazhong University of Science and Technology Jiefang Avenue Wuhan Hubei Peoples Republic of China Received April Accepted August ReferencesLang BH Ng SH Lau LL Cowling BJ Wong KP Wan KY A systematic reviewand metaanalysis of prophylactic central neck dissection on shorttermlocoregional recurrence in papillary thyroid carcinoma after totalthyroidectomy Thyroid So YK Seo MY Son Y Prophylactic central lymph node dissection forclinically nodenegative papillary thyroid microcarcinoma influence onserum thyroglobulin level recurrence rate and postoperative complicationsSurgery Hughes DT Rosen JE Evans DB Grubbs E Wang TS Solórzano CCProphylactic central compartment neck dissection in papillary thyroid 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Cancer and effect on Locoregional recurrence Ann Surg Oncol McHenry CR Is prophylactic central compartment neck dissection indicatedfor clinically nodenegative papillary thyroid Cancer the answer isdependent on how the data are interpreted and the weight given to therisks and benefits Ann Surg Oncol Selberherr A Riss P Scheuba C Niederle B Prophylactic firststep centralneck dissection level does not increase morbidity after Totalthyroidectomy Ann Surg Oncol Hartl DM Leboulleux S Al Ghuzlan A Baudin E Chami L Schlumberger M Optimization of staging of the neck with prophylactic central andlateral neck dissection for papillary thyroid carcinoma Ann Surg McAlister ED Goldstein DP Rotstein LE Redefining classification ofcentral neck dissection in differentiated thyroid cancer Head Neck Miller JE AlAttar NC Brown OH Shaughness GG Rosculet NP Avram AM Location and causation of residual lymph node metastasis aftersurgical treatment of regionally advanced differentiated thyroid CancerThyroid Sun G Wang Y Zhu Y Wang Y Xu K Wei W Lymph node metastasisbetween sternocleidomastoid and sternohyoid muscle in clinically nodepositive papillary thyroid carcinoma Head Neck Homma A Hatakeyama H Mizumachi T Furusawa J Kano S Sakashita T Lymph node metastasis in the suprasternal space from thyroidpapillary cancer Int Cancer Conf J Yu S Ge J Sun B Wei Z Lei S Lymph node metastasis in suprasternal spacein pathological nodepositive papillary thyroid carcinoma Eur J Surg Oncol Wu G Kong D Thyroidectomy with Wu Gaosong's ProcedureVideoEndocrinologr httpsdoi101089ve20150050 Wu G Wang K Intraoperative Neuromonitoring and Protection of theSuperior Laryngeal Nerve with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160070 Wu G Cui Q Wang K Carbon Nanops for Identifying Lymph Nodesand Protecting Parathyroid Glands in Thyroid Lobectomy with IpsilateralCentral Compartment Lymph Nodes Dissection VideoEndocrinology httpsdoi101089ve20160064Kong D Cui Q Gaosong W A Novel Classification of Parathyroid Glands andTheir Preservation in Thyroidectomy VideoEndocrinology httpsdoi101089ve20170093 Wang K Wu G Intraoperative Neuromonitoring in Selective Neck Dissectionfor Thyroid Cancer SND IIa Vb with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160082 Yuan Q Wu G Hou J Liao X Liao Y Chiang F Correlation betweenelectrophysiological changes and outcomes of vocal cord function in recurrent laryngeal nerves with visual integrity during thyroidectomyThyroid Cui Q Li Z Kong D Wang K Wu G A prospective cohort study of novelfunctional types of parathyroid glands in thyroidectomy Medicine 9552e5810Tavares MR Cruz JA Waisberg DR Toledo SP Takeda FR Cernea CR et alLymph node distribution in the central compartment of the neck ananatomic study Head Neck Wang K Cai H Kong D Cui Q Zhang D Wu G The identificationpreservation and classification of the external branch of the superiorlaryngeal nerve in thyroidectomy World J Surg Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
"deltalike protein DLL3 may be related with prognosis inpatients with small cell lung cancer SCLC However this finding remains controversial in small cell lung cancerThis metaanalysis was systematically performed to evaluate the prognostic value of DLL3 in SCLCMethods The PubMed EMBASE and Web of Science databases were retrieved to collect the eligible referencesThrough Stata software we pooled hazard ratios HR with confidence intervals CI by using random orfixedeffects models to evaluate the association between DLL3 and SCLC survival resultsResults A total of interrelated studies including patients were qualified After we removed study theremaining studies including patients were pooled to testify that high expression of DLL3 was an inferiorprognostic for patients with SCLC in Asian populations HR CI I2 p Thepooled results showed that DLL3 might be higher expression in advanced metastasis SCLC in Asian populations RR CI I2 p But the expression of DLL3 was not correlated with sex RR CI I2 p smoking history RR CI I2 p and tumour stage RR CI I2 p Conclusions Our metaanalysis confirms that in Asian populations high expression of DLL3 was a potential poorprognostic biomarker for SCLC and DLL3 highly expressed in advanced stage SCLC in Asian populationsKeywords Deltalike protein DLL3 Prognostic Small cell lung cancer SCLC MetaanalysisIntroductionLung cancer has the highest morbidity and mortality ofall malignant tumours which is one of the most common cancers worldwide [] The major histologic subtypes oflung cancerNSCLC and small cell lung cancer SCLC [] SCLC is an invasive highgrade malignancy withearly development and a bad prognosis Most patientswith SCLC are advanced with widespread metastasiswhen they are diagnosed and systemic chemotherapy islung cancer are nonsmall cell Correspondence ligaofenghl126comDepartment of Thoracic Surgery The Third Affiliated Hospital of KunmingMedical University Yunnan Cancer Hospital Kunming Yunnan Chinathe most effective therapy [] Few therapeutic methodsare available after SCLC relapse and the prognosis is especially poor Therapeutic treatments for patients withSCLC have essentially remained unchanged in recentyears [] Thus further research into the mechanism ofSCLC and the exploration of new therapeutic targets forSCLC is imperativeDeltalike protein DLL3 is a transmembrane protein that promotes the development of neuroendocrinetumours through its reciprocity with the Notch pathway[] It is expressed in cancer tissues in approximately of patients with SCLC and other neuroendocrineis not expressed in nonneuroendocrinecancer but 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 World Journal of Surgical Oncology Page of tumours or normal tissues [] Most studies have shownthat DLL3 is a latent treatment target point for SCLC[] Recently many researches initiate to focus on theprognostic value of DLL3 in SCLC Some of the researchers demonstrated that DLL3 had no correlationwith prognosis in SCLC patients [ ] but other investigators showed that DLL3 was associated with thesurvival of patients with SCLC [] So previous studiesof the prognostic value of DLL3 in SCLC remain controversial we performed this systematic metaanalysis forresolving this controversialPublished s discussing the prognostic value ofDLL3 in SCLC were systematically reviewed in ourmetaanalysis We aimed to include all correlationalstudies to assess the prognostic value of DLL3 andattempted to identify an accurate biomarker to guideprognosis and treatment for SCLC in the futureMethodsSearch strategyThe PRISMA guidelines were followed for our metaanalysis see Additional file [] The literatures werecollected through retrieving the PubMed EMBASE andWeb of Science databases from the initial date to February The search strategy was DLL3 OR deltalike protein all fields AND nonsmall cell lung cancer OR NSCLC OR small cell lung cancer OR SCLC ORLung Adenocarcinoma OR Lung Squamous cell carcinoma OR lung cancer OR lung tumour OR lung neoplasm OR lung carcinoma all fields There was nolimitation on the publication status All eligible studieswere retrieved and the headlines and s of all thereference lists of the reviews or studies were independently filtrated through three authors based on the criteria Differences between two authors were resolved bythe third authors opinionsInclusion criteriaResearches were included by following the standards studies reported the prognostic value of DLL3 in SCLC studies were published as original s studiesreported the data of HR and CI or provided survivalcurve and studies in which the prognostic value wasinvestigated by survival analysis with overall survivalOS diseasefree survival DFS progressionfree survivalRFS ordiseasespecific survival DSS We excluded the animalresearches and other review literaturesPFS relapserecurrencefree survivalData extractionTwo researchers independently extracted the data included the author country edition year sample capacitypatients sex smoking history distant metastasis tumourstage histologic subtype biomarkers scoring methodsboth univariateand multivariatecutoffs value and the data of HR and CI We extracted the multivariate analysis outcome if one study includedanalysisoutcomes When we could not extract the data of HRand CI directly in the but the survival curveswere reported we extracted and digitized the survivalcurves by operating the Engauge Digitizer softwarehttpdigitizersourcefenet and we estimated thedata of HR and CI by the Excel programme files asexploited by Tierney [] When the data of HRand survival curves were not reported we contacted thecorresponding authors of eligible s by email to obtain the original data these s were excluded ifthere was no responseAssessment of study qualityTwo researchers independently used the Quality InPrognosis Studies QUIPS tool to assess risk of bias ofall the publications [] According to the criteria every was evaluated as low risk moderate risk or highrisk by following six different areas study participationstudy attrition prognostic factor measurement outcomemeasurement study confounding and statistical analysisand reporting [] Differences were settled throughdiscussionStatistical analysisTwo authors independently extracted the HR and CI from the original s The HR was used to describe the high DLL3 expression versus low DLL3 expression We took the reciprocal of the HR if the sshowed the low DLL3 expression vs high DLL3 expression We observed that high expression of DLL3 portended a worse prognosis when HR and that HR indicated preferable prognosis For the analysis results p was considered statistically significant Statisticalheterogeneity was assessed by calculating the I2 statistic[] The presence of heterogeneity was indicated whenI2 and then a randomeffects model was appliedto poolthe results [] a fixedeffects model wasemployed to pool the results when I2 [] Weperformed further subgroup or sensitivity analysis toanalyse the heterogeneity The publication bias was estimated by Beggs and Eggers tests when p indicates no publication bias [] Through Stata software we performed this metaanalysis and acquiredthe forest plotsResultsSearch results and study characteristicsUsing the searching strategy described above a total of original documents were identified from the databases with approximately studies remaining after excluding duplicates Afterandscreening thetitles 0cChen World Journal of Surgical Oncology Page of s of the publications publications werenot related to evaluating the prognosis role of DLL3in SCLC Finally we were left with eligible studiesafter screening the full text among which swere included in our final analysis Fig []Ten s were excluded for the following reasonsseven s were review researches and commentaries one did notreport hazard ratios andKaplanMeier curves and s were s anddid not report relevant outcomes The eligible s characteristics are shown in Table All theses came out from the initial date to February and the patients were diagnosed as SCLC byhistopathology Among these studies all studies investigated DLL3 byinparaffinembedded tissueimmunohistochemistryIHCQuality assessment of the included studiesWe performed quality evaluations of the s following the QUIPS tool and two authors independently evaluated allthe literature Differences were resolved bydiscussion After screening all included s we foundthat there were no researches that reported study attritionand there were no standardization of cutoffs value forevaluating DLL3 expression And then studies were evaluated as low risk were evaluated as moderate risk and study was evaluated as high risk Table This outcomeindicated that most of the studies we included were of amedium or high qualityPrognostic value of DLL3 in SCLCAs shown in Fig eligible s were pooled foranalysing the prognostic value of DLL3 in SCLC TheFig The flow diagram of studies selection 0cChen World Journal of Surgical Oncology Page of Table Study characteristics of the eligible sStudyYear Country No ofsamplesSexmalefemaleSmokinghistorynonyesYan [] ChinaDistantmetastasisnegativepositiveXie [] USATanaka [] JapanRegzedmaa [] ChinaHuang [] ChinaFuruta [] JapanNANANANANANANATumourstageIIIIIIIVHistologicsubtypeMethod Outcomes BiomarkersCutoffvalueNANASCLCSCLCSCLCSCLCSCLCSCLCIHCIHCIHCIHCIHCIHCOSOSOSOSDLL3TTF1DLL3DLL3DLL3CTLA4MSTNscore ¥ ¥ ¥ score ¥ OSPFSDLL3OSDLL3ASCL1score ¥ ¥ SCLC small cell lung cancer IHC immunohistochemistry OS overall survival PFS progressionfree survival DLL3 deltalike protein CTLA4 cytotoxic T lymphocyteassociated protein MSTN mesothelin ASCL1 achaetescute homologue1 NA not applicableresults showed that high expression of DLL3 indicatedno prognostic value in patients with SCLC HR CI I2 p Howeverprominent heterogeneity existed in the pooled results sowe conducted further subgroup analysis Among the sixstudies three s were from China two were fromJapan and one was from America We divided the studies into the Asian group and the American group forsubgroup analysis As shown in Additional file FigA1we found that the heterogeneity was in the Asiangroup and we believed that the main cause of the heterogeneity was the study from American So we decidedto eliminate the study from American and only analysedthe remained studies After we removed the Americanstudy the outcomes indicated that high DLL3 expressionwas a poor prognosis marker in SCLC HR CI I2 p Fig Then wedivided the studies into two groups according to thesize of the sample and the final outcomes indicated thathigh DLL3 expression was a poor prognosis marker inthe group with sample size greater than HR CI p Fig But there was nosignificant relationship between DLL3 expression andprognosis in the group with sample size less than HR CI I2 p Fig Beggs test p and Eggers test p there was nodemonstratedresultsthatpublication bias in our metaanalysis Fig Finallysensitivity analysis showed that allthe studies werestable see Additional file FigA6The clinical characteristics of patients with DLL3expression in SCLCThe pooled results showed that the expression of DLL3was not correlated with sex RR CI I2 p Table Additional file FigA2 smoking history RR CI I2 p Table Additional file FigA3 and tumour stage RR CI I2 p Table Additional file FigA4 But DLL3 was highly expressed in patients withdistant metastasis RR CI I2 p Table Additional file FigA5DiscussionMany studies have shown that DLL3 played a significantprognosis value in patients with cancer For examplehigh DLL3 expression was associated with shorter OSand PFS in small cell bladder cancer [] High DLL3expression was associated with bad OS and usuallyexpressed in older patients and advanced stage in endometrial cancer [] Xie reported that high DLL3expression predicted a better OS in patients with SCLC[] and other studiesforeboded that high DLL3Table Quality assessment of included studiesStudyStudyparticipationYan []Xie []Tanaka []Regzedmaa []Huang []Furuta [] indicates low risk of bias StudyattritionPrognostic factormeasurementOutcomemeasurementStudy confoundingStatistical analysisand reportingTotal riskof biasLowModerateLowLowHighModerate moderate risk of bias and high risk of bias 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in SCLC patients DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneityexpression was an inferior prognostic marker for SCLC[ ] Thus the results of different studies remainedcontroversial We included a total of studies with patients with SCLC to assess the prognostic value ofDLL3 in SCLC by pooling the data of HR and CIFirst we performed this metaanalysis using studiesBut heterogeneity was observed in our pooled resultsThrough further subgroup analysis we found that astudy from the American was the main cause of heterogeneity As we removed the American study we foundFig Forest plots of prognostic value of DLL3 in Asia patients with SCLCDLL3 deltalike protein USA the United States of America HR hazardratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneity 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in different studies with different sample sizes DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneitythat high expression of DLL3 is a marker of poor prognosis in SCLC But we also noticed that the results ofthe American study were contrary to our conclusionsthey reported that high expression of DLL3 is a markerof good prognosis in SCLC One interpretation of thisresult was that DLL3 expression varies between differentpopulations The American study indicated that the highexpression of DLL3 was a marker of good prognosis[] and there was no significant correlation betweenDLL3 expression and prognosis in several Japan studies[ ] while the high expression of DLL3 is a markerof poor prognosis in China studies [ ] These results showed that the expression of DLL3 might be different in different populations However only one studyFig Funnel plot of DLL3 present on overall survival DLL3 deltalike protein HR hazard ratios 0cChen World Journal of Surgical Oncology Page of Table Summary of the correlation between DLL3 expression and the clinical characteristics of patients with SCLCClinical characteristicsSex malefemaleRR CI No of studiesI2 p Smoking historynonyesDistant metastasis negativepositive P valueTumour stage IIIIIIIVSCLC small cell lung cancer RR relative ratios CI confidence intervals DLL3 deltalike protein I2 percentage heterogeneity between studies p testfor heterogeneity researched the correlation with DLL3 expression andprognosis in SCLC outside of Asia so the results needto be treated with caution In addition more studieswere suggested for the future to further verify the existence of such differencesOur other explanation of this result was that the expression of DLL3 was the same in different populationsAmong the included studies the sample size of studieswas less than including the America and Japanstudies Therefore we speculated that insufficient sample size might cause bias in the results We conducted asubgroup analysis according to the sample size and theresults showed that the high expression of DLL3 in thestudies with large sample sizes N ¥ was associatedwith poor prognosis while the pooled results of thestudies with low sample sizes N showed no significant correlation between the expression of DLL3 andprognosis Therefore the sample size may be one of thereasons for the differences in DLL3 expression in eachstudy Moreover immunohistochemistry was used to detect DLL3 expression in all of the studies Immunohistochemical staining is a semiquantitative method and isevaluated with great subjectivity [] Different antibodies and different cutoff values for DLL3 expressionwere employed in all the included studies and thus couldalso be another cause of the differences in results TheAmerican study also explained their different results byclaiming that many of the other studies were performedusing mRNA expression instead of protein expression orused different cutoffs value of DLL3 expression []Researches havereported that DLL3 is highlyexpressed in SCLC [ ] which suggested that DLL3might promote the development of SCLC Therefore wealso discussed the correlation between DLL3 expressionand the clinical characteristics of patients with SCLCThe pooled results showed that DLL3 expression had nosignificant correlation with patients sex smoking statusand stage while DLL3 often highly expressed in metastasis patients of SCLC Our survival analysis outcomeswere consistent with this result which suggested thathigh expression of DLL3 might be one of the factorscontributing to poor prognosis in patients with advancedmetastatic SCLC in Asia However only a few studiesreported the correlation between the expression of DLL3and clinical characteristics and the methods and cutoffs values used to detect the DLL3 expression in eachstudy were not uniform Therefore more reliable studiesare needed to further verify our outcomes Our resultssuggest that it is valuable to further investigate the correlation between DLL3 and the clinical characteristics ofpatients with SCLCOur metaanalysis is the first to focus on the prognostic value of DLL3 in SCLC The significance of thismetaanalysis lies in providing a basic direction and evidence for further research into the mechanism of DLL3in SCLC For SCLC Notch1 over expression could induce G1 cell cycle arrest [] Previous studies reportedthat DLL3 downregulated the Notch receptor expression thereby the Notch signalling pathway was inhibitedwithin the cell [] Therefore high expression of DLL3can promote the development of SCLC by inhibiting theNotch signalling pathway Studies also have shown thatthe high expression of DLL3 may reduce the sensitivityof chemotherapy drugs [] These studies have demonstrated that DLL3 may be associated with the prognosisof SCLC and also consistent with our metaanalysis results Thus studies of the corresponding targeted drugsof DLL3 can effectively inhibit the expression of DLL3and thus improve the survival of SCLC Rovalpituzumabtesirine RovaT is a new antibodydrug conjugate directed against DLL3 in SCLC [] A phase I trial foundthat patients with high DLL3 expression in SCLCshowed a better response to RovaT than those with alow DLL3 expression [] However disappointingly thephase III TAHOE trial has been stopped because theRovaT group showed a worse OS compared to the control group [] But more clinical trials are recruitingparticipator to investigate RovaT as maintenance therapy in advanced stage SCLC The lack of progress withthis drug does not prevent us from making a breakthrough with other similar drugs Some researches foundthat the intratumoural and intertumoural distributionof DLL3 protein in SCLC is homogeneous [] supporting the conclusion that biopsy specimens are a reliablesource for DLL3 evaluation for targeted therapy Inaddition most studies have demonstrated that DLL3 is 0cChen World Journal of Surgical Oncology Page of highly expressed in SCLC while it is not or is lessexpressed in other types of lung cancer and normal tissues [] Therefore the expression of DLL3 can be detected by biopsy as an indicator for diagnosis predictingtherapeutic efficacy and monitoring recurrence or metastasis of SCLC in the futureAlthough our study fully explains the prognostic valueof DLL3 in SCLC our analysis still has several limitations Firstlarge heterogeneity was observed in thepooled results This is explained by the observation thatthe evaluation criteria for the expression of DLL3 areparticularly mixed and there are no international standards for cutoffs values to determine the expression ofDLL3 Thus the scoring methods and cutoffs values ofDLL3 should be unified to strengthen our conclusionsOtherwise the detection method of DLL3 in most studies is mainly immunohistochemistry at present which isa semiquantitative subjective and inaccurate detectionmethod Different studies show different prognosticvalues of DLL3 Therefore we need other more precisedetection methods to evaluate the expression of DLL3 inSCLC in the futureSecond the therapy method is also a key limitationThe current studies only focus on tissue specimens frompatients with SCLC after surgery or biopsy and fewstudies reported the treatment methods in their researches However the prognostic value of DLL3 may liein the therapeutic method Therefore every study shouldpay attention to the impact of patienttreatmentmethods on prognosis in the futureThird some of the original studies did not report thedata of HR and CI The HR and CI resultswere measured from survival curves an evaluationmethod with certain deviation and subjectivity whichmight influence the authenticity of the resultsConclusionIn summary our metaanalysis confirmed that high expression of DLL3 was a potential poor prognostic biomarker for SCLC in Asian populations moreover DLL3expression was correlated with advanced metastasisSCLC in Asian populations However the relationshipbetween DLL3 expression and the prognostic or clinicalcharacteristics of patients with SCLC in European andAmerican populations need to be further verified Thusdetecting the expression of DLL3 in tumour tissue willbe helpful to guide therapy in Asian patients of SCLCFor our research other highquality studies especiallyfrom European and American countries are required toconfirm our findings about the prognosis value of DLL3in SCLC in the future In view of the limitations of ouranalysisthe conclusions should be interpreted withcautionSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12957020020045Additional file The PRISMA checklistAdditional file FigA1 Forest plots of prognostic value of DLL3 inSCLC SCLCsmall cell lung cancer HRhazard ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA2 Forestplots of the correlation between DLL3 expression and sex of patientswith SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA3 Forestplots of the correlation between DLL3 expression and smoking history ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA4 Forestplots of the correlation between DLL3 expression and tumour stage ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA5 Forestplots of the correlation between DLL3 expression and metastasis ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA6Sensitivity analysis of all the studiesAbbreviationsCI Confidence intervals DLL3 Deltalike protein DFS Diseasefree survivalDSS Diseasespecific survival HR Hazard ratios IHC ImmunohistochemistryOS Overall survival PFS Progressionfree survival QUIPS Quality In PrognosisStudies RFS Relapserecurrencefree survival RR Risk ratio SCLC Small celllung cancerAcknowledgementsNot applicableAuthors contributionsAll authors contributed to the study conception and design Materialpreparation data collection and analysis were performed by Benchao ChenHeng Li and Chao Liu The first draft of the manuscript was written byBenchao Chen and all authors commented on previous versions of themanuscript All authors read and approved the final manuscriptAuthors informationNot applicableFundingThis work was supported by the National Natural Science Foundation ofChina [No ]Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived July Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwide 0cChen World Journal of Surgical Oncology Page of Regzedmaa O Li Y Li Y Zhang H Wang J Gong H Prevalence ofDLL3 CTLA4 and MSTN expression in patients with small cell lung cancerOnco Targets Ther httpsdoi102147OTTS216362 Huang J Cao D Sha J Zhu X Han S DLL3 is regulated by LIN28B and miR518d5p and regulates cell proliferation migration and chemotherapyresponse in advanced small cell lung cancer Biochem Biophys ResCommun 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radiation induces epithelialmesenchymaltransition in human bronchial epithelial cellsBo Tang123 Yue Xi121State Key Laboratory of Radiation Medicine and Protection School of Radiation Medicine and Protection Soochow University Suzhou China 2Collaborative InnovationCenter of Radiation Medicine of Jiangsu Higher Education Institutions Suzhou China 3Department of Radiation Protection Safety Shandong Center for Disease Controland Prevention Jinan ChinaFengmei Cui12 Jin Gao12 Huiqin Chen12 Wentao Yu12 andYu Tu12Correspondence Yu Tu tuyusudaeducnObjective The present study aimed to analyze the mechanism by which longterm occupational exposure of workers to lowdose ionizing irradiation induces epithelialmesenchymaltransition EMT of the human bronchial epithelial cells using transcriptome profilingMethods RNAseq transcriptomics was used to determine gene expression in blood samples from radiationexposed workers followed by bioinformatics analysis Normal bronchialepithelial cells 16HBE were irradiated for different durations and subjected to immunofluorescence Western blotting scratch healing and adhesion assays to detect the progressionof EMT and its underlying molecular mechanismsResults Transcriptomics revealed that exposure to ionizing radiation led to changes in theexpression of genes related to EMT immune response and migration At increased cumulative doses ionizing radiationinduced significant EMT as evidenced by a gradual decrease inthe expression of Ecadherin increased vimentin elevated migration ability and decreasedadhesion capability of 16HBE cells The expression of fibronectin FN1 showed a gradualincrease with the progression of EMT and may be involved in EMTConclusion Ionizing radiation induces EMT FN1 may be involved in the progression of EMTand could serve as a potential biomarker for this processIntroductionPeople are inevitably exposed to ionizing irradiation in their daily lives and at work The uncertaintiesabout the health risks associated with longterm exposure to radiation at a lowdose in occupationallyexposed workers have been the focus of research for many years In their International Nuclear WorkersStudy INWORKS published in the International Journal of Epidemiology in Hamra specifically evaluated the radiation hazards and risk in a cohort of nuclear workers in countries []Abbott A explored the risk of lowdose irradiation in [] Longterm general and occupational exposure to radiation has been the focus of research in the past Keil conducted a cohort mortalitystudies of underground miners from the Colorado Plateau [] Kamiya conducted a cohort study ofnuclear workers in USA and reported the ERR values of cancers and cardiovascular diseases in []A recent epidemiological study emphasized the harmful effects of lowdose radiation exposure on humanhealth and reported elevated cancer mortality among nuclear workers exposed to radiation at a cumulative dose mSv and dose rate mSvyear [] However the biomarkers and molecular mechanismthat predict the longterm and lowdose effect of radiation are not clearThe carcinogenic effects of ionizing radiation rank first among other potential hazards of occupational exposure to radiation It is of great clinical significance to detect and evaluate the carcinogenicrole of radiation In the early stages of exposure to radiation several biological changes occur of whichepithelialmesenchymal transition EMT is an important indicator of malignant change EMT refers toThese authors contributedequally to this workReceived February Revised June 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 BSR20200453101042BSR20200453Table General information pertaining to the participantsAge yearWorking age yearMarital statusEducationIncome per month RMBGroup¥MarriedUnmarriedOtherElementary or lessJuniorSeniorUndergraduate or more¥Casesthe biological process in which epithelial cells are transformed into cells with interstitial phenotype characterized byloss of cell adhesion and enhanced ability to migrate [] Based on the specific biological environment in which MToccurs there are three subtypes of which type EMT is associated with epithelial malignancies Whether bronchialepithelial cells undergo EMT following longterm and lowdose radiation exposure and its mechanism are the focusof the present studyThe present study aimed to analyze the biological changes after exposure to longterm and lowdose ionizing radiation Highthroughput sequencing was used to analyze the mRNA expression profiles of workers with or withoutradiation exposure Normal bronchial epithelial cells underwent EMT following multiple rounds radiation and thedifferentially expressed mRNAs were validated using a cellular model These findings provide an experimental basisfor radiationinduced carcinogenesisSubjects and methodsSubjectsThe present study was approved by the ethic committee of the Soochow University and informed consent was obtainedfrom all participants The study included a total of participants as shown in Table which included workerswith no radiation exposure and workers exposed to radiation at a total dose of mSv for an average of yearsReagent and cell lineHuman normal bronchial epithelial cells 16HBE culture medium bronchial epithelial growth factor and penicillinstreptomycin were purchased from Shanghai Zhongqiao Xinzhou Biological Company Antibodies against FN1ab32419 vimentin VIM ab92547 Ecadherin Ecad ab40772 and the goat antirabbit IgG HL Alexa secondary antibody ab150080 were purchased from AbcamTranscriptome sequencingRNA library construction and sequencingThe ribosomal RNA rRNA was removed using the NEBNext rRNA Depletion Kit New England Biolabs Inc Massachusetts USA The RNA library was constructed using the NEBNext® Ultra¢ II Directional RNA Library PrepKit New England Biolabs Inc Massachusetts USA Quality control and quantification of the library were performed using a BioAnalyzer system Agilent Technologies USA and sequenced using the bp pairedendmethod in an Illumina Hiseq The reads were subject to quality control Q30 and checked using the Cutadaptprogram v193 The highquality reads were mapped to the human reference genome UCSC HG19 using theHisat2 program v204 Guided by the annotation from the TGF file the Cuffdiff program was used to generate thetranscript abundance of LncRNA and mRNA known as FPKM Fragments per kilobase of exon per million fragments mapped The foldchanges between two groups were calculated and the differentially expressed mRNAs wereidentified based on Pvalue for analysis of GO and KEGG pathway 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 BSR20200453101042BSR20200453Table Primers used in the present studyGene IDGene nameReferenceLAMB1KRT6BFN1ADAM9RNF182GAPDHPrimerForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseSequence 5cid23cid2AAATCTTGTGCTTGCAATCCTCCGCAAAGCAACTGTTGTTTAAGGTACCAGACAAAGTACGAGGAGGCTTGTTCTTAGCATCCTTGAGAATAGATGCAACGATCAGGACAGCAGGTTTCCTCGATTATCCTTAGGCTGAAGGAAAAGAGCATATAAGTCCCTTCCTTGTTGTAAGTCCGTAGAGACAAAGCCGCCTAAAGGCCACATGAAGGGTCTGGCCTCCAAGGAGTAAGACCAGGGGAGATTCAGTGTGGTGGO and KEGG analysisThe Gene Ontology GO project httpwwwgeneontology provides a set of structured controlled vocabulariesfor community use in annotating genes gene products and sequences which is divided by molecular function MFbiological process BP and cell component CC GO terms with Pvalue are considered to be statistically significant By mapping multiple types of omics data such as genomics transcriptomics proteomics and metabolomicsinto KEGG pathway the biological function of these genes can be interpreted Pvalue was considered to bestatistically significantRealtime PCRThe plasma sample was thawed out from ¦C and centrifuged at g for min at ¦C Samples μlwere transferred to another centrifuge tube ml and TRIzol LS Reagent μl and acetic acid μl wereadded The homogenized samples were incubated with chloroform ml After shaking the tube manually for s and incubating at to ¦C for min the sample was centrifuged at g for min at ¦C Followingcentrifugation the solution separated into a red phenol chloroform phase in the lower layer and a colorless aqueousphase in the upper layer All the RNA were distributed in the aqueous phase The volume of the aqueous phase wasapproximately of the TRIzol LS Reagent added during homogenization The aqueous phase was transferred to anew centrifuge tube and mixed with isopropanol for min to precipitate the RNA After centrifugation at g for min at ¦C the RNA became visible on the bottom and walls of the tube The supernatant was removed andthe RNA was washed with at least ml of ethanol After centrifugation at g for min at ¦C the RNAwas airdried for min The concentration and purity of RNA were determined using a NanoDrop® ND1000RNA was reverse transcribed into cDNA using the SuperScript¢ III Reverse Transcriptase Invitrogen RealtimePCR was performed using a qPCR SYBR Green master mix kit CloudSeq using the following condition ¦C min cycles ¦C s ¦C s The melting curve was then obtained with the condition ¦C s ¦C s ¦C s gradually from ¦C to ¦C ¦Cs The relative expression of the genes were calculated using theääCT method The primers used are shown in Table Cell culture and radiationHuman normal bronchial epithelial cells were purchased from Shanghai Zhongqiao Xinzhou Biological Companyand cultured in medium supplemented with bronchial epithelial growth factor and penicillinstreptomycin SciencellUSA When the cells were in logarithmic growth period the culture dish was placed in a biological Xray irradiatorRS2000Pro for radiation dose of Gy at a dose rate of Gymin After passage on the other day the cells wereexposed to radiation at the accumulative dose of Gy The radiationexposed cells were divided into and Gy groupsImmunoï¬uorescenceCells in the five groups were fixed with paraformaldehyde Meilunbio China cleared in Triton X100Meilunbio China for min and then blocked in the serum for min The cells were incubated with the primary antibody Abcam USA at ¦C overnight and then incubated with the secondary antibody Abcam 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 BSR20200453101042BSR20200453Figure Differentially expressed mRNAs in workers with or without radiation exposureA Cluster analysis of differentially expressed mRNA based on FPKM values via heatmap2 In the picture A is the control groupand B is the exposed group B Scatter plot based on the expression of the two groups Red dots upregulated genes greendots downregulated genes purple dots no significant change Foldchange was C Volcano plot based on foldchange andPvalue The red rectangle represents the differentially expressed mRNA P foldchange ¥ at room temperature for h in the dark The nuclei were counterstained using DAPI and examined by fluorescence microscopy Images were acquired using a scanning laser confocal microscope FV1200 and the results weresemiquantitatively analyzed using the ImageJ softwareWestern blottingCells were lysed in lysis buffer containing PMSF μl on ice for min transferred to a centrifuge tube ml and centrifuged at rpm for min at ¦C Protein concentration was determined using the BCA methodSolarbio China and the lysates were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresisSDSPAGE The proteins were transferred to polyvinylidene difluoride PVDF membrane and the membranes wereblocked with skim milk Oxoid UK for h The membranes were then incubated with the primary antibodyAbcam USA at ¦C overnight and the secondary antibody Abcam USA at room temperature for h Theproteins were detected using enhanced chemiluminescence ECL immunoassay Beyotime China and visualizedin an AI680 ultrasensitive multifunctional analyzer 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 BSR20200453101042BSR20200453 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 BSR20200453101042BSR20200453Figure GO analysis of differentially expressed mRNA in workers with or without radiation exposureA The top GO terms with the largest number of genes B The ratio of the number of differentially expressed genes in the top enriched GO terms with high fold enrichment to the total number of differentially expressed genes The left column pertains toupregulated mRNAs the right column pertains to downregulated mRNAs PTable Baseline characteristics of participantsAgeSmokeAlcoholTeaRiceVegetableOilSugarChiliSaltedFruitSaltMeatControl Radiation PScratch assayCells in the five groups were plated in a sixwell plate and cultured at ¦C When the cells grew to confluencea uniform scratch was made in the cell monolayer using a ml pipette tip Then the cells were washed with PBS andcultured with growth factorfree medium Images were collected at and h using a microscope IX73 andwere analyzed quantitatively using the ImageJ softwareAdhesion assayCell adhesion was tested using a cell adhesion detection kit BestBio China according to the manufactures protocolCells in the five groups were plated in 96well plates at the density of cellswell in triplicates for each groupAfter h of incubation at ¦C the medium was changed and μl of the staining solution B was added to the wellsThe OD was measured at nm after h using a microplate reader Synergy The formula used for calculatingthe cell adhesion rate was cell adhesion rate OD of test cells OD of the blank OD of the control ODof the blank CCK8 assayCells in the five groups were plated in a 96well plate at the density of per well in triplicates On the next day μl of CCK8 solution Meilunbio China was added to each well and the plates were incubated at ¦C After h a microplate reader Synergy was used to determine the OD at nm The formula used for calculating cellviability was cell viability OD of test cells OD of cellfree medium OD of control cell OD of cellfreemedium Statistical analysisThe general information of the participants was subjected to a normality test and expressed as mean standard deviation SD The data were analyzed using SPSS For the quantitative data with normal distribution and homogeneity of variance analysis of variance was used For the qualitative data chisquare tests were used The significancelevel was set to α and P005 was considered statistically significantResultsBaseline characteristics of participantsAll the participants were male Except for salt intake there were no significant differences among the other parameters including age smoking history and eating habits P005 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 BSR20200453101042BSR20200453Figure KEGG analysis of differentially expressed mRNA in workers with or without radiation exposureA The top pathways with high enrichment scores associated with the upregulated mRNA B The top pathways with highenrichment scores associated with the downregulated mRNADifferentially expressed mRNA in radiationexposed workersThe differentially expressed mRNA between the two groups were calculated using the Cuffdiff software with thefoldchange ¥ ie log FC ¥ Pvalue and FPKM value ¥ in at least one sample as the thresholdFinally we screened a total of differentially expressed genes including upregulated and downregulatedgenes Figure and Supplementary Material S1GO and KEGG analysis of differentially expressed mRNAWe then performed GO analysis on the differentially expressed mRNAs For the upregulated genes there were subclasses in BP in CC and in MF For the downregulated genes there are subclasses in BPs inCCs and in MFs Figure In BPs the upregulated mRNAs were involved in intracellular protein transportcellular protein localization ectoderm development etc In CCs the upregulated mRNAs were involved basal laminabasement membrane laminin complex etc In the MFs the upregulated mRNAs were involved in cell adhesionmolecule binding and integral binding For the downregulated mRNA the BPs mainly included immune responseimmune system process immune effector process and defense response among others the CCs mainly includedmembranebounded anelle cytoplasm and extracellular anelle and the MFs mainly included protein bindingidentification of protein binding and cofactor binding among othersKEGG analysis Figure revealed that pathways were related to the upregulated mRNA mainly including pro 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 BSR20200453101042BSR20200453Figure Validation of RNAseq by RTPCRP005 vs controltein export bacterial invasion of epithelial cells small cell lung cancer and ECMreceptor interaction among othersThere were pathways associated with the downregulated mRNA mainly including pertussis and transcriptionalmisregulation in cancerThrough the analysis of GO and KEGG we found that the GO terms with higher enrichment and KEGG pathwaywere closely related to EMT migration and ECMreceptor interaction Basement membrane laminin complex andcell adhesion molecule binding have been reported to participate in changes in cell morphology or cell migration Insummary we believe that exposure to a certain dose of radiation may cause EMT or EMTrelated regulatory processesRTPCR analysis of differentially expressed genesGO and KEGG pathway analysis revealed that EMTrelated regulatory processes cell migration and ECMreceptorinteraction were closely associated with these differential expressed mRNAs Based on these results five candidategenes were selected including LAMB1 KRT6B FN1 ADAM9 and RNF182 RTPCR analysis was performed toanalyze the expression of these genes in the blood samples which were also used for RNAseq The results showedthat data from the RTPCR assay were consistent with those from RNAseq Figure Establishment of EMT model in 16HBE cellsTo further explore the function of FN1 we first observed the changes of EMT in human bronchial epithelial cellsinduced by longterm and lowdose radiation As shown in Figure 5AB exposure to the cumulative dose of radiationresulted in a gradual decrease in the protein expression of Ecad and increase in VIM levels indicating that these cellsacquired EMT The migration capacity and proliferation activity were also examined Scratch healing assay revealedthat the irradiated cells had a significantly higher migration capacity than normal 16HBE cells Figure 5CD Celladhesion assay showed that the adhesion rates of the irradiated cells were significantly lower than those withoutradiation exposure which indirectly supported the observation that their migration ability was improved Figure5E Moreover CCK8 assay show that the activity of the irradiated cells increased to a certain extent Figure 5FFN1 expression in 16HBE cells during EMTWestern blotting was performed to analyze the expression of FN1 in 16HBE cells at different cumulative doses ofradiation As shown in Figure the expression of FN1 gradually increased with increasing cumulative doses of radiation The expression of FN1 was significantly increased at the radiation dose Gy showing that FN1 was involvedin the EMT of 16HBE cells and may be used as a potential biomarker of the cumulative doses of radiation 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 BSR20200453101042BSR20200453Figure Changes in VIM Ecad migration and proliferation of 16HBE cells following irradiationImages of Ecad A and VIM B expression at different cumulative doses of radiation observed under a confocal microscope Cand D Wound healing assay was performed to determine the cell migration capability E and F Cell migration and activity weredetermined by adhesion and CCK8 assays respectively P005 vs Gy 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 BSR20200453101042BSR20200453Figure Expression of FN1 in 16HBE cells following different cumulative doses of radiationP005 vs 0vGyDiscussionLung cancer has always been an area of intensive research in the biological effects caused by ionizing radiation andnot just the epidemiological study of radiation [] Owing to the occupational specificity and small sample size ofthe present study for example in some s the work content of many radiation workers involves confidentialityissues which brings great difficulties to the preinvestigation work and the information of the personnel cannot befully grasped so the sample size is generally small Studies using larger cohorts are still necessary to obtain moreinformation such as Mayak cohorts The conclusions from these studies need further corroboration due to the differences of the research applied and statistical methods used in the study [] In addition genderspecific analysiswas also limited due to the low number of female workers and the low exposure dose []To identify radiationrelated factors highthroughput sequencing was performed using peripheral blood of healthyradiationexposed workers RNAseq results revealed differently expressed genes between participants with orwithout radiation Among these genes the upregulated genes were mainly located on chromosome and while thedownregulated genes were in chromosome and However we did not identify any correlation between thedistribution of chromosomal positions of these differential genes and ionizing radiation Then bioinformatics analyses GO and KEGG were conducted to identify the potential functions of these differential mRNAs We found thatthe differential genes induced by radiation were mainly involved in the ECMreceptor interaction laminin complex 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 BSR20200453101042BSR20200453cell adhesion molecule binding and immune reaction Based on these results five differential genes were selectedand validated using the same samples Although there was no statistical difference in the smoking history betweenthe two groups smoking is a known highrisk factor for lung cancer and cannot be eliminated as a factor Studieshave shown that smokers and nonsmokers have different molecular mechanisms of lung cancer development suchas hypermethylation of gene promoters [] Therefore the abnormal expression of some genes needs to be furtherstudied to determine their correlation with ionizing radiationAmong the upregulated genes SEC62 which is reported to be overexpressed in lung cancer prostate cancer andthyroid cancer [] is involved in endoplasmic reticulum stress tolerance and cell migration and is identified as aprognostic marker for nonsmall cell lung cancer [] Moreover overexpression of SEC62 is also reported in the atypical fibrous xanthomas AFX [] CDC9 a member of tetraspanin superfamily is involved in the regulation of manydisease and physiological processes including cell migration and adhesion [] PLD1 is the mediummembraneprotein that is overexpressed in various cancers such as lung cancer breast cancer and kidney cancer [] It alsoplays a key role in LAinduced breast cancer cell migration and invasion [] Thus most of the upregulated genesidentified in our study are related to cancerSeveral prior studies used A549 as an in vitro model to study lung cancer [] However we used normalbronchial epithelial cells 16HBE in our study EMT in 16HBE cells can be induced by silicon dioxide [] smokingextract CSE [] and transforming growth factor β1 TGFβ1 [] The novelty of the present study was inthe use of the 16HBE cells and the induction of EMT using ionizing radiation Among the upregulated genes FN1was found to be closely associated with EMT Subsequently RTPCR and Western blotting further demonstrated thatthe expression of FN1 was increased in 16HBE cells following EMT FN1 a member of the glycoprotein family isinvolved in cell migration and adhesion [] Many studies have reported that FN1 has an indispensable role in EMT[] FN1 together with Ecad and VIM serves as a marker for EMT in nonsmall cell lung cancer MoreoverFN1 may play an important role in the pathogenesis of nasopharyngeal carcinoma [] and the drug resistance oflung cancer []In the present study the mRNA expression of workers with or without radiation exposure was profiled usinghighthroughput sequencing and bioinformatics analysis An EMT model of the normal bronchial epithelial cellswas established after repeated irradiation of the cells The differentially expressed mRNAs were selected based on thebioinformatics analyses and validated in the cellular model We found that FN1 was involved in EMT and could beused as a potential biomarker Our research provides evidence for the possible mechanism through which longtermexposure to ionizing radiation induces EMTCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThe work was supported by the Nuclear Energy Development Project [grant number ]Author ContributionConceptualization FMCand YT Investigation BT YX HQC and WTY Formal analysis BT YX and JG Funding acquisition YT Project administration YT Writing original draft BT YX and FMC Writing review editing BT YX FMCand YX All authors have read and agreed to published version of the manuscriptAbbreviations16HBE human normal bronchial epithelial cells EMT epithelialmesenchymal transition FN1 fibronectin rRNA ribosomalRNAReferences Hamra GB Richardson DB Cardis E Daniels RD Gillies M OHagan JA Cohort Proï¬le The International Nuclear Workers StudyINWORKS Int J Epidemiol 101093ijedyv122 Abbott A Researchers pin down risks of lowdose radiation Nature 101038523017a Keil AP Richardson DB and Troester MA Healthy worker survivor bias in the Colorado Plateau uranium miners cohort Am J Epidemiol 101093ajekwu348 Kamiya K Ozasa K Akiba S Niwa O Kodama K Takamura N Longterm effects of radiation exposure on health 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" The adopted strategy was the same as that used in prior years [] and is based on four exclusive queries that return four disjoint citation subsets The first query QPub_plain is based on a plaintext search in PubMed titles and s using keywords The second query QPub_indexed relies on the PubMed indexing scheme using MeSH terms and results are made exclusive of the previous set The third one QWoS_restricted is based on a plaintext search in WoS restricted to the two research areas Medical Informatics and Health Care Sciences Services The fourth query QWoS_filtered is based on the same plaintext search used in WoS but filtered by nonrelevant research areas eg Archeology Dance Zoology etc and the two research areas of the previous query It is of note that the two WoS queries select only nonPubMedindexed papers that are supposed to be caught by the two PubMed queriesA first review of the four subsets of retrieved citations was performed by the two section editors to select candidate best papers Following the IMIA Yearbook protocol these candidate best papers were then individually reviewed and rated by both section editors the chief editor of the Decision Support section and external reviewers from the international Medical Informatics community Based on the reviewers ratings and comments the Yearbook editorial committee then selected the best papers of the year in the decision support domainIMIA Yearbook of Medical Informatics IMIA and Ge Thieme Verlag KG 0cReview Results The literature search has been performed on January A total of unique references were obtained distributed as follows for QPub_plain for QPub_indexed for QWoS_restricted and for QWoS_filtered yielding subtotals of references from PubMed and from WoS Compared to the previous year the global query retrieved more papers After a first individual screening independently performed by both section editors based on the title and of papers not rejected by both section editors were discussed by the two editors to achieve a final selection of candidate best papers After the external review of these s the editorial committee finally selected three of them as best papers for [] Table They are discussed in the next section and summaries of their contents are available in the AppendixDiscussion and OutlookIn the first paper Hendriks [] propose an approach to the modeling of clinical practice guidelines which certainly builds on already existing approaches but which is systematically conducted in order to be scalable and used to represent complex guidelines They promote the formalism of clinical decision trees CDTs as they are both clinically interpretable by healthcare professionals and computerinterpretable thus suitable for implementation in datadriven CDSSs The disambiguation of textual guidelines is supported first by the formal unequivocal specification of data items used as decision criteria using international coding systems to enforce interoperability and second by the representation of guideline knowledge as CDTs The method is applied to the Dutch breast cancer guidelines Sixty CDTs were built involving a total of data items among which could not be linked to standard terminologies The authors report the ambiguity of certain criteria which could be subjective or had multiple definitions The resulting knowledge base was implemented in a decision support application where it can be interactively browsed or automatically executed By modeling guidelines in such a way this work is a step forward in the sharing of encoded knowledgeIn the second paper KamiÅ¡aliÄ [] tackled the issues linked to the formalization of the medical processes used for managing chronic diseases and their execution in CDSSs They analyzed the decisionmaking dimensions of the therapeutic management of chronic diseases like those known to increase the cardiovascular risk and identified three basic levels therapy strategy dosage adaptation and intolerance management To handle these different aspects consistently they propose a formalism called extended Timed Transition Diagram eTTD With eTTDs they illustrate the multilevel and finegrained modeling required to capture the contents of arterial hypertension management guidelines This detailed demonstration on how procedural knowledge for hypertension management can be formalized to develop a CDSS could certainly be used in other medical domainsThe third paper by Khalifa [] presents a conceptual and practical framework to help assess confidence in predictive tools GRASP for Grade and Assess Predictive Tools is both a method to look for evidence from the published literature and an analysis grid It standardizes the assessment of the available literature associated to a predictive tool and the grading of its level of proof Three phases of evaluation are considered i before the implementation of the tool to assess both its internal and external validity ii during the implementation to assess its potential effect and usability and iii after the implementation to assess its effectiveness and safety In each phase the level of evidence can be assessed from the study design A qualitative summarizes the direction of evidence positive negative mixed This grid can be considered as similar to existing grids for instance the CONSORT statement for clinical trials However it gives a rigorous methodology for a critical appraisal of predictive tools and could be extended to all kind of CDSSs It might be a useful tool to extend the evidencebased culture in the field of medical informaticsBesides the three best papers selected for the Decision Support section of the edition of the IMIA Yearbook several other works retrieved from the literature review deserve to be cited Some of them deal with the personalization of decisions Laleci [] propose a scientific and technical approach to develop personalized care plans that comply with clinical practice guidelines for the management of complex polypathology situations Jafarpour [] propose a solution to dynamically manage the conflicts that can rise in this type of complex contexts Ben Souissi [] introduce the use of health information technology involving multiple criteria decision to support the choice between antibiotics alternatives Interestingly other works promote the creation and sharing of operational knowledge bases as exemplified by Hendriks [] Thus Huibers [] transform the textual STOPPSTART criteria into unambiguous definitions mapped to medical terminologies Canovas et al [] formalize EUCAST expert rules as an ontology and production rules to detect antimicrobial therapies at risk of failure M¼ller [] propose an diagnostic knowledge base that can compete with commercial ones Replacing humans is another topic of research and Spnig [] work on two aspects to virtualize a doctor the automatic acquisition of data through sensors and speech recognition and the automation of diagnostic reasoning Rozenblum et al[] propose a machine learning method to generate clinically valid alerts to detect errors in prescriptions Acceptability of CDSS is another key point Kannan [] propose a method for a CDSS design to best meet a precisely specified and assessable user purpose Design alerts may also avoid rejection of CDSSs by caregivers Fernandes [] created algorithms able to aggregate filter and reduce the notifications delivered to healthcare professionals Amrose et al [] tried to understand in real life the impact of alerts on users and to find the actions they triggered Finally it is always interesting to obtain varied evaluation results of controversial CDSSs In this respect Kim [] evaluated Watson for Oncology in thyroid carcinoma and reported a concordance rate with local practices considered as too low to adopt the tool As evidenced by the number and the variety of works around decision support research in the field is very active This years selection highlighted pragmatic works that promote the transparency and sharing of the IMIA Yearbook of Medical Informatics 2020Duclos 0cTable Best paper selection of s for the IMIA Yearbook of Medical Informatics in the section 'Decision Support' The s are listed in alphabetical order of the first authors surname Section Decision Support\uf0a7 Hendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Zonderland HM Smorenburg CH Jager A Siesling S Transformation of the National Breast Cancer Guideline Into DataDriven Clinical Decision Trees JCO Clin Cancer Inform \uf0a7\t KamiÅ¡aliÄ\tA\tRia±o\tD\tKert\tS\tWelzer\tT\tNemec\tZlatolas\tL\tMultilevel\tmedical\tknowledge\tformalization\tto\tsupport\tmedical\tpractice for chronic diseases Data Knowledge Engineering \uf0a7 Khalifa M Magrabi F Gallego B Developing a framework for evidencebased grading and assessment of predictive tools for clinical decision support BMC Med Inform Decis Mak knowledge bases used by decision support tools as well as the grading of their utility The ultimate goal is that users could trust such tools to then use themAcknowledgementWe would like to thank all the present and past editorial boards of the IMIA Yearbook especially Martina Hutter and Adrien Ugon for their support as well as the reviewers for their participation to the selection of the best papers for the Decision Support section We cannot end this synopsis without a meaningful thought for our colleague and friend Vassilis Koutkias who started this year again to tackle the tasks of a Decision Support section coeditor but passed away in last December and unfortunately could not finishReferences Jankovic I Chen JH Clinical Decision Support and Implications for the Clinician Burnout Crisis Yearb Med Inform Koutkias V Bouaud J Contributions on Clinical Decision Support from the Literature Yearb Med Inform Aug2811357 Hendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Transformation of the National Breast Cancer Guideline Into DataDriven Clinical Decision Trees JCO Clin Cancer Inform KamiÅ¡aliÄ A Ria±o D Kert S Welzer T Nemec Zlatolas L Multilevel medical knowledge formalization to support medical practice for chronic diseases Data Knowledge Engineering Khalifa M Magrabi F Gallego B Developing a framework for evidencebased grading and assessment of predictive tools for clinical decision support BMC Med Inform Decis Mak Laleci GB Yuksel M Sarigul B Arvanitis TN Lindman P Chen R A Collaborative Platform for Management of Chronic Diseases via GuidelineDriven Individualized Care Plans Comput Struct Biotechnol J Jafarpour B Raza Abidi S Van Woensel W Raza Abidi SS Executiontime integration of clinical practice guidelines to provide decision support for comorbid conditions Artif Intell Med Ben Souissi S Abed M El Hiki L Fortemps P Pirlot M PARS a system combining semantic technologies with multiple criteria decision aiding for supporting antibiotic prescriptions J Biomed Inform Huibers CJA Sallevelt BTGM de Groot DA Boer MJ van Campen JPCM Davids CJ Conversion of STOPPSTART version into coded algorithms for software implementation A multidisciplinary consensus procedure Int J Med Inform C¡novasSegura B Morales A Juarez JM Campos M Palacios F Impact of expert knowledge on the detection of patients at risk of antimicrobial therapy failure by clinical decision support systems J Biomed Inform M¼ller L Gangadharaiah R Klein SC Perry J Bernstein G Nurkse D An access medical knowledge base for community driven diagnostic decision support system development BMC Med Inform Decis Mak Spnig S EmbergerKlein A Sowa JP Canbay A Menrad K Heider D The virtual doctor An interactive clinicaldecisionsupport system based on deep learning for noninvasive prediction of diabetes Artif Intell Med Rozenblum R RodriguezMonguio R Volk LA Forsythe KJ Myers S McGurrin M Using a Machine Learning System to Identify and Prevent Medication Prescribing Errors A Clinical and Cost Analysis Evaluation Jt Comm J Qual Patient Saf Kannan V Basit MA Bajaj P Carrington AR Donahue IB Flahaven EL User stories as lightweight requirements for agile clinical decision support development J Am Med Inform Assoc Fernandes CO Miles S Lucena CJP Cowan D Artificial Intelligence Technologies for Coping with Alarm Fatigue in Hospital Environments Because of Sensory Overload Algorithm Development and Validation J Med Internet Res 20192111e15406 Amroze A Field TS Fouayzi H Sundaresan D Burns L Garber L et al Use of Electronic Health Record Access and Audit Logs to Identify Physician Actions Following Noninterruptive Alert ing Descriptive Study JMIR Med Inform 201971e12650 Kim M Kim BH Kim JM Kim EH Kim K Pak K Concordance in postsurgical radioactive iodine therapy recommendations between Watson for Oncology and clinical practice in patients with differentiated thyroid carcinoma Cancer Correspondence toPr Catherine DuclosLIMICS INSERM Facult© L©onard de Vinci rue Marcel Cachin Bobigny FranceEmail catherineduclosaphpfr IMIA Yearbook of Medical Informatics 2020Pragmatic Considerations on Clinical Decision Support from the Literature 0cAppendix Content Summaries of Best Papers for the Decision Support Section of the IMIA YearbookHendriks MP Verbeek XAAM van Vegchel T van der Sangen MJC Strobbe LJA Merkus JWS Zonderland HM Smorenburg CH Jager A Siesling STransformation of the National Breast Cancer Guideline into datadriven clinical decision treesJCO Clin Cancer Inform May3114Since clinical practice guidelines are still narrative and described in large textual documents the aim of this work was to model complex guidelines as datadriven clinical decision trees CDTs that could be still humaninterpretable while computerinterpretable for implementation in decision support systems The Dutch national breast cancer guidelines were translated into CDTs Data items which characterize the patient and the tumor and represent decisional criteria were encoded unambiguously using existing classifications and coding systems related to breast cancer when feasible In total CDTs were necessary to cover the whole guidelines driven by data items Of all data items could be coded using existing classification and coding systems All CDTs represented unique patient subpopulations Complex guidelines could be transformed as systematically constructed modular datadriven CDTs that are clinically interpretable and executable in a decision support applicationKamiÅ¡aliÄ A Ria±o D Kert S Welzer T Nemec Zlatolas LMultilevel medical knowledge formalization to support medical practice for chronic diseasesData Knowledge Engineering This research is focused on knowledge representation to support the medical processes involved in chronic diseases management which can be viewed as a procedural and sequential application of knowledge An intuitive easy and effective mechanism for medical knowledge formalization is proposed through a formalism called extended Timed Transition Diagram eTTD This formalism allows for the consistent representation of three basic levels of decision making that should be taken into account in the prescription and adaptation of longterm treatment therapy strategy dosage and intolerances The methodology can be manually applied to build eTTDs from clinical practice guidelines eTTDs implementation is demonstrated by modeling clinical practice guidelines for the therapeutic management of arterial hypertension The obtained models can be used as a baseline framework for the development of decision support systems involving medical proceduresKhalifa M Magrabi F Gallego BDeveloping a framework for evidencebased grading and assessment of predictive tools for clinical decision supportBMC Med Inform Decis Mak Oct Deciding to choose a clinical predictive tool in clinical practice should be guided by its correctly assessed effectiveness The objective of this work is to developp a conceptual and practical framework to Grade and Assess Predictive tools GRASP and provide clinicians with a standardised evidencebased system to support their search for and selection of efficient predictive tools The GRASP framework grades predictive tools based on published evidence across three dimensions phase of evaluation level of evidence and direction of evidence The final grade of the tool is based on the phase of evaluation that gets the hightest grade supported by the highest level of positive or mixed evidence that supports a positive This framework was successfully applied to five predictive tools GRASP report updates could be a way to maintain a data base that documents the evidence of predictive tools IMIA Yearbook of Medical Informatics 2020Duclos 0c" | Thyroid_Cancer |
"Meningiomas are the most common primary central nervous system tumors Potential risk factorsinclude obesity height history of allergyatopy and autoimmune diseases but findings are conflicting This studysought to assess the role of the different risk factors in the development of meningioma in adolescentsyoungadultsMethods The cohort included Jewish men and women who had undergone compulsory physicalexamination between and at age to years prior to and independent of actual military enlistmentTo determine the incidence of meningioma the military database was matched with the Israel National CancerRegistry Cox proportional hazard models were used to estimate the hazard ratios for meningioma according to sexbody mass index BMI height and history of allergic or autoimmune diseaseResults A total of subjects females were diagnosed with meningioma during a followup of personyears Median age at diagnosis was ± years range On univariate analysis female sex p and height p were associated with risk of meningioma When the data were stratified by sex heightremained a significant factor only in men Spline analysis of the male subjects showed that a height of m wasassociated with a minimum disease risk and a height of meters with a significant riskConclusions This large population study showed that sex and adolescent height in males m wereassociated with an increased risk of meningioma in adulthoodKeywords Allergy Autoimmune disease Height Meningioma SexBackgroundMeningiomas are the most common primary centralnervous system tumors They originate from the meninges which are the membranous layers surrounding thebrain Most meningiomas are grade I benign are grade II atypical and are grade IIIanaplastic [] Benign meningiomas have a female predominance or which is not found in the more Correspondence matanbe4gmailcom1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center Beilinson Hospital Petach Tikva IsraelFull list of author information is available at the end of the aggressive types [] In the USA meningiomas werefound to be more common in blacks than in whites witha ratio of [] The risk of acquiring a meningiomaincreases with age The median age at diagnosis is years []The only established external nongenetic risk factorfor brain tumors is exposure to ionizing radiation []An Israeli study revealed abnormally high rates of meningioma in patients treated with lowdose radiation tothe scalp for tinea capitis during the 1950s [] Otherpotential risk factors include obesity height history ofallergyatopy and history of autoimmune diseases but 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 0cBenZion Berliner BMC Cancer Page of the results are conflicting [] Establishing risk factors for meningioma can help identify individuals whomight benefit from risk reduction strategies and possiblyearly screening methodsThe aim of the present study was to assess potentialrisk factors for the development of meningioma in adolescence and early adulthoodMethodsStudy populationIsraeli adolescents undergo a compulsory medicalexamination at age to assess their fitness for military service regardless of whether they are drafted ornot Arab and Orthodox Jewish females and malesand Druze females are exempted Together with thephysical examination sociodemographic and psychobehavioral data are collected and the medical historyis thoroughly reviewed using documents provided byeach subjects primary care physician At the end ofthe process recruits are assigned a Functional Classification Code FCC that describes their medical status and occupational medical ranking The medicaldata and FCC are stored in the armys main databasewhich was computerized in []The population for the present study was derived from subjects born in who underwentpreenlistment medical examination between whenthe database was computerized and at age years Subjects with missing data on height and weightwere excluded n We also excluded subjects of North African and Asian origin born before many of whom had been exposed to radiation forthe treatment of tinea capitis after immigrating to Israelduring the 1950s [] These communities were laterfound to have a particularly high rate of meningioma []The final cohort consisted of subjects Fig Records were reviewed from the date of the initialmedical examination for military fitness to the date of afirst diagnosis of any cancer death or the predeterminedend of followup December Study variablesAtthe preenlistment medical examination demographic variables for each recruit were recorded in thearmy database as follows date of birth age at examination country of origin education socioeconomic status height weight and body mass index BMI Originwas defined by fathers country of birth or if the examinees father was born in Israel by paternal grandfatherscountry of birth and categorized as Europe includingNorth and South America Australia and SouthernAfrica Asia predominantly the Middle East Africaoverwhelmingly North Africa and Israel third or latergeneration Education was categorized as Fig Selection of the study populationand ¥ years of schooling Socioeconomic status wasdetermined by place of residence at the time of examination coded on a scale of and categorized intolow score middle score and high score [] Height and weight were measured by trainedmedics using a stadiometer and a beam balance with examinees barefoot and in underwear BMI was calculatedas weight in kilograms divided by height squared inmeters and categorized according to the WHO asunderweight kgm2 healthy weight kgm2 overweight kgm2 and obese ¥kgm2 Height was categorized according to the Centersfor Disease Control and Prevention below 25th percentile 25th to 50th percentile 50th to 75th percentile and75th percentile and aboveCognitive function including language skills and intellectual performance [] was assessed by a general 0cBenZion Berliner BMC Cancer Page of intelligence test administered by trained personnel Thetest is scored on a 90point scale that is adjusted fromtime to time scores are categorized as low medium and high []inflammatory bowel disease pemphigusMedical history was assessed according to the FCCslupus vascuAutoimmune diseases diabetes mellituslitisthyroiddisease celiac rheumatoid arthritis Addison disease andidiopathic thrombocyt ia purpura and allergic diseases asthma urticaria eczema allergic rhinitis atopicdermatitis allergic conjunctivitis and anaphylaxis weregrouped together for the present analysisAscertainment of meningioma incidenceTo determine the incidence of meningioma we matchedthe subjects who underwent the preenlistment medicalexamination during the study years to the Israel National Cancer Registry INCR a national populationbased registry established in In Israeli lawmandated the reportage of all diagnoses of malignant insitu and invasive borderline and certain benign brainand central nervous system tumors The estimated rateof reportage for solid tumors is which meets thestandards of the International Association of CancerRegistrieswwwhealthgovilPublicationsFilesICDC_365_EN_summarypd The INCR data includethe date of diagnosis site affected the InternationalClassification of Diseases code and the histologic description of the tumor according to the third edition ofthe International Classification of Diseases for OncologyICDO3 codes and At the time ofmatching the INCR had been updated until the end ofStatistical analysisCategorical variables are presented as number and percentage and continuous variables as mean and standarddeviation SD median 25th and 75th percentiles minimum and maximum were also calculated The association between risk factors and time to meningiomadiagnosis was assessed using Cox proportional hazardmodels hazard ratios HR confidence intervals CI and pvalues were calculated Log minus logfigures were inspected to confirm the proportionality ofthe hazard Crude rates were also determined Independent variables were initially entered individually into theCox model After sex and the interaction of sex andheight were found to be statistically significant separatemodels were established for men and women A Cox regression cubic spline function with three equally spacedknots positioned between the minimum and maximumvalues of height was fit to the data to estimate the heightvalue associated with minimum risk of meningioma inmen SASSTAT and SASGRAPH software version SAS Institute Inc Cary NC USA Other statistical analyses were performed with SPSS Statistics for Windowsversion IBM Armonk NY USA Twosided pvalues of were considered statistically significantResultsStudy populationThe baseline characteristics of the study population arepresented in Table The mean age at initial examination was ± years of the cohort was female The mean duration of followup was ± years median which represent in this study population a follow up of personyears The characteristics of the medical history of the subjects arepresented in the supplementary table Table 1SLinkage of the military database with the INCR yieldeda diagnosis of meningioma in of the subjects who underwent medical examination in to at age years grade I atypical anaplastic not specified and one patient with meningiomatosisTable The mean age at diagnosis ofmeningioma was ± years range andat the end of followup ± years median Univariate analysisOverall as expected meningiomas were more common infemales cases crude rate per personyears than in males cases crude rate per personyears p HR CI However there was no sex difference in the incidence for themore aggressive meningiomas atypical and anaplasticcrude rate per personyears for males andfemales On univariate analysis only sex and height weresignificantly associated with the risk of meningioma in thewhole study population p for both variables Afterstratification by sex height remained significant only inmales Table The risk of meningioma was minimalwhen height was up to m and statistically significantwhen height was greater than m Fig BMI was notassociated with an elevated risk of meningioma even whenanalyzed separately by sex Table Past medical history of asthma diabetes and otheratopic or autoimmune diseases was not associated withrisk of meningioma Even when autoimmune and allergic diseases were analyzed as a group there was no association with lower risk of meningioma Table andSupplemental Table When the subjects of African and Asian origin whowere excluded from the main analysis were included inthe cohort there was a significant interaction betweenperiod of birth vs and Asianand African origin representing the Middle East andNorth Africa as opposed to European and Israeli origin 0cBenZion Berliner BMC Cancer Page of Table Baseline characteristics of the study population total and by sexMaleCharacteristicsNumberBirth yearTotalLowMediumHigh years years years years 25th percentile25th50th percentile50th75th percentile 75th percentileEuropeAsiaAfricaIsraelEuropeAsiaAfricaIsraelSocioeconomic statusEducationCognitive indexaBMI category Kgm2Height category CDC percentileCountry of birthOriginAge at time of medical examination yearsBMIHeight metersaRated on a 90point scaleFemaleNumberSDMeanSDTotalNumberMeanSDMeanThe conjoined effect of birth year and origin showedthat origin North Africa and Asia was significant onlyfor subjects born between and SupplementalTable DiscussionIn this nationwide populationbased study we analyzedthe association of the development of meningioma insubjects born between and with baseline variables obtained for the subjects at the average age of years As expected meningiomas were found to be associated with sex female and taller stature None of theother sociodemographic and medical variables assessedincluding BMI and a diagnosis of asthma or diabetes atage years was associated with an increased risk ofmeningioma 0cBenZion Berliner BMC Cancer Page of Table Meningioma type and rate total and by sexMeningioma typeMeningioma NOSMeningiomatosisGrade Atypical AnaplasticTotalPersonyearsNOS Not otherwise specifiedMalesNumberPer FemalesNumberPer TotalNumberPer It is well accepted that benign meningioma is morecommon in females than males but the sex predilectiondisappears with the more aggressive meningiomas []The female predominance might be explained by thefinding that meningiomas harbor receptors for estrogenand progesterone []We discovered an association between the risk ofmeningioma and height in men but not with BMI inmen or women The results of previous studies for thesetwo factors were conflicting A large Norwegian studyincluding million subjects found that height was associated with meningioma in both men and women butBMI was not [] whereas another study of postmenopausal females revealed an association of meningiomawith both BMI and height [] A metaanalysis of studies supported the correlation of BMI and meningioma It is worth noting that the Norwegian study exceeds the metaanalysis in size and power and that inthe Norwegian study a subgroup analyses for womenand men as well as different age groups was performedwithout finding convincing evidence of a strong association between overweight obesity and risk for meningioma [] In our study BMI was measured when thesubjects were years old much younger than theTable Univariate analysis association of potential risk factors with diagnosis of meningioma by sexVariablesMalesNCases Crude rate HR CILower UpperpCases Crude rate HR CILower UpperpFemalesNHeightHeight continuousBMIPercentile Kgm2 Autoimmune diseasesa NoAllergic diseasesbAsthmaDiabetesYesNoYesNoYesNoYes aAutoimmune disease diabetes mellitus lupus vasculitis IBD pemphigus thyroid disease celiac rheumatoid arthritis Addison disease and idiopathic thrombocyt icpurpurabAllergic disease including asthma urticaria eczema allergic rhinitis atopic dermatitis allergic conjunctivitis and anaphylaxis 0cBenZion Berliner BMC Cancer Page of Fig Spline analysis in the men group showing the minimum risk for meningioma at a height of m and a statistically significant increase inthe risk for meningioma at heights taller than mstudies included in the metanalysis which might explainthe discordant results []Height has been associated with different types of cancer melanoma thyroid testis breast and lymphomaSuggested mechanism for the greater risk of meningioma in taller people is their higher levels of circulatinginsulinlike growth factors IGFs which may influencecell proliferation and tumor growth [] Moreoveroverexpression of IGFI and IGFII mRNA transcriptshas been demonstrated in meningioma [] Circulatinglevels of IGFs are highest during puberty They decreaserapidly in the third decade of life in the general population but seem to stay consistently higher in taller adults[] It is not clear why this association was evident onlyin males in our study maybe in women the influence ofthe hormonal status blurred the influence of the heightSeveral earlier studies reported an inverse associationbetween a history of allergic diseases including asthmaand meningioma [ ] However this finding wasnot supported by others [ ] We failed to demonstrate an association between meningiomas and allergicdiseases including asthma urticaria eczema allergicrhinitis atopic dermatitis conjunctivitis and anaphylaxisand allergy to beesSimilarly a recent study reported an inverse association between hyperglycemia and the risk of meningioma [] whereas another found a positive associationwith a history of diabetes mellitus [] In the presentstudy diabetes was not associated with the risk of meningioma This was true for other autoimmune diseasesas welllimitationThis analysis also has certain limitations The followup period in this study was limited to years such thatthe study population was still young when the studyended Subsequently the median age of those who developed meningioma in our study was younger than themedian age of patients with meningioma in the generalpopulation [] With a more extensive followup wemight find more latent tumor growths that could potentially increase or shift the incidence of intracranial neoplasms Anotherisunderreporting of meningiomas that are diagnosed onlyaccording to radiographic findings without histologicalfindings As it is well known that in some cases meningiomas diagnosed radiographically mayjust befollowed by repeat scanningcohortits prospectivepopulationbased design large sample size high degreeof completeness of the cancer registry data throughoutthe study period and the ability to carefully control forpotential confounders such as exposure to radiation Itshould be noted that in a study that was published recently and examined the same cohort the median heightremained almost stable during the study period theStrengths of ourofthestudyinclude 0cBenZion Berliner BMC Cancer Page of median height of males increased by cm and that offemales remained stable despite environmental socialand nutritional changes []ConclusionThis large populationbased study showed that sex female and tall stature in adolescent males was associatedwith an increased risk of meningioma in adulthoodSupplementary informationSupplementary information accompanies this paper at doi101186s12885020072924Additional file Supplementary Table Medical historycharacteristics of the study populationAdditional file Supplementary Table Univariate analysisassociation of potential risk factors with diagnosis of meningioma by sexAdditional file Supplementary Table Interaction between birthperiod and origin whole population adjusted for sexAbbreviationsCNS Central nervous system BMI Body mass index FCC FunctionalClassification INCR Israel National Cancer Registry ICDO InternationalClassification of Diseases for Oncology SD Standard deviation HR Hazardratio CI Confidence interval IGF Insulinlike growth factorAcknowledgmentsNot applicableAuthors contributionsMBZB and SYK analyzed the preliminary database extracted the relevantinformation to allow hypothesis testing and prepared the manuscript andtables Statistical analysis and figure preparation were performed by LHK andED HL LKB YL AH JM and GT participated in the preliminary preparationand conceptual design and revised the final manuscript ABA OG AK and TSreviewed the neurological proof of concept and revised the final manuscriptand supplementary materials All authors read and approved the finalmanuscriptFundingThe study was funded by the Israel Cancer Association by the Lillia andJacob Alther donation financial support without any role in the manuscriptpreparationAvailability of data and materialsThe datasets used during this study are available from the correspondingauthor on reasonable requestEthics approval and consent to participateAll procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional andor nationalresearch committee and with the Helsinki declaration and its lateramendments or comparable ethical standards The study was approved bythe IDF Israel Defense Forces Medical Corps Institutional Review Boardwhich waived the requirement for informed consent because the data usedwere obtained from medical records without patient participation referencenumber Consent for publicationNot applicableAuthor details1NeuroOncology Unit Davidoff Cancer Center Rabin Medical Center Beilinson Hospital Petach Tikva Israel 2Department of GastroenterologyHadassah University Hospital Ein Kerem Jerusalem Israel 3Sackler Facultyof Medicine Tel Aviv University Tel Aviv Israel 4Braun School of PublicHealth and Community Medicine Hadassah University Hospital Ein KeremJerusalem Israel 5Israel Center for Disease Control Israel Ministry of HealthRamat Gan Israel 6School of Public Health University of Haifa Haifa Israel7Department of Neurosurgery Rabin Medical Center Beilinson HospitalPetach Tikva Israel 8Medical Corps Israel Defense Forcesand Department ofMilitary Medicine Hebrew University of Jerusalem Faculty of MedicineJerusalem Israel 9Institute of Endocrinology and Talpiot Medical LeadershipProgramSheba Medical Center Tel Hashomer IsraelReceived April Accepted August ReferencesOstrom QT Gittleman H Fulop J Liu M Blanda R Kromer C Wolinsky YKruchko C BarnholtzSloan JS CBTRUS statistical report primary brain andcentral nervous system tumors diagnosed in the United States in NeuroOncology 201517Suppl 4iv1iv62 doi101093neuoncnov189Claus EB Bondy ML Schildkraut JM Wiemels JL Wrensch M Black PMEpidemiology of intracranial meningioma Neurosurgery doi10122701neu000018828191351b9Braganza MZ Kitahara CM Berrington de González A Inskip PD Johnson KJRajaraman P Ionizing radiation and the risk of brain and central nervoussystem tumors a systematic review NeuroOncology doi101093neuoncnos208 Modan B Baidatz D Mart H Steinitz R Levin SG Radiationinduced headand neck tumours Lancet doi101016s0140 Wiedmann MKH Brunb C Di Ieva A Lindemann K Johannesen TBVatten L Helseth E Zwart JA Overweight obesity and height as risk factorsfor meningioma glioma pituitary adenoma and nerve sheath tumor alarge populationbased prospective cohort study Acta Oncol doi1010800284186X20171330554Johnson DR Olson JE Vierkant RA Hammack JE Wang AH Folsom ARVirnig BA Cerhan JR Risk factors for meningioma in postmenopausalwomen results from the Iowa Women's health study NeuroOncology doi101093neuoncnor081 Michaud DS Bové G Gallo V Schlehofer B Tjønneland A Olsen A OvervadK Dahm CC Teucher B Boeing H Steffen A Trichopoulou A Bamia CKyrozis A Sacerdote C Agnoli C Palli D Tumino R Mattiello A BuenodeMesquita HB Peeters PH May AM Barricarte A Chirlaque MD DorronsoroM José Sánchez M RodrÃguez L Duell EJ Hallmans G Melin BS Manjer JBquist S Khaw KT Wareham N Allen NE Travis RC Romieu I Vineis PRiboli E Anthropometric measures physical activity and risk of glioma andmeningioma in a large prospective cohort study E Cancer Prev Res Phila doi10115819406207CAPR110014Niedermaier T Behrens G Schmid D Schlecht I Fischer B Leitzmann MFBody mass index physical activity and risk of adult meningioma andglioma a metaanalysis Neurology doi101212WNL0000000000002020Brenner AV Linet MS Fine HA Shapiro WR Selker RG Black PM Inskip PDHistory of allergies and autoimmune diseases and risk of brain tumors inadults Int J Cancer doi101002ijc10320 Wang M Chen C Qu J Xu T Lu Y Chen J Wu S Inverse associationbetween eczema and meningioma a metaanalysis Cancer Causes Control doi101007s1055201198086 Gal R The selection classification and placement process in a portrait ofthe Israeli soldier Westport CT Greenwood Press p YustKatz S Bar Oz A Derazne E Katz LH Levine H KeinanBoker L Amiel ACompeting interestsThe authors declare that they have no financial or nonfinancial competinginterestsKanner A Laviv Y Honig A Shelef I Siegal T Twig G Kark J Echoes fromthe past changing associations between brain tumors and ethnicity JNeurol Sci doi101016jjns2019116552 [Epubahead of print]Israel Central Bureau of Statistics Characterization and classification of localauthorities by the socioeconomic level of the population Jerusalem Israelcentral Bureau of Statistics 0cBenZion Berliner BMC Cancer Page of Twig G Gluzman I Tirosh A Gerstein HC Yaniv G Afek A Derazne E Tzur DKarasik A Gordon B Fruchter E Lubin G Rudich A CukiermanYaffe TCognitive function and the risk for diabetes among young men DiabetesCare Nov37112982 doi102337dc140715 Guevara P EscobarArriaga E SaavedraPerez D MartinezRumayor A FloresEstrada D Rembao D Calderon A Sotelo J Arrieta O Angiogenesis andexpression of estrogen and progesterone receptors as predictive factors forrecurrence of meningioma J NeuroOncol doi101007s110600172662y Gunnell D Oliver SE Donovan JL Peters TJ Gillatt D Persad R Hamdy FCMeal DE Holly JMP Do heightrelated variations in insulinlike growthfactors underlie the associations of stature with chronic diseases J ClinEndocrinol Metab doi101210jc2003030507 Zumkeller W Westphal M The IGFIGFBP system in CNS malignancy MolPathol Crowe FL Key TJ Allen NE A crosssectional analysis of theassociations between adult height BMI and serum concentrations of IGFIand IGFBP1 and in the European prospective investigation intocancer and nutrition EPIC Ann Hum Biol doi BergBeckhoff G Schüz J Blettner M Münster E Schlaefer K Wahrendorf JSchlehofer B History of allergic disease and epilepsy and risk of glioma andmeningioma INTERPHONE study group Germany Eur J Epidemiol doi101007s1065400993556Schneider B Pülhorn H Röhrig B Rainov NG Predisposing conditions andrisk factors for development of symptomatic meningioma in adults CancerDetect Prev doi101016jcdp200507002Linos E Raine T Alonso A Michaud D Atopy and risk of brain tumors ametaanalysis J Natl Cancer Inst doi101093jncidjm170 Bernardo BM Orellana RC Weisband YL Hammar N Walldius G MalmstromH Ahlbom A Feychting M Schwartzbaum J Association betweenprediagnostic glucose triglycerides cholesterol and meningioma andreverse causality Br J Cancer doi101038bjcPublishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjonThis 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 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partners occupation and economic statusPatients occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatients marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patients hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patients family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet 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 Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0c" | Thyroid_Cancer |
"Ovine pulmonary adenocarcinoma OPA is a neoplastic disease caused by exogenous Jaagsiekte SheepRetrovirus exJSRV The prevalence of JSRVrelated OPA in Eastern European countries including Romania is unknownWe aimed to investigate the prevalence and morphological features of OPA classical and atypical forms in theTransylvania region Romania the immunophenotype of the pulmonary tumors and their relationships with exJSRVinfection A total of adult ewes slaughtered between and in two private slaughterhouses fromTransylvania region Romania was evaluated Lung tumors were subsequently assessed by cytology histologyimmunocytochemistry immunohistochemistry electron microscopy and DNA testingContinued on next page Correspondence mariantaulescuusamvclujro1Department of Veterinary Pathology University of Agricultural Sciences andVeterinary Medicine Calea Manastur ClujNapoca Romania2Laboratory of Genomics Biodiversity Animal Breeding and MolecularPathology Institute of Life Sciences University of Agricultural Sciences andVeterinary Medicine ClujNapoca RomaniaFull 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 0cToma BMC Veterinary Research Page of Continued from previous pageResults Out of examined sheep had OPA prevalence The diaphragmatic lobes were the most affectedGrossly the classical OPA was identified in of investigated cases and the atypical OPA in that includedsolitary myxomatous nodules Histopathology results confirmed the presence of OPA in all suspected cases which wereclassified into acinar and papillary types Myxoid growths MGs were diagnosed in classical OPA cases and in cases ofatypical form Lung adenocarcinoma was positive for MCK and TTF1 and MGs showed immunoreaction for VimentinDesmin and SMA Ki67 expression of classical OPA was higher than atypical OPA and MGs JSRVMA was identified by IHC in both epithelial and mesenchymal cells of OPA Immunocytochemistry and electron microscopy alsoconfirmed the JSRV within the neoplastic cells ExJSRV was identified by PCR in of analyzed samples Phylogeneticanalysis revealed the presence of the exJSRV type MT8096781 in Romanian sheep affected by lung cancer andshowed a high similarity with the UK strain AF1052201Conclusions In this study we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPAin sheep Additionally we described the first report of atypical OPA in Romania and to the best of our knowledge inEastern Europe Finally we showed that MGs have a myofibroblastic originKeywords Atypical OPA Epidemiology Jaagsiekte sheep retrovirus type Myxoid growths Ovine pulmonaryadenocarcinomaincluding JSRV []BackgroundOvine pulmonary adenocarcinoma OPA represents avirusrelated neoplastic disease caused by an exogenousbetaretrovirus [] The disease was first reported inSouth Africa in the late 1800s as an important cause ofchronic respiratory distress in sheep [] The etiologicalagent of OPA is exogenous Jaagsiekte Sheep RetrovirusexJSRV A specific U3 long terminal repeatLTRsequence of exJSRV was detected in lungs from affectedanimals [ ] The LTR region varies among differentretrovirusessheep endogenousretrovirus [] and ovine enzootic nasal tumour virus []Based on the U3 sequence and restriction profiles of thevirus some authors suggest that there are two types ofexogenous retroviral sequencesKenyan andSouth African and type II Wyoming USA and UK isolates [ ] ExJSRV has a specific tropism for the differentiated epithelial cellstype II pneumocytes andnonciliated bronchiolar Clara cells of the lung and it isthe only virus known to cause pulmonary adenocarcinoma in naturally infected animals [] ExJSRV is mainlytransmitted by infected aerosols [ ] and in naturalcases the incubation period is very long ranging from to years Therefore it is most often encountered inadult sheep [] but the lambs can also manifest clinicalsigns [] There is no evidence for breed or sex relatedOPA susceptibility [] Other animal species includinggoats and moufflons were occasionally diagnosed withOPA [ ]type IOPA shares many histological similarities with the human pulmonary adenocarcinoma representing an important animal model for understanding the mechanisms ofviral oncogenesis [] Although JSRV was found in humanpulmonary neoplastic cells its role in the development oflung cancer in humans is not fully elucidated []There are two recognized forms of OPA which showgross histological and immunohistochemical differences[ ] The lesions in classical OPA predominantlyaffect all pulmonary lobes and are located in the cranioventral area They can be either nodular or exhibit a diffuse growth type showing a grey moist appearance on thecross section Atypical OPA consists of hard nodulespearlywhite that have a dry cut surface the tumors arewelldelimited by the surrounding pulmonary tissue []such asThe classical form is more common than the atypicaltype In Europe the classical OPA has been reported inseveral countriesIreland [] UK []Scotland [] Italy [] Germany [] Spain [] Theincidence of the disease is usually low but in some geographical areas it reaches up to [] More than of the affected animals usually die because of progressive respiratory failure [] therefore causing importanteconomic losses The atypical OPA is less contagious thanclassical form [] The atypical OPA has been reported inSpain Peru Iran and India [ ] but there is noevidence of its occurrence in other countries where OPAis commonly foundClinically the affected sheep develop chronic and progressive respiratory distress especially when exercised Acommon sign of classical OPA is mucous nasal discharge because of production of large fluids amounts inthe lung [] This fluid might be absent in some casesparticularly in the atypical form of OPA where the tumors remain incidentally found in abattoir studies orwhen the animals are necropsied for other reasons []Currently there are no efficient methods to clinicaldiagnose the disease and a full diagnosis can be obtainedonly postmortem by histological examination [] Polymerase chain reaction PCR analysis of bronchoalveolarlavage BAL samples collected from living animals may 0cToma BMC Veterinary Research Page of be useful for preclinical identification of infected individuals with exJSRV However this technique is not ableto identify all the early stages of classical OPA and atypicalform where the mucus production is absent orfewer infected cells are present in the BAL sample [] Currently this method is not extensively used because the sample collection requires sedation Furthermore intravitam diagnosis of exJSRV infection by PCRfrom blood offers many false negative results probablybecause the numbers of infected white blood cells is verylow [ ] Additionally the viral genetic material orproteins can also be detected by PCR and immunohistochemistry IHC respectively in pulmonary and lymphoid tissues [ ]Romania is an important European country in sheepfarming with over million heads reported in according to Romanian Minister of Agriculture There areonly two studies regarding OPA presence in Romaniawith reported incidences of and [ ] However no evidence of exJSRV infection in relation withOPA nor atypical OPA had been previously describedCurrently no information about the prevalence of classical OPA in Romania is available Furthermore to theauthorâs knowledge exJSRVrelated OPA has not beenreported so far in other countries from Eastern Europein the last decadeIn this study we aimed to investigate the prevalenceand morphological features of OPA classical and atypicalforms in Turcana sheep breed in the Transylvania regionRomania immunohistochemical features of the neoplastic epithelial components and myxoid growths MGsand identification of exJSRV by electron microscopyimmunocytochemistry IHC and PCR methods A comparison between nucleotide sequences of exJSRV identified in sheep lung tumors and other reported exJSRVstrains from different geographical regions was also previewed in order to identify the strain present in RomaniaResultsPrevalence distribution patterns and gross features ofOPAOut of examined slaughtered ewes cases weresuspected of OPA after gross examination Howeverhistological examination confirmed the presence of OPAin cases therefore a prevalence of of the disease in Transylvania Romania Data of sheep includedin the study and confirmation of JSRV infection aresummarized in Table In the remaining cases n the diagnoses ofchronic suppurative bacterial bronchopneumonia andverminous bronchopneumonia associated with extensivefibrosis and atelectasis were histologically differentiatedfrom OPA All diagnosed sheep belonged to Turcanabreed and were adult females the age ranging from to yearsOn postmortem examination the distribution patterns of pulmonary lesions were divided into three majroups Fig 1a Group I in cases the lesions were focal or solitary multifocal to coalescing anddiffuse large masses affecting a single pulmonary lobewith higher prevalence in the left diaphragmatic lobe cases Fig 1b Group II in cases the lesionswere located unilateral but affecting or more pulmonary lobes Fig 1c Group IIIin the other cases neoplastic processes were bilaterally locatedmainly affecting the diaphragmatic lobes and involvingup to of the pulmonary parenchyma Fig 1d Inthese severe cases the lungs failed to collapse and theywere heavy and denseGrossly the morphological features of pulmonary lesions were classified according to GarciaGoti []into two main pathologic forms classical OPA and atypical OPA The classical OPA was identified in cases and consisted of pink to light grey or whitesolitary nodules of varying size cm or large denseconfluent masses reaching up to cm in diameter Insome cases the neoplastic masses were delimited fromthe normal parenchyma by a fine line of atelectasia or bya zone of emphysema On the cut surface the tumorsincluded in this form were moist had a homogenous appearance and the airways usually contained a viscousfrothy fluid Fig 1d In two cases large areas of lytic necrosis and cavitation were randomly distributed withinthe neoplastic tissue In most cases multifocal to diffusepleural fibrosis over the neoplastic masses was a common gross findingThe atypical OPA was encountered in four cases and characterized by white dried nodular orconfluent lesions of approximately â cm in diametermainly located in the subpleural area of the left diaphragmatic lobe Fig 1e and e1 No or small amount of lungfluid was detected in these cases In two of the cases thepulmonary lesions consisted of whitegrey solitary welldelimited and unencapsulated nodules Fig 1f on the cutsurface these nodules showed a multilobular soft to denseand gelatinous appearance and consisted with myxomalike masses Fig 1gCytological and histopathological featuresCytological examination of both classical and atypicalforms of OPA revealed numerous well differentiated cuboidal or polygonal neoplastic epithelial cells arranged insmall acini clusters or individually Fig 2a The neoplastic cells had a moderate amount of pale blue finelygranular cytoplasm moderate nuclear cytoplasmic NC ratio and round to oval centrally located nuclei withfinely stippled chromatin and â distinct blue nucleoli 0cToma BMC Veterinary Research Page of Table Data of sheep included in the study and confirmation of JSRV infectionCase samples115MAY17Pathologic formClassicalGenderageFHistological typePapillaryBreedTurcanaIHCJRSVPCRJSRVICCNPTEMNP225MAY17356JUN17433DEC17534DEC17636DEC17702JAN18803JAN189012JAN18101JAN18112JAN18125JAN181310JAN18143JUL18155JUL18166JUL18177JUL181802AUG181903AUG18201AUG18212AUG18223AUG18235AUG18246AUG18251OCT18262OCT18273OCT18284OCT18295OCT18306OCT18317OCT18328OCT18333MAY19TurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaTurcanaFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFClassicalAtypicalClassicalClassicalClassicalAtypicalClassicalClassicalAtypicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalAtypicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassicalClassical344MAY19Age adult sheep â years F female MGs myxoid growths NP not performedClassicalTurcanaFPapillaryMGsAcinarAcinarAcinarAcinarAcinarAcinarAcinarPapillaryPapillaryAcinarPapillaryAcinar MGsPapillaryAcinarPapillaryPapillary MGsAcinar MGsPapillaryAcinarAcinarMGsAcinarPapillary MGsAcinarAcinarAcinarAcinar MGsAcinarAcinarPapillary MGsAcinarâââNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPNPââNPNPâNPAnisocytosis and anisokaryosis were mild to moderatewith rare mitotic figures Large numbers of macrophagesand mature lymphocytes were admixed with the neoplastic cells OPA containing MGs were poorly cellular and composed of spindle stellate and elongatedcells with round to oval nuclei and inserted in apale blue slightly vacuolar extracellular myxoid material Alltumors diagnosed by cytological examwere subsequently confirmed by histopathology andimmunohistochemistryHistologically all pulmonary tumors classified as classical form of OPA n were composed of a singlelayer of neoplastic cuboidal columnar or polyhedral epithelial cells lining the alveolar lumina or bronchiolessurrounded by a fine to moderate fibrovascular stromaThe predominated histologicaltypes of pulmonaryadenocarcinoma were represented by acinar n Fig 2b and papillary n Fig 2c The diameter ofindividual neoplastic epithelial cells ranged from to μm showed moderate amount of pale acidophilic 0cToma BMC Veterinary Research Page of Fig Gross features of spontaneous OPA in sheep a Distribution of pulmonary lesions b Classical form of OPA involving the diaphragmaticlobe arrow c Classical form of OPA affecting multiple pulmonary lobes arrows d Classical form of OPA on cut surface showing the junctioninterrupted line between the tumor and normal tissue e and e1 Atypical form of OPA showing a subpleural whitegrey nodule arrow On cutsurface the tumor is pearly white and dried arrow f Gross aspects of pulmonary myxomalike nodule arrow g the myxomatous mass shows amultilobular whitegray gelatinous feature on cut surfacefinely granular cytoplasm and intermediate NC ratioAnisokaryosis and anisocytosis were low to moderatethe nuclei were round to oval centrally located with afine granular to lacy chromatin with and â distinctbasophilic nucleoli The average of mitotic rate was per HPF without atypical features Multifocal thestroma was heavily infiltrated with macrophages lymphocytes and few plasma cells The adjacent parenchymaof the neoplastic mass showed atelectasis and at the periphery of the tumors the neoplastic cells were occasionally arranged in a lepidic growth pattern The myxoidgrowths consisting of sparsely cellular structures of spindles cells embedded in an abundant extracellular matrixwere observed in cases of classical OPA The MGswere admixed with the neoplastic epithelial componentor in some areas were disposed as multiple poorly demarcated masses Fig 2d eThe histological features of the atypical form n were similar to those described in classical form andtwo cases were classified as acinar type The distinguishof atypical OPA features were represented by moreprominent limits between normal tissue and neoplasticmasses due to higher fibroblast proliferation and inflammatory infiltrates predominated by mature lymphocytesand macrophages Fig 2fIn the other two cases of pulmonary solitary nodulesthe neoplastic epithelial component was absent and theneoplasm was composed by short bundles and streamsof spindle stellate or individual cells Fig 2g The mesenchymal cells were embedded into an abundant myxoidmatrix that contains moderate amounts offoamyamphophilic ABPAS positive material mucin Fig 2hand i The individual cells had variable distinct bordersintermediate NC ratio and moderate amounts of paleacidophilic homogenous cytoplasm The nuclei wereoval to elongated â μm in diameter in transversesection centrally located with clumped chromatin andindistinct nucleoli No mitotic figures were present anisocytosis and anisokaryosis were low Based on thehistological features a presumptive diagnosis of pulmonary myxomas was madeOverall mesenchymal proliferations and myxomatouschanges named as myxoid growths MGs were identified in out of tumors cases of classical 0cToma BMC Veterinary Research Page of Fig Microscopical findings of OPA a Cytological exam of pulmonary tumors showing multiple nest of cubical to polygonal epithelial cellsinterpreted as type II alveolar cells DQ stain b Histological appearance of OPAclassical form tubular type and c papillary type HE stain d and eThe microphotographs of classical OPA showing myxoid growths white arrow interspersed with neoplastic epithelial component blue arrowHE stain f Histological exam of atypical OPA showing a welldelimited neoplastic nodule surrounded by fibrous tissue and infilammatoryinfiltrates arrow HE stain g Myxomalike tumor without neoplastic epithelial component The mass is multinodular white arrow andcomposed of spindle to stellates cells and abundant extracellular matrix the inset HE stain h and i ABPAS stained sections from myxomalikenodules showing abundant pale blue extracellular myxomatous matrix the Goblet cells of bronchial lining epithelium are used as positivecontrol for ABPAS stainform and cases of atypical form consistent with pulmonary nodules without epithelial cell neoplasia myxomalike nodulesImmunohistochemical features of pulmonary massesImmunohistochemically in both forms of OPA the neoplastic epithelial component showed a strong and diffusereaction for MCK Fig 3a and TTF1 Fig 3b the epithelial cells were negative for vimentin Fig 3c Thecells of MGs were diffusely and intense positive forvimentin Fig 3d desmin Fig 3e selective positive foralphaSMA Fig 3f and negative for MCK Fig 3g suggesting their true mesenchymal origin probably frompulmonary interstitial myofibroblasts All mesenchymalcomponents were also negative for S100 protein Fig3h and TTF1 The proliferative index ki67 of theepithelial component of classical OPA was higher meanvalue of Fig 3i than atypical OPA mean valueof whereas in the MGs this labelling was low inall forms mean value of classical form and in atypical OPA consisting of myxomalike noduleswithout neoplastic epithelial component Fig 3jstainingJSRV identification methodsImmunocytochemistry and immunohistochemistryImmunocytochemicalsmearsimprintsshowed a positive reaction consistent with the presenceof intracytoplasmic JSRV antigen within the neoplasticcells Fig 4a and a1 No immunocytochemical expression of JSRVMA was detected in the imprints smearsfrom normal lungsof 0cToma BMC Veterinary Research Page of Fig Immunophenotypical characterization of OPA a The neoplastic epithelial cells of classical form are diffusely and intensely immunopositivefor MCK b TTF1positive nuclei of the epithelial cells are present within the neoplastic masses Inset the bronchial epithelium was used aspositive control for TTF1 c All epithelial cells lining neoplastic acini are negative for vimentin blue arrow in contrast to immunopositive stromalcells white arrow d Myxoid growths showing an intense immunopositive reaction for Vimentin Inset detail of diffuse cytoplasmic labeling forVimentin e Mesenchymal cells of myxoid growths have diffuse and strong cytoplasmic labeling for Desmin Inset The smooth muscle cells ofpulmonary arteries and bronchioles are strongly positive for desmin and served as internal positive control f The cells of myxoid growths haveselective and moderate cytoplasmic labeling for alphaSMA Inset The bronchiolar smooth muscle cells are positive for SMA internal positivecontrol g The myxoid component of OPA showing a negative immunoreaction of MCK blue arrow compared to the neoplastic epithelial cellswhich are strongly immunopositive h Neoplastic cells of myxomalike nodules showing a negative reaction for S100 protein Inset the bronchialcartilage is diffusely positive for S100 protein internal positive control i The proliferative index characterized by ki67 immunopositive nuclei washigher in the epithelial neoplasia compared to the MGs and myxomalike nodules blue arrows j Inset Bronchialassociated lymphoid tissueBALT hyperplasia was used as internal positive control for ki67 DAB and hematoxylin counterstainIn both classical and atypical OPAimmunohistochemical evaluation of pulmonary tumors showed a diffuse positive JSRVMA reaction of neoplastic cells in cases The positive reaction was characterizedby a finely granular brown staining of the cellular cytoplasm of both neoplastic epithelial component Fig 4band MGs Fig 4c Additionally a positive reaction wasalso observed for the inflammatory cells mainly macrophages and lymphocytes Moreover the intensity andnumber of JSRVpositive cells in atypical tumours werereduced compared to the classicalform of OPA Anegative JSRVMA reaction was found in all normal pulmonary tissues n selected as negative controlTransmission electron microscopy TEMUltrastucturally the neoplastic type II pneumocytes havebeen recognized by the presence of microvilli basal orcentrally located nuclei numerousintracytoplasmicmoderately electronodense round and lamellar structures of approximately â nm in diameter interpreted as lamellar bodies Fig 4d Additionally roundoval structures of â μm in diameter delimited by a 0cToma BMC Veterinary Research Page of Fig JSRV detection from neoplastic tissues a and a1 Immunocytochemistry showing intracytoplasmic JSRVMA expression within the neoplastic epithelialcells DAB and hematoxylin counterstain B Diffuse expression of the JSRVMA marker by neoplastic epithelial cells forming tubular structures c Expression ofthe JSRVMA marker by MGs and epithelial tumor Left inset Cell cytoplasm of MGs white arrow is strongly labelled with JSRVMA antibody Right insetNeoplastic epithelial cells have diffuse cytoplasmic labelling for JSRVMA bue arrow DAB and hematoxylin counterstain d JSRVinfected type pneumocytes Note the presence of intracytoplasmic lamellar body white arrow and aggregates and solitary intracytoplasmic viral ps grey arrowTEM Bar μm Inset Detail of a viral replication site showing specific virions compatible with JSRV white arrow TEM Bar nm e Electrophoresis profile ofthe amplified DNA fragments bp evidencing the presence of proviral Jaagsiekte DNA in ovine pulmonary adenocarcinoma OPA L DNA ladder PCRpositive samples PCR negative â samples M1 and M2 control samples amplified from healthy ovine lung tissue NTC no template control negativecontrol f Phylogenetic analysis of different JSRV strains and other retroviruses based on the LTR region The Romanian strains of exJSRV JSRVRO GenBankMT8096781 showed homology at nucleotide level of with UK strain GenBank AF1052201 and with the South African strain GenBank M80216 0cToma BMC Veterinary Research Page of moderately electrondense membrane and containing anabundant electron lucent material intracytoplasmic vacuoles were also identified within the cytoplasm of typeII alveolar cells JSRV ps were found within theaforementioned intracytoplasmic vacuoles or in the cytosol of the neoplastic cells in out of examined samples The viral ps were arranged into small groupsâ virions or individually measured about ânm in diameter and showed a moderately electrondense central area Fig 4d Extracellular JSRV pswere not observed in none of the examined examplesPCR DNA sequencing and phylogenyAll pulmonary lesions histologically diagnosed as OPAwere submitted for PCR analysis The expected pbamplicons obtained from exJSRV proviral DNA werevisualized in out of analysed samples while no bands were visible in the negative controlsFig 4e The unaffected tissues adjacent to the neoplastic masses were also negative for the virus presenceBased on the obtained LTR nucleotide sequences ofJSRV deposited in GenBank under accession numberMT8096781 from lung tumours and their comparisonwith other retroviral strains available in GenBank weevidenced a homology at nucleotide level of withexJSRV UK strain GenBank AF1052201 and only with the South African strain GenBank M80216Fig 4f Therefore we concluded that affected animalsare infected with Jaagsiekte Sheep Retrovirus type which is the most probable responsible for the adenocarcinoma found in Romanian Turcana sheepDiscussionsPrimary respiratory tract and lung tumors in animalshave a lower incidence compared with other systems aswell as compared to human patients In contrast thelung is the preferential site of metastases in all animals[] In sheep the most common tumor of the respiratory system is ovine pulmonary adenocarcinoma OPAalso known as jaagsiekte or ovine pulmonary adenomatosis []This work confirms the presence ofJSRVinfectedsheep in Romania To the authorâs knowledge JSRVrelated OPA has not been reported in other countries ofEastern Europe in the last decadeFor this study the samples were collected from twoslaughterhouses which are representative for Transylvania not for the whole country Unfortunately due tospecific slaughtering procedures we collected the pulmonary tissue samples only at the end of slaughteringAs a consequence an exact match between examinedlungs and ear tag for each slaughtered sheep was notpossible to be determined Therefore neither the exactorigin and age of each sheep were not possible to beestablished However according to the records of theslaughterhouses all slaughtered individuals were adultfemales and originated from different counties of Transylvaniaincluding Cluj BistritaNasaud Mures Sibiuand Alba Thus the authors can relate that all the positive OPA cases originated from Transylvania a historicalregion that is located in center of Romania The presentstudy shows that the prevalence of OPA is about and is higher compared to the previous reports regarding OPA situation in Romania and respectively[ ] ExJSRV presence was not determined in any ofthese two previous studies Furthermore the prevalenceof OPA in Romania is higher than in other Europeancountries including UK [] and Ireland [] We can think about two possible explanations ofthis prevalence of OPA in Romanian Turcana sheepFirst it can be related with a high number of sheep thatare raised in Romania According to the Ministry ofAgriculture data the number of sheep in was over million heads This places the Romanian sheep industry in the third position in Europe after Spain and UKAnother important causative factor could be related withsheep management in Romania The majority of animalsare raised in free pastures and there is a lot of sheepmovements between regions to find fresh grasslandsThis is a traditional sheep breeding management and itis a way of life for the sheep breeders that is still verypresent in Romania These two factors might explain ahigher OPA prevalence in Romanian sheep comparedwith other European countries In contrast to the prevalence of OPA in Romania the disease is quite commonin South America South Africa and Scotland where â of infected animals develop pulmonary tumors []OPA represents a continuous issue regarding propermethods of diagnosis and prevention [ ] Despitesome specific clinical signs of classical OPA includingdyspnea and tachypnea in combination with progressive weight loss and nasal mucous discharge [] andsuggested imaging modalities radiography computedtomography and ultrasonographic examination [ ]currently the gold standard diagnostic method for bothclassical and atypical OPA relies in gross and histologyexams performed during post mortem evaluation [] Inour study data about clinical signs of slaughtered sheepwith OPA were not available Postmortem evaluation including gross and histological exams of the affectedsheep is essential for confirmation of OPA Althoughgross changes are relatively specific for OPA in somecases chronic bronchopneumonia should be included inthe differential list A recent study showed that numerous cases of suspected OPA based on grosschanges were false positive lesions due to its similaritieswith chronic bronchopneumonia moreover wereexJSRVnegative by RTPCR and IHC In some cases 0cToma BMC Veterinary Research Page of in sheepare usuallyOPA and bronchopneumonia can occur concurrentlyand the pneumonic lesions may obscure the characteristic histological features of OPA [] Lesions of chronicbronchopneumonialocallyextensive dense greydark red in colour affecting thecranioventral region ofthe lung [] In our studychronic suppurative bacterial bronchopneumonia without neoplastic lesions was histologically diagnosed onlyin four cases of suspected OPA characterized by cranioventral consolidation of the pulmonary lobes Our findings showed that the distribution of both classical andatypical forms of OPA was different than those causedby chronic bacterial bronchopneumonia and the diaphragmatic lobe was the most affected part of the pulmonary parenchyma In the other two cases OPA hadconcurrent pulmonary necrosis fibrosis atelectasis andabscesses caused by Corynebacterium pseudotuberculosis and these findings are in agreement with a previousstudy []The histological features of OPA consist of proliferation of epithelial cells into acini papillary and solidstructures supported by a fibrovascular stroma In atypical form in addition to the epithelial neoplasia markedfibroblast proliferation collagen deposition and mononuclear cellinfiltration are characteristic microscopicchanges [] These inflammatory changes may be causedby intercurrent infections or a specific immune responseof the host [] Occasionally small nodules of neoplasticloose mesenchymal tissue appear admixed with the neoplastic component [] Both epithelial and mesenchymalcomponents showed rare mitoses and a ki67 index meanvalue of [] and this is in good agreement withour findings where ki67 index of the epithelial componentof classical OPA was higher mean value of thanatypical OPA mean value of Another subtype of solitary tumor associated withexJSRV in sheep is grossly characterized by round orelongated whiteâgrey nodules of bright gelatinous appearance and sharply demarcated from the surroundingparenchyma [] These nodules are histologically classified as myxoid nodules or myxoid growths or mesenchymal growths MGs [ ] MGs resemble characteristicfeatures of benign mesenchymal tumors and have beendescribed in a variable proportion of tumoursintermingled with the neoplastic epithelial component [] insome cases they were identified in metastases [] TheMGs are not an uncommon feature encountered in OPAbut interestingly they were not reported in experimentalstudies []In the current study out of ovine pulmonarytumors presented MGs accounting of the samples although there are reports of up to of theOPAs that contain also the neoplastic mesenchymalcomponent | Thyroid_Cancer |
Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso and Lucia Altucci Department of Precision Medicine University of Campania Luigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespeciï¬cdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneï¬cial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientiï¬c interest focusing on theidentiï¬cation of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol ï¬setin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modiï¬cations are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modiï¬cations Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT are enzymes classiï¬ed as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identiï¬ed inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespeciï¬c degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair OCallaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context speciï¬city BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deï¬ciency DAngelo Bioactive compounds in the diet can act as antioxidantand antiammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and ï¬setin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health beneï¬ts Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneï¬cial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneï¬cial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and antiammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin ï¬setin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classiï¬cation of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid proï¬le antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidproï¬le antioxidant antiammatoryAntioxidant antiammatoryAnticancer antioxidantantiammatoryAnticancercardiovascularpreventive antiammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesDAngelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonï¬avonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has antiammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger antiammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits proammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modiï¬es SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneï¬cial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneï¬cial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneï¬cial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress ï¬brosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together theseï¬ndings highlight the beneï¬cial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneï¬cialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been conï¬rmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective beneï¬t through improvement ofendothelial functionammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well deï¬ned Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientiï¬c research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efï¬cacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimers disease Parkinsonsdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood ï¬ow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneï¬cial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe ï¬avonoid polyphenol quercetin Que ²²pentahydroxyï¬avone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and antiammatory properties Nabavi Wu In recent years the scientiï¬c community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inï¬ammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inï¬ammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inï¬ammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the bodysantioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseasesammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable conï¬guration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cells antioxidant defense mechanisms moderateoxidative stress can in fact increase the cells antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These ï¬ndings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientiï¬c community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere signiï¬cantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpeciï¬cally in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats conï¬rming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these ï¬ndings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneï¬cial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ammatoryconditions is reported in Table Speciï¬cally a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and antiammatory capacities this ï¬avonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary ï¬brosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclariï¬cation Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneï¬cial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer antiammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneï¬cial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInï¬ammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efï¬cacy of Provex CV Supplement toReduce Inï¬ammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an antiammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a signiï¬cant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These ï¬ndings were also conï¬rmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Speciï¬cally BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and antiammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ammation signaling Speciï¬cally BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is | Thyroid_Cancer |
Inflammation plays a leading role in the pathogenesis of nephrolithiasis The association of the dietary inflammatory index DII with urinary lithogenic factors is unclear This study aimed to evaluate the relation of DII to urinary risk factors of kidney stones formationResults Of participants n n n n and n had hyperoxaluria hypercreatininuria hypercalciuria hyperuricosuria hypocitraturia respectively There was a significant increasing trajectory in urinary calcium uric acid and creatinine as well as a decreasing trend in urinary citrate across tertiles of DII score all P After multivariate adjustment for energy intake age physical activity and body mass index high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI Ptrend hypercalciuria OR 95CI Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend No association was identified between DII and hyperoxaluriaKeywords Dietary inflammatory index Kidney stones Hypercalciuria Hypocitraturia Hyperoxaluria Hyperuricosuria HypercreatinuriaIntroductionNephrolithiasis is one of the most prevalent urologic disorders and impose a substantial burden on human health globally [] The high recurrence rate of kidney stones is yet unsolved [ ]Thus there is an urgent need to target modifiable risk factors to prevent the development and recurrence of renal stonesHigher urinary excretions of oxalate calcium creatinine and uric acid as well as lower excretions of citrate are potential modifiable a0 urinary lithogenic risk factors Correspondence mina_mirzaei101yahoocom Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box IranFull list of author information is available at the end of the involved in the formation of kidney stones [] Inflammation is also another mechanism which plays a leading role in the pathogenesis of nephrolithiasis [] Dietary modifications toward decreasing inflammation may have a potential to prevent kidney stones or their recurrence Several micronutrients or foods such as magnesium vitamin E vitamin C carotenoids fruits and fish had an antiinflammatory impact [] In contrast simple sugars red meats highfat dairy products and refined grains are associated with elevated inflammatory markers [] Nevertheless nutrients or foods are not consumed separately but as part of the whole diet [] The Dietary Inflammatory Index DII is developed to measures the overall inflammatory potential of diets [] which has been recognized to be related to the biomarkers of inflammation [] A proinflammatory diet has 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 0cMaddahi a0et a0al BMC Res Notes Page of been found to be related to the reduced kidney function [] However there is no study investigating the relation of DII to urinary lithogenic factors Therefore this study aimed to assess the association of DII with hypercalciuria hypocitraturia hyperoxaluria hyperuricosuria and hypercreatinuria in patients with nephrolithiasisMain textMethodsSubjectsThis crosssectional study was performed on a total of stone former men aged a0years in Tehran Iran in Participants were recruited from the Urology Research Center of Sina Hospital Tehran Iran Inclusion criteria for this study were having a history of kind stone formation and age ¥ a0years People with a history of thyroid disease fatty liver disease malignancy stroke diabetes cardiovascular disease and hypertension were excluded Participants who were on medications such as corticosteroids diuretics anticancer drugs multivitamins potassium citrate calcium and vitamin D or C supplements were not eligible for this study Furthermore all alcohol drinkers and drugs abusers were excluded Patients were included in the study after signing written informed consentsDietary assessmentUsual food intake of patients during the previous year was measured by a validated semiquantitative 168item food frequency questionnaire FFQ[] DII was calculated using the method reported by Shivappa et a0al [] The DII is based on scientific papers scoring food parameters based on whether they elevated reduced or had no impact on six inflammatory biomarkers [Creactive protein interleukin IL1 beta IL10 IL4 IL6 and tumor necrosis factoralpha As mentioned Shivappa et a0 al calculated DII according to the food parameters As dietary patterns of different populations are different with each other some food parameters used in the study by Shivappa may not be available in different FFQs Hence researchers calculate DII according to available foods in the FFQ by modification of the method used by Shivappa et a0al [] In the current study the DII score was calculated using the corresponding food parameters available from the FFQ used in our study This approach has been used broadly in the previous studies [] The DII score was calculated with the use of the corresponding nutrients or food parameters available from the FFQ including energy protein total fat carbohydrate dietary fiber monounsaturated fatty acids n3 fatty acids n6 fatty acids polyunsaturated fatty acids saturated fatty acids cholesterol trans fatty acids vitamin A thiamin niacin riboflavin Vitamin B6 folate vitamin B12 vitamin E vitamin C Vitamin D bcarotene iron magnesium zinc selenium as well as caffeine onion greenblack tea paper and garlic The inflammatory effect scores for dietary components used for calculation of DII in this study are reported in Additional file a0 Table a0S1 To calculate the DII score for each participant the mean intake of each nutrient or food parameter was standardized by subtracting mean global intake of food items from the actual individuals intake and dividing it by the global SD to create a zscore Zscore is used to express an individuals exposure relative to the standard global exposure This approach both anchors the individuals exposure to a robust range of dietary patterns in a variety of cultural traditions and obviates completely the problem of noncomparability of units because the a0Zscores is independent of the units of measurement These zscores then were converted to proportions and centered by multiplying values by and subtracting to normalize the scoring system and to avoid skewness The centered percentile values for food items were then multiplied by the corresponding food itemspecific inflammatory effect scores to obtain the food itemspecific DII scores Calculation of DII for carbohydrate intake in a participant in our study as an example for DII calculation is presented in Additional file a0 Table a0S2 similar approach was followed for the calculation of DII for other nutrients Information about global daily mean intake standard deviation for global intake and overall inflammatory effect score of all nutrientsfood items used for DII calculation is reported in the study by Shivappa et a0al [] The overall DII score for each individual was calculated by summing food itemspecific DII scores [] Higher DII scores indicate a more proinflammatory diet while lower DII scores indicate a more antiinflammatory dietMeasurements of a0study outcomesThe 24h urine samples were collected from all participants and urine was analyzed using an AutoAnalyzer as described previously [] Hyperoxaluria a0 was a0 defined a0 as a0 the urinary oxalate Ë a0 mgday hypocitraturia as a0urinary citrate of a0mgday hyperuricosuria as urinary uric acid over a0 gday hypercreatininuria as urinary creatinine of Ë a0 mgday and hypercalciuria as a0a urinary calcium ¥ a0mgday []Measurement of a0other variablesGeneral Information was obtained using interview Physical activity was measured using of a0 the a0 International a0Physical a0Activity a0Questionnaires a0IPAQ [] Body weight was measured in minimal clothing after removal of shoes by a digital scale Seca Germany with a precision about a0kg Height of individuals was assessed in 0cMaddahi a0et a0al BMC Res Notes Page of standing position without shoes using a calibrated stadiometer Seca Germany to the nearest a0cm BMI was calculated as weight divided by the square of height kgm2Statistical analysesDII was categorized into tertiles T1 to T2 to T3 to Analysis of variance ANOVA and Chi square tests were used to compare continuous and nominalordinal variables across tertiles of DII respectively Continuous variables are reported as mean ± SE and nominalordinal variables as frequency Odds ratio OR and confidence interval CI for the relation of DII to study outcomes was calculated using the logistic regression analysis Statistical significance was set at p for all tests All analyses were undertaken using the statistical Package for Social Science Version SPSS Inc Chicago IL USAResultsParticipants in the highest tertile of the DII had significantly higher total daily energy intake P and lower physical activity P than those in the other tertiles There was a significant increasing trajectory in urinary calcium P uric acid P and creatinine P and a decreasing trend in urinary citrate P across tertiles of DII score Table a0DII score and a0urinary lithogenic factorsIn the crude model it was found that higher adherence to the DII was significantly related to the increased odds of hypercreatininuria OR 95CI Ptrend hypercalciuria OR 95CI Table Characteristics of a0the a0study participants across a0tertiles of a0the a0DII scoreAge yearHeight cmWeight kgBMI kgm2Physical activity scoreEnergy intake kcaldayUrinary creatininekg weight mgdaykgTotalN ± ± ± ± ± ± ± ± ± ± ± Dietary inflammatory index scoreTertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± P value Urinary citrate mgdayUrinary oxalate mgdayUrinary uric acid gdayUrinary calcium mgdayJob status Engineerphysician Clerk Student Teacher Selfemployed Retired Worker UnemployedMarital status Married SingleEducation level Illiterate Diploma University degreeCategorical variables are presented as frequency n and continuous variables as mean ± SE Oneway ANOVA was used for continuous variables and persons Chi square test for categorical variables 0cMaddahi a0et a0al BMC Res Notes Page of Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend After multivariate adjustment for energy intake age physical activity and BMI high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI hypercalciuria OR 95CI Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend The relation of DII to hypercreatininuria hyperoxaluria and hyperuricosuria was not significant after adjustment for carbohydrate fiber and protein intake Table a0 Ptrend adjustment for covariates dietary intakes of protein and fiber were slightly related to the decreased odds of hypocitraturia Protein OR 95CI fiber OR 95CI and hypercalciuria Protein OR 95CI fiber OR 95CI while the intake of protein and fiber was not to associated with hypercreatininuria hyperoxaluria and hyperuricosuriaDiscussionWe revealed that in stone former men a diet with a high DII is significantly related to the increased odds of having hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia but not to hyperoxaluriaIt has been confirmed that kidney stone formers could be susceptible to recurrence in stones formation We also evaluated the association of dietary protein and fiber intake on urinary risk factors Table a0 After Table Univariate and a0 multivariate logistic regression models for a0 the a0 relation of a0 DII score to a0 urinary risk factors of a0kidney stone formationDietary inflammatory index scoreModel Crude modelModel Model Model Odds ratio CIOdds ratio CIOdds ratio CIOdds ratio CIHypercreatininuria T1 T2 T3 P value for trendHypocitraturia T1 T2 T3 P value for trendHyperoxaluria T1 T2 T3 P value for trendHyperuricosuria T1 T2 T3 P value for trendHypercalciuria T1 T2 T3 P value for trend Model adjusted for energy intakeModel additionally adjusted for age BMI and physical activityModel adjusted for carbohydrate fiber and protein intake 0cMaddahi a0et a0al BMC Res Notes Page of Table Multivariate logistic regression models for a0the a0relation of a0dietary fiber and a0protein intake as a0continues variable to a0urinary risk factors of a0kidney stone formationFiberOdds ratio CIProteinOdds ratio CIModel Model Model Model HypercreatininuriaHypocitraturiaHyperoxaluriaHyperuricosuriaHypercalciuriaModel adjusted for energy intakeModel additionally adjusted for age BMI and physical activitybecause of unhealthy dietary patterns [] Inconsistent with our finding a study did not report any significant difference in creatinine across tertiles of DII in subjects with chronic kidney disease [] A randomized controlled trial a0 study by Noori et a0 al [] on recurrent stone formers showed that a a0DASH diet which in contrast to a diet with a high DII is featured by a high intake of whole grains fruits lowfat a0 dairy products and vegetables and a low intake of total fat cholesterol saturated fat meat and refined grains is significantly associated with a decrease in calcium oxalate supersaturation and an increase in citrate excretion Moreover another study reported that greater adherence to the Mediterranean a0dietary pattern a0is related to the reduced risk for incident kidney stones [] The relationship between systemic inflammation and nephrolithiasis has been identified previously [] Since both DASH and Mediterranean diets attenuate a0 inflammation [ ] the protective effects of these dietary patterns on kidney stones formation may be mediated at least partly by reducing systemic inflammation A crosssectional study conducted on diabetic patients also reported that higher intake of vegetable and fish dietary pattern is related to a lower creatinine rates [] Vegetables and fish as components of DII are identified to have antiinflammatory effects [ ] The DII is a tool to assess the overall impact of a diet on inflammatory potential [] and is associated with markers of systemic inflammation including such as IL6 [] and CRP [] [] IL6 and CRP are two of the inflammatory biomarkers considered in the calculation of DII [] It has been revealed that the DII score is inversely related to the Dietary Approaches to Stop Hypertension Score DASH Mediterranean Diet Score and Healthy Eating Index2010 [ ] Taken together these findings support that a likely mechanism for the relation of DII scores to hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia could be explained by the higher systemic inflammation level among people following a proinflammatory dietSince DII positively depends on protein intake that is also a metabolic risk factor for hypercalciuria hyperuricosuria and hypocitraturia and on contrary dietary fiber has a negative impact on DII and is a factor that can reduce calcium absorption we also adjusted the analysis for protein carbohydrate and fiber intake to differentiate the metabolic impact of DII from its proinflammatory impact It was found that DII is related to hypercalciuria and hypocitraturia independent of dietary intake of protein carbohydrate and fiber however the relationship between DII and hyperuricosuria disappeared showing that this association may be resulted from metabolic effects of DII Nevertheless protein and fiber intake was inversely associated with hypercalciuria and hypercalciuriaConclusionIn conclusion this study found that a diet with high inflammatory property might be unfavorably associated with urinary risk factors of kidney stone formation in men with a history of nephrolithiasisLimitationFirst since the participants of the current study were limited to men our findings may not be generalizable to women therefore it is essential to conduct such a study on women too Third causation cannot be inferred the crosssectional design of the present investigation Finally the calculation of DII by FFQ has a potential recall bias for the evaluation of dietary intake 0cMaddahi a0et a0al BMC Res Notes Page of Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 yAdditional file a0 Table a0S1 Inflammatory effect scores for dietary components used for calculation of DII Table a0S2 calculation of DII for carbohydrate intake in a participant in our study as an example for total DII calculationAbbreviationsIPAQ International physical activity questionnaires DII Dietary inflammatory index PUFAs Polyunsaturated fatty acids CRP C reactive protein FFQ Food frequency questionnaire ANOVA Analysis of variance OR Odds ratio CI Confidence interval BMI Body mass indexAcknowledgementsWe would like to thank the Tehran University of Medical Sciences This work was supported by Tehran University of Medical Sciences Grant ID13951046Authors contributionsSMKA designed the research and collected the samples NSM and SHA wrote the paper HY and MSY analyzed data KhM conducted research and had primary responsibility for final content All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsThe data are not publicly available due to containing information that could compromise the privacy of research participantsEthics approval and consent to participateEthics approval for the study protocol was granted by The Human Ethics Committee of Tehran University of Medical Sciences Grant ID IRTUMSVCRREC13951046 All participants signed written informed consent formsConsent for publicationNot ApplicableCompeting interestsAll authors declared that they have no competing interestsAuthor details Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box Iran Department of Urology Sina Hospital Tehran University of Medical Sciences Tehran Iran Department of Epidemiology and Biostatistics School of Public Health Tehran University of Medical Sciences Tehran Iran Received March Accepted July References Li Y Zhang J Liu H et al Curcumin ameliorates glyoxylateinduced calcium oxalate deposition and renal injuries in mice Phytomedicine Fink HA Akornor JW Garimella PS et al Diet fluid or supplements for secondary prevention of nephrolithiasis a systematic review and metaanalysis of randomized trials Eur Urol Littlejohns TJ Neal NL Bradbury KE Heers H Allen NE Turney BW Fluid intake and dietary factors and the risk of incident kidney stones in UK Biobank a populationbased prospective cohort study European urology focus Ong CN Minerals from drinking water Bioavailability for various world populations and health implications Nutrients in Drinking Water Yagisawa T Chandhoke PS Fan J Metabolic risk factors in patients with firsttime and recurrent stone formations as determined by comprehensive metabolic evaluation Urology Grases F Melero G CostaBauza A Prieto R March J Urolithiasis and phytotherapy Int Urol Nephrol Khan SR Reactive oxygen species inflammation and calcium oxalate nephrolithiasis Translational Androl Urol Bo S Durazzo M Guidi S et al Dietary magnesium and fiber intakes and inflammatory and metabolic indicators in middleaged subjects from a populationbased cohort Am J Clin Nutri Chrysohoou C Panagiotakos DB Pitsavos C Das UN Stefanadis C Adherence to the Mediterranean diet attenuates inflammation and coagulation process in healthy adults the ATTICA Study J Am Coll Cardiol Upritchard JE Sutherland W Mann JI Effect of supplementation with tomato juice vitamin E and vitamin C on LDL oxidation and products of inflammatory activity in type diabetes Diab Care Neale E Batterham M Tapsell LC Consumption of a healthy dietary pattern results in significant reductions in Creactive protein levels in adults a metaanalysis Nutri Res Esmaillzadeh A Kimiagar M Mehrabi Y Azadbakht L Hu FB Willett WC Dietary patterns and markers of systemic inflammation among Iranian women J Nutri Mohseni R Mohseni F Alizadeh S Abbasi S The Association of Dietary Approaches to Stop Hypertension DASH Diet with the risk of colorectal cancer a metaanalysis of observational studies Nutrition and cancer Alizadeh S Djafarian K Alizadeh M ShabBidar S The relation of healthy and Western dietary patterns to the risk of endometrial and ovarian cancers a systematic review and metaanalysis Int J Vitamin Nutrition Res Alizadeh S ShabBidar S Mohtavinejad N Djafarian K A posteriori dietary patterns and risk of pancreatic and renal cancers Nutrition Food Science Mohseni R Abbasi S Mohseni F Rahimi F Alizadeh S Association between dietary inflammatory index and the risk of prostate cancer a metaanalysis Nutr Cancer Shivappa N Steck SE Hurley TG et al A populationbased dietary inflammatory index predicts levels of Creactive protein in the seasonal variation of blood cholesterol study SEASONS Public Health Nutrition Xu H Sjögren P Ãrnlöv J et al A proinflammatory diet is associated with systemic inflammation and reduced kidney function in elderly adults J Nutri Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relative validity of food group intake in a food frequency questionnaire developed for the Tehran lipid and glucose study J Epidemiol Shivappa N Steck SE Hurley TG Hussey JR Hébert JR Designing and developing a literaturederived populationbased dietary inflammatory index Public Health Nutri Bondonno NP Blekkenhorst LC Bird AL et al Dietary inflammatory index and the aging kidney in older women a year prospective cohort study Eur J Nutri Maddahi NS Mirzaei K Aghamir SMK Modaresi SS Yekaninejad MS Major Dietary Patterns and kidney stone formation among Iranian men J Nutri Sci Dietetics Moghaddam MB Aghdam FB Jafarabadi MA Allahverdipour H Nikookheslat SD Safarpour S The Iranian version of international physical activity questionnaire IPAQ in Iran content and construct validity factor structure internal consistency and stability World Appl Sci J Trinchieri A Mandressi A Luongo P Longo G Pisani E The influence of diet on urinary risk factors for stones in healthy subjects and idiopathic renal calcium stone formers Br J Urol Rouhani MH Najafabadi MM Surkan PJ Esmaillzadeh A Feizi A Azadbakht L Dietary inflammatory index and its association with renal function and progression of chronic kidney disease Clin Nutri ESPEN 0cMaddahi a0et a0al BMC Res Notes Page of Noori N Honarkar E Goldfarb DS et al Urinary lithogenic risk profile in sgÃ¥rd R Rytter E Basu S AbramssonZetterberg L Möller L Vessby B recurrent stone formers with hyperoxaluria a randomized controlled trial comparing DASH Dietary Approaches to Stop Hypertensionstyle and lowoxalate diets Am J Kidney Dis Leone A FernándezMontero A de la FuenteArrillaga C et al Adherence to the Mediterranean dietary pattern and incidence of nephrolithiasis in the Seguimiento Universidad de Navarra Followup SUN cohort Am J Kidney Dis Saneei P Hashemipour M Kelishadi R Esmaillzadeh A The Dietary Approaches to Stop Hypertension DASH diet affects inflammation in childhood metabolic syndrome a randomized crossover clinical trial Ann Nutr Metab Hsu CC Jhang HR Chang WT et al Associations between dietary patterns and kidney function indicators in type diabetes Clin Nutr Duda MK OShea KM Tintinu A et al Fish oil but not flaxseed oil decreases inflammation and prevents pressure overloadinduced cardiac dysfunction Cardiovasc Res High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type diabetes Scand J Food Nutri Wood LG Shivappa N Berthon BS Gibson PG Hebert JR Dietary inflammatory index is related to asthma risk lung function and systemic inflammation in asthma Clin Exp Allergy Hodge A Bassett J Shivappa N et al Dietary inflammatory index Mediterranean diet score and lung cancer a prospective study Cancer Causes Control Wirth MD Hébert JR Shivappa N et al Antiinflammatory dietary inflammatory INDEX scores are associated with healthier scores on other dietary indices Nutri Res Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims 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"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 |
distribution and reproduction in any medium provided the original work isproperly citedLacrimal gland neoplasms comprise up to of all orbital masses clinically and histologically Much of our current coreknowledge regarding lacrimal gland tumors stems from prior study of their more common counterparts the salivary glandsThe prognosis for each lacrimal gland tumor is contingent upon proper clinical evaluation and ultimately the histopathologicdiagnosis We describe a case of an invasive carcinoma expleomorphic adenoma CaexPA with a cystadenocarcinomacomponent arising from the lacrimal gland in the absence of any previously diagnosed pleomorphic adenoma benign mixedtumor or prior incisional surgery This case illustrates the importance ofincludingimmunohistochemistry and genetic testing to narrow a diï¬erential diagnosis and potentially aid or guide therapy in the futureOur ï¬nding suggests that carcinoma of the lacrimal gland may be derived from previously undiagnosed and perhaps evensubclinical pleomorphic adenomathe histopathologic assessment IntroductionMalignant mixed tumor of the lacrimal gland also knownas carcinoma expleomorphic adenoma CaexPA is thethird most frequent epithelial lacrimal gland tumor afterpleomorphic adenoma PA and adenoid cystic carcinomaACA [] Clinically aï¬ected patients tend to be to years older than those with PA and often present with aninsidiously progressive mass of a lacrimal fossa that suddenly becomes symptomatic Other reported clinical settings are patients without any previously known lacrimalgland tumor who develop a rapidly growing mass associated with pain and patients with one or more recurrencesof PA who undergo malignant transformation [] Diagnostic imaging is critical for the clinical diagnosis of a lacrimalgland neoplasm Computed tomography CT and magnetic resonance imaging MRI are ideal but MRI remainsthe preferred method to visualize surrounding tissue anddetect radiographic features concerning aggressive malignancy [] The incidence of a malignant transformation ofPA increases with the duration of the tumor Prior studiesreport that approximately less than of PA undergoesmalignant change within years after the ï¬rst treatmentand by the end of years [] CaexPA can arisein patients without a known preexisting lacrimal glandtumor The malignant component is most often adenocarcinoma not otherwise speciï¬ed however other histologicsubtypes have been described such as adenoid cystic carcinoma ductal carcinoma mucoepidermoid carcinoma andsquamous cell carcinoma 0cCase Reports in PathologyHerein we report a case of an invasive CaexPA with anepithelial component consistent with a cystadenocarcinomain the absence of any previously diagnosed PA and supportedby immunohistochemical and molecular studies Case PresentationA 64yearold male patient presented to the emergencydepartment due to left lateral canthal pain tearing and ipsilateral hearing loss over four months The initial examinationrevealed a visual acuity of bilaterally The pupils werereactive without an aï¬erent pupillary defect Intraocularpressures were and mmHg respectively Confrontational visual ï¬elds and color plates were unremarkable Therewas a complete abduction restriction of the left eye Theexternal examination revealed edematous upper and lowereyelids predominantly overlying the lateral orbital rim associated with temporal sloping and a nontender palpable andï¬xed mass of the temporal fossa a There wasptosis of the left upper eyelid with a reduced marginal reï¬exdistance of two and a half millimeters mm Exophthalmometry measured mm and mm with a base measurement of mm The anterior segment was otherwisenormal The fundus exam revealed symmetrically sharp andpink disc margins without pallor or edemaMaxillofacial CT scan with contrast showed a lytic lesion ofthe left orbital wall with associated heterogeneous soft tissuemass measuring cm medially displacing the left lateralrectus muscle b Magnetic resonance imaging of thebrain and orbits revealed an enhancing ltrating mass of theleft lateral orbital wall extending into the left supra zygomaticmasticator space c A core guided needle biopsywas performed and the hematoxylineosin HE stainedslide revealed a moderately diï¬erentiated adenocarcinomainvolving ï¬brous connective tissue and demonstrating a cribriform architectural pattern with moderate cytologic atypia andindividual cell necrosis Figures 2a and 2bPositron emission tomography and CT of the chest abdomen and pelvis did not reveal any underlying malignancy orevidence of metastases Subsequently the patient underwentleft orbital exenteration with eyelid sparing Grossly the specimen included orbital contents frontal bone portions of thefrontal sinus and zygomatic bone Serial sectioning revealeda cm multilocular cystic mass involving the lacrimalgland fossa abutting the globe superotemporallyHistopathologically the HEstained sections disclosedpredominantly neoplastic cystic structures in the proximityof the lacrimal gland acini measuring to mm in diameterltrating ï¬brous connective tissue and bone cA small focus of pleomorphic adenoma was identiï¬ed associated with a lowgrade ductal carcinoma in situ The invasivecystic component revealed intraluminal papillary architecture and cribriform arch formations of the lining epitheliumd The neoplastic epithelium was composed ofmedium to largesized cuboidal cells with intercalated ductcell appearance eosinophilic cytoplasm and apocrine features Small foci of invasive solid components were observeddemonstrating cribriform architecture moderate to severenuclear pleomorphism and up to mitotic ï¬gures per highpower ï¬eld Columnar cells with pseudostratiï¬ed nucleiwere also present with moderate nuclear atypia Foci of ruptured cysts with hemorrhage granulation tissue lymphocyticltrate and dystrophic calciï¬cation were also seen No lymphovascular or perineural invasion was identiï¬edImmunohistochemical studies using monoclonal antibodies were performed with appropriate positive controls encompassing Gata3 mouse monoclonal antibody predilute Cell Marque Rocklin CA gross cystic disease ï¬uidprotein GCDFP15 mouse monoclonal antibody dilute Thermo Fisher Scientiï¬c Fremont CA androgen receptor AR rabbit monoclonal antibody dilute Cell Marque Rocklin CA SOX10 rabbit polyclonalantibody dilute Cell Marque Rocklin CAprostatic speciï¬c antigen PSA mouse monoclonal antibody predilute Cell Marque Rocklin CAthyroid transcription factor TTF1 mouse monoclonalantibody predilute at micrograms Ventana Medical Services Tucson AZ p63 mouse monoclonal antibody predilute Biocare Medical Concord CA p53mouse monoclonal antibody predilute at microgramsVentana Medical Services Tucson AZ high molecularweight cytokeratin HMWK903 mouse monoclonalantibody predilute Cell Marque RocklinCA CAM mouse monoclonal antibody predilute Ventana Medical Services Tucson AZ estrogen receptor ERmouse monoclonal antibody diluted VectorLaboratories Burlingame CA progesterone receptor PRmouse monoclonal antibody prediluted Leica BiosystemsNewcastle UK Her2neu rabbit monoclonal antibody predilute Ventana Medical Services Tucson AZ and Ki67rabbit monoclonal antibody predilute Ventana MedicalServices Tucson AZ Immunohistochemical studies wereperformed on an automated stainer Leica BiosystemsInc BOND III System Buï¬alo Grove IL All antibodiesand testing were performed in a CLIAcertiï¬ed laboratoryThe invasive component of the tumor was positive fata3 AR HMWK903 and CAM52 and focally positivefor GCDFP15 while negative for p63 ER PR SOX10 PSAand TTF1 stains Figures 2e2h p53 was positive in lessthan of the tumor cells The Ki67 proliferative index ofthe tumor cells was low p63 and SOX10 stainshighlighted the PA and the in situ component of the tumorDetection of HER2 status by immunohistochemistry wasequivocal and reï¬ex testing using ï¬uorescence in situhybridization FISH was negative for HER2neu geneampliï¬cation based on the updated guidelines of theAmerican Society of Clinical OncologyCollege of AmericanPathologists criteria for HER2neu testing in breast cancerMolecular proï¬ling using a nextgeneration sequencingNGS based assay Foundation One was performed onthe lacrimal gland tumor paraï¬nembedded tissue to identify genomic alterations within cancerrelated genes Thefollowing genomic alterations were detected HRAS G13RPIK3CA E542K and BCORL1 H215fs Microsatellitestatus MDstable and tumor mutation burden TMB low1MutsMbThe combined histopathologic immunohistochemicaland molecular ï¬ndings supported the diagnosis of invasive 0cCase Reports in PathologyaabcbcFigure a Clinical photo of the patient at time of presentation direct view b Computed tomography maxillofacial axial cut Illustrating a cm mass of the lateral orbit eroding the lateral orbital wall with a soft tissue component extending into the orbit causing proptosis andnonaxial displacement of the globe c MRI brain and orbit T1 fatsuppressed image with gadolinium showing a lobulated mass withmixed cystic and solid components and inhomogeneous enhancement involving the left lateral orbital wall and suprazygomatic leftmasticator space Additionally the mass demonstrates edema and enhancement suggesting ltration by the massCaexPA disclosing a carcinomatous component with apredominant cystic growth pattern and focal solid areasreminiscent of a cystadenocarcinoma The surgical marginswere negative In addition the microscopic examination ofthe left eye globe revealed pseudoexfoliation syndrome andFuchs endothelial dystrophyThe patient underwent postoperative adjuvant chemoradiation followed by excision of the eyelids with no residualtumor Followup examination showed no evidence of recurrence or metastatic disease nine months after completingadjuvant therapy DiscussionMuch remains unknown about both the natural histologicprogression and malignant transformation of CaexPAWe know that lacrimal gland neoplasms comprise oforbital masses clinically [ ] and of orbital masseshistologically [ ] and nearly half of epithelial tumorsare malignant [] Although uncommon malignant transformation of PA can occur with incomplete excision InvasiveCaexPA can represent malignant transformation of PAmany of which develop recurrence or distant metastasis tothe lung bone abdomen and CNS [] This casehighlights the importance of an immunohistochemical andgenetic evaluation in complex lacrimal tumorsThe malignant epithelial component of CaexPA hasmorphological varieties including adenocarcinoma adenoidcystic carcinoma squamous cell carcinoma mucoepidermoid carcinoma and ductal carcinoma It could be likelya mixture of subtypes [] Rarely the only evidence ofpleomorphic adenoma is the presence of large areas of hyalinized stroma composed of myoepithelial cells and a fewductal structuresIn the current case the carcinomatous component of thetumor discloses predominantly an ltrative cystic growthpattern reminiscent of a cystadenocarcinoma previouslydescribed in neoplasms of the salivary and lacrimal glandsThe term cystadenocarcinoma of the salivary gland hasevolved It encompasses a variety of tumors depicting a cysticpattern of growth to a subset of papillary and cystic malignant neoplasms that have indolent behavior This is alsoobserved in other lowgrade salivary gland carcinomashowever it is important to note that they can demonstrateltrative growthlocal recurrence and metastasize toregional lymph nodes at the time of diagnosisPreviously Foss [] in a review of cases ofcystadenocarcinoma of the salivary gland used the followingdiagnostic criteria occurrence within a salivary gland invasive growth a predominantly cystic pattern ofgrowth with or without a papillary component and theabsence of acinar or mucoepidermoid diï¬erentiation or evidence of origin in a PA In the same review the predominantcell type varies among tumors and includes small cuboidalintercalated ductlike cellslarge cuboidal cells and tallcolumnar cells The subgroups of the large cuboidal cells 0cCase Reports in PathologyababcHMWCK903Androgen receptordedp53ep63fcfghghFigure a Moderately diï¬erentiated adenocarcinoma involving cores of ï¬brous connective tissue HE 2x b High power view of thetumor revealed a cribriform pattern moderate nuclear pleomorphism and scattered mitoses HE 40x c Neoplastic cystic structures inclose proximity to lacrimal gland tissue star in a hyalinized stroma and lymphoid proliferation HE 2x d Tumor that arose from afocus of pleomorphic adenoma star depicting cystic structures with small papillae HE 4x Inset papillary projections lined by largecuboidal cells with moderate nuclear atypia and apocrine features HE 20x e Tumor cells were strongly positive for HMWK903 4xf Nuclear expression of androgen receptor was present in the tumor 20x g p53 positive in less than of tumor cells 4x h p63was negative within the invasive component of the tumor 4xhave central nuclei abundant eosinophilic cytoplasm largenucleoli and apocrine features which are similar to thoseobserved in this case The fourth group has a combinationof cell types Furthermore Foss suggest that tumorswith predominantly columnar cells are associated withincreased metastatic potentialThe cystic growth pattern characteristic of this tumor isoften associated with an ammatory response due toruptures of the dilated structures as noted in this case Cystformation in neoplasias of the salivary and lacrimal glandscan behave as a mimicker of an ammatory process Pakdel report a case of a spontaneous rupture of a PAmasquerading as orbital cellulitis [] Histologically aruptured cystic space of a PA surrounded by a monocyticltrate and foreign bodytype granulomas is described Inthis paper the authors consider the spontaneous rupture ofthe cystic space as an underlying mechanism for the acutepresentation of this tumorCystadenocarcinoma can have a broad diï¬erentialdiagnosis The principal considerations include papillary cystic acinic cell adenocarcinoma secretory carcinoma mucoepidermoid carcinoma MEC and ductal carcinoma Aciniccell carcinoma and secretory carcinoma are typically indolent monotypic tumors that can disclose a papillary cysticarchitecture Histologicallysecretory carcinoma sharesnearly identical growth patterns to acinic cell carcinoma 0cCase Reports in Pathologybut instead shows a multivacuolated eosinophilic cytoplasmoften with luminal and intracytoplasmic mucin and no truezymogen granules Immunohistochemically secretory carcinoma is S100 and mammaglobinpositive and typicallynegative for DOG1 while acinic cell carcinoma shows theopposite staining proï¬le Additionally secretory carcinomaharbors t1215p13q25 resulting in an ETV6NTRK3 genefusion [] MEC is usually composed of a mixture ofpredominantly epidermoid squamoid cells abundant intermediate cells ranging from small basal cells with basophiliccytoplasm to larger cells with eosinophilic cytoplasm andmucous cells Welldiï¬erentiated MEC is a circumscribedtumor that can disclose glandular and cystic structures linedby a single layer of mucussecreting cells however the intermediate and highgrade tumors show solid nests or sheets ofcells composed of primarily epidermoid cells with a scant cystic component and obvious invasion severe pleomorphismnecrosis and increased mitosesOn the other hand primary ductal adenocarcinoma ofthe salivary gland SDC originates from the excretoryportion of the salivary duct is a rare aggressive malignantepithelial tumor and accounts for only of epithelial lacrimal gland tumors Histologically the tumor is highly ltrative and usually solid with occasional cystic areas disclosingbreastlike ductal carcinoma features with central necrosisOccasionally cystadenocarcinoma with large cuboidal cellseosinophilic cytoplasm and highgrade nuclear atypia bearssome similaritiesto ductal adenocarcinoma Howeverpapillarycystic invasive growth is not usually seen in ductaladenocarcinoma [] The invasive component features trabeculae ducts and sheets of neoplastic cells in a desmoplasticstroma with perineural and vascular invasion The latter isnot commonly observed in cystadenocarcinoma Immunohistochemically the tumor cells are positive for a low molecular weight cytokeratin CAM52 CK7 CEA EMA andGCDFP15 Other than epithelial markersthis tumorexpresses AR in up to of invasive cases HER2 positivityin of cases and p53 overexpression in all reportedcases The Ki67 proliferative index is over The tumoris ER and PR negativeFurthermore in this case the tumor also demonstratescytomorphologicalfeatures similar to that of a low tointermediategrade ductal carcinoma in situ of the breast Thelatter ï¬ndings might correlate with the previously describedlowgrade cribriform cystadenocarcinoma of the salivary glandalso known as lowgrade salivary ductal carcinoma and salivaryductal carcinoma in situ currently categorized as intraductalcarcinoma low grade and high grade respectively These typesof tumors show a variety of growth patterns both solid and cystic ranging from cribriform to solid to micropapillary and arereminiscent of lowgrade ductal carcinoma in situ of the breastFocal ltration may be noted []The exact pathogenesis of CaexPA remains controversial Prior studies have demonstrated that PA and CaexPA of the salivary and lacrimal glands share similar genomicproï¬les and frequently overexpress the PLAG1 oncoprotein[ ] Harrison [] in their review indicate that thedevelopment of CaexPA follows a multistep model of carcinogenesis with the progressive loss of heterozygosity at 8qthen 12q and ï¬nally 17p Alterations including ampliï¬cationgene fusion and translocations in 12q genes such as HMGICHMGA2 and MDM2 may play a role in the malignant transformation In the same study the authors also mention thatloss of 17p is usually common in CaexPA indicating tumorsuppressor gene p53 loss as this tumor evolves Additionallyit appears that CaexPA and other malignant epithelialtumors other than ACC do not harbor MYB gene rearrangements or fusions It is important to note that there does notappear to be a correlation between rearrangement statusand clinical outcome []Additional mutational analysis of the lacrimal gland carcinomas has been also evaluated [] demonstrating that RASKRAS NRAS PIK3CA and MET mutations are frequent indiverse epithelial neoplasms of the lacrimal gland with thehighest proportion of mutations found in adenoid cystic carcinoma PIK3CA and MET mutations can coexist with RASmutationsPIK3CA and HRAS mutations are detected in this casewhich correlates with alterations already described in lacrimal gland carcinomas however alterations in MDM2HMGA2 NTRK3 p53 PLAG1 and ETV6 among others werenot observedIn summary this case demonstrates an invasive CaexPA with a malignant epithelial component that resemblesthe cystadenocarcinoma mixed cell type described by Foss [] Immunohistochemically the tumor was ARpositive while negative for ER PR and Her2 with expression of p53 in less than of the tumor cells Theprevalence of AR varies between the diï¬erent subtypes ofsalivary gland carcinomas SGC In recent medical literature data AR expression has been detected in as many as of SDC [] Dalin [] in the same study alsoindicate that adenocarcinoma and acinic cell carcinoma ofthe salivary gland express AR in and of the casesrespectivelyOur ï¬ndings emphasize the importance ofThe current classiï¬cation of the salivary gland tumors[] and the combined histopathologic and immunohistochemical features suggest that this tumor could representthe results of the natural course of a lowgrade cystadenocarcinoma with focal transformation into an intermediatehighgrade invasive ductal carcinoma expleomorphic adenomafurtherexploration of CaexPA pathogenesis especially the extentof disease and the histologic subtype In additiontheimmunohistochemical and genetic testing provides important support for the diagnosis as well as potentially guidesfuture therapy and prognostic evaluation Correctly identifying the type of malignant epithelial component is a signiï¬cant factor in the survival of aï¬ected patients To ourknowledge this patient represents the ï¬rst case in whichCaexPA of the lacrimal gland reveals an indolent epithelial malignancy with a low proliferative index arising froman undiagnosed PA after many likely consecutive molecular alterationsLastly due to the rarity of these tumors arising from thelacrimal gland further studies are necessary to evaluate theirbiologic behavior and determine any correlation betweenCaexPA and pseudoexfoliation syndrome 0cCase Reports in PathologyEnd Results International Journal of Ophthalmology vol no pp [] F A Jakobiec J R Bily and R L Font Orbit in OphthalmicPathology An Atlas and Textbook W H Spender Edpp WB Saunders Co Philadelphia 4th edition[] M H Devoto and J O Croxatto Primary cystadenocarcinoma of the lacrimal gland Ophthalmology vol no pp [] R H Simpson Salivary duct carcinoma new developmentsmorphological variants including pure in situ high gradelesions proposed molecular classiï¬cation Head and NeckPathology vol Supplement pp [] R D Foss G L Ellis and P L Auclair Salivary gland cystadenocarcinomas a clinicopathologic study of cases TheAmerican Journal of Surgical Pathology vol no pp [] L Barnes J W Eveson P Reichart and D Sidransky Pathology and Genetics of Head and Neck Tumours IARC [] J Antony V Gopalan R A Smith and A K Y Lam Carcinoma ex pleomorphic adenoma a comprehensive review ofclinical pathological and molecular data Head and NeckPathology vol no pp [] F Pakdel N Pirmarzdashti S Soltani Z Nozarian F AAmoli and A Kassaee Spontaneous rupture of lacrimalgland pleomorphic adenoma Ophthalmic Plastic and Reconstructive Surgery vol no pp e41e43 [] R R Seethala Head and neck pathology Surgical PathologyClinics vol no [] S L von Holstein A Fehr M Persson Lacrimal GlandPleomorphic Adenoma and Carcinoma ex PleomorphicAdenoma Genomic Proï¬les Gene Fusions and Clinical Characteristics Ophthalmology vol no pp [] T Y Chen M G Keeney A V Chintakuntlawar et alAdenoid cystic carcinoma of the lacrimal gland is frequentlycharacterized by MYB rearrangement Eye vol no pp [] M Dalin P Watson A Ho and L Morris Androgen receptor signaling in salivary gland cancer Cancers vol no p Conflicts of InterestNone of the authors declare any competing interests Currently Dr Del Valle Estopinal is the Director of OphthalmicPathology and Assistant Clinical Professor of the Departments of Ophthalmology and Pathology at University ofCalifornia Irvine The City Dr S Orange CA Authors ContributionsDr Maria Del Valle Estopinal was the ocular pathologistthat performed the histopathologic immunohistochemicaland genetic evaluation Dr Bryant Carruth wastheoculoplasticstrained ophthalmologist who performed thecore biopsy The other authors participated in the clinicalcare and preparation of this manuscript All authors readand approved the ï¬nal paperAcknowledgmentsThe authors acknowledge the cooperation of the patient Theyacknowledge the help of the SUNY Upstate Departmentof Pathology and Eye Plastic and Reconstructive Surgeonsof Central New York in the contribution to evaluationand treatment of this patientReferences[] C L Shields J A Shields R C Eagle and J P Rathmelllacrimal glandClinicopathologic review of cases oflesions Ophthalmology vol no pp [] R L Font J O Croxatto and N A Rao Tumors of the Eye andOcular Adnexa American Registry of Pathology WashingtonDC [] W Harrison P Pittman and T Cummings Pleomorphicadenoma of the lacrimal gland a review with updates ontransformation and molecular genetics SaudimalignantJournal of Ophthalmology vol no pp [] SO Baek YJ Lee SH Moon YJ Kim and YJ JunPrimary adenocarcinoma of the lacrimal gland Archives ofPlastic Surgery vol no pp [] D Bell M C Sniegowski K Wani V Prieto and B EsmaeliMutationallacrimal gland carcinomas andimplications for treatment Head Neck vol Supplement pp E724E729 landscape of[] J A Shields C L Shields J A Epstein R Scartozzi and R CEagle Jr Primary epithelial malignancies of the lacrimalgland the Ramon L Font lecture Ophthalmic Plastic Reconstructive Surgery vol no pp [] Lacrimal Gland Tumor Study Group An epidemiologicalsurvey of lacrimal fossa lesions in Japan number of patientsand their sex ratio by pathological diagnosis Japanese Journalof Ophthalmology vol no pp [] S L von Holstein M H TherkildsenJ U PrauseG Stenman V D Siersma and S Heegaard Lacrimal glandlesions in Denmark between and Acta Ophthalmologica vol no pp [] W M Hassan M S Bakry H M Hassan and A S AlfaarIncidence of orbital conjunctival and lacrimal gland malignant tumors in USA from Surveillance Epidemiology and 0c' | Thyroid_Cancer |
Human pyruvate dehydrogenase phosphatase PDP1 plays an important physiological role in energy metabolism however its expression and function in human pancreatic adenocarcinoma PDAC remain unknown This study aimed to investigate the expression pattern and mechanisms of action of PDP1 in human PDACMethods The expression pattern of PDP1 in PDAC was determined and its correlation with patient survival was analyzed Ectopic expression or knockdown of PDP1 was performed and in vitro proliferation and migration as well as in vivo tumor growth of PDAC were measured The mechanism was studied by biochemical approachesResults PDP1 was overexpressed in human PDAC samples and high expression of PDP1 correlated with poor overall and diseasefree survival of PDAC patients PDP1 promoted the proliferation colony formation and invasion of PDAC cells in vitro and facilitated orthotopic tumor growth in vivo PDP1 accelerated intracellular ATP production leading to sufficient energy to support rapid cancer progression mTOR activation was responsible for the PDP1induced tumor cell proliferation and invasion in PDAC AMPK was downregulated by PDP1 overexpression resulting in mTOR activation and cancer progressionConclusion Our findings suggested that PDP1 could be a promising diagnostic and therapeutic target for antiPDAC treatmentKeywords Pancreatic adenocarcinoma Pyruvate dehydrogenase phosphatase ATP mTOR AMPKIntroductionPancreatic adenocarcinoma PDAC which accounts for of pancreatic cancer cases is among the top lifethreatening cancers with a very high death rate and a survival rate of only approximately [] The malignancy of PDAC has been increasing in recent years Correspondence zhaojiangang78163com lianyu_chenhotmailcom Ye Li and Jia Shen contributed equally to this work Department of Integrated Oncology Fudan University Shanghai Cancer Center Shanghai China Department of Oncology Shaoxing Central Hospital Zhejiang ChinaFull list of author information is available at the end of the and this disease is estimated to be the second leading cause of cancerrelated death in the USA [] The dismal prognosis of PDAC could be due to the lack of sensitive detection at its early stage although many biomarkers have been suggested as indicators of PDAC in various experimental studies [] Effective treatments for PDAC are unavailable Although surgical resection is recommended as the only curative treatment [] only a very low number of patients who have small tumors that are detected early are suitable for surgery [] Chemotherapy as the firstline treatment is applied to nonsurgical PDAC patients [] however the response rate is low and these drugs can only prolong survival by a few months even in 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 0cLi a0et a0al Cell Biosci Page of responding patients [] Identifying new prognostic and therapeutic biomarkers for PDAC patients is importantThe human PDP1 gene encodes pyruvate dehydrogenase phosphatase PDP one of the two PDP isoforms in mammalian cells [] Physiologically PDP1 serves as a critical regulator of the pyruvate dehydrogenases complex PDC PDP1 positively regulates the catalytic activities of PDC by the removal of phosphates from the serine sites on E1α of the complex [] Upon activation by PDP1 the PDC irreversibly triggers oxidative decarboxylation of pyruvate into acetylCoA which is the main substrate for cellular energy production [] Mutation of PDP1 in some patients may cause PDC deficiency a genetic disorder characterized by neurodegeneration and abnormal metabolism [] Loss of PDP1 may also cause intolerance to exercise and mild developmental delay in patients [ ] and may produce a lethal phenotype in infants [] The expression of PDP1 in muscle cells of obese and diabetic subjects was reduced and could be reversed by endurance training [ ] The loss of PDP1 in obesity was found to signal insulin resistance that could be reversed by plasma insulin supplementation [] Overexpression of PDP1 was also found in human prostate cancer and could promote cell proliferation and tumor growth [] The expression pattern and function of PDP1 in PDAC remain unclearIn this study we attempted to identify the expression and functional role of PDP1 in human PDAC The expression pattern of PDP1 in human PDAC samples was illustrated by extracting data from the GEO database The correlation between PDP1 expression and patient survival was profiled to evaluate its prognostic value The functional role of PDP1 in the proliferation growth and invasion of PDAC cells was then evaluated in cellular and animal models The signaling pathway involved in the regulation of PDP1 was elucidated We believe this study will shed light on the prognostic and therapeutic value of PDP1 in PDACMaterials and a0methodsReagents plasmids and a0antibodiesSodium acetate and compound C were obtained from SigmaAldrich USA Human and murine ORF clones of PDP1 and shRNA against human PDP1 were purchased from Origene USA Antibodies against PDP1 phosphomTOR mTOR phosphoAMPKα AMPKα PCNA and actin were purchased from Cell Signaling Technologies USACell line and a0cell cultureThe PDAC cell line KP3 was purchased from the Japanese Collection of Research Bioresources Cell Bank Cells were cultured in RPMI medium supplemented with fetal bovine serum FBS and penicillinstreptomycin under humid conditions of CO2 and a0°C Panc2 cells were obtained from the Frederick National Laboratory for Cancer Research Frederick MD USA Cells were cultured in DMEM supplemented with FBS and penicillinstreptomycin under humid conditions of CO2 and a0°CAnimal studyThe animal study was performed in accordance with the approved protocol by Fudan University Ref No 2019FUSCCJS017 In brief Panc2 cells tagged with a luciferase reporter were mixed with Matrigel Matrix BD Bioscience USA A a0μl mixture containing Panc2 cells was injected into the pancreas of C57BLJ mice Measurement of orthotopic tumor size was performed once per week from a0week postinjection using the IVIS Spectrum live animal imager PerkinElmer USA with luciferin a0mgkg ip as a substrate At the end of the study the mice were sacrificed and the pancreas was dissectedCell proliferation and a0colony formation assaysCell proliferation was measured by cell counting In brief cells were seeded for growth and were counted at and a0days of cultivation The formation of colonies was measured by colony formation assays A total of cells were seeded for growth for a0days Cells were fixed with paraformaldehyde and stained with crystal violet The number of colonies formed was countedCell invasion assayThe invasiveness of PDAC cells was measured by chamber assays In brief cells were seeded onto the upper chamber of inserts precoated with Matrigel matrix BD Bioscience USA The receiving chamber was filled with DMEM supplemented with FBS Fortyeight hours later the medium on the upper chamber was removed and cells that migrated through the Matrigel matrix were dissociated with calceinAMcontaining buffer The fluorescence intensity representing the number of invaded cells was measured by a fluorescence microplate readerImmunoblottingTotal protein was extracted from the cell pellets using RIPA buffer and separated by SDSPAGE Protein was then transferred onto the PDVF membrane which was blocked in BSA blocking buffer for a0h at room temperature Then the membrane was incubated with primary antibodies overnight at a0°C followed by secondary 0cLi a0et a0al Cell Biosci Page of antibody incubation for a0 h at room temperature The membrane was visualized with chemiluminescence BioRad USA using ECL Select as a substrate GE Healthcare GermanyIntracellular ATP measurementIntracellular ATP measurement was performed with a commercial kit Biovision USA In brief cells were lysed with a0μl of ATP assay buffer followed by deproteinization through a a0 kDa spin column Then a0μl of flow through was mixed with a0μl of ATP assay buffer a0μl of ATP probe a0μl of ATP converter and a0μl of developmental reagent for reaction in the dark for a0min at room temperature The absorbance was then read at a0 nm The concentration of ATP in the cell samples was calculated with a standard curve plotted using an ATP standardHistologyParaffinembedded pancreatic sections were prepared and 5mm slices were generated For histological measurement of PDAC the slides were stained with hematoxylin eosin HE The image was captured under a light microscopeStatistical analysisData are presented as the mean ± SD Data were analyzed by oneway ANOVA and p was considered statistically significant All experiments were performed in triplicate unless stated otherwiseResultsPDP1 was a0overexpressed in a0PDAC and a0overexpression of a0PDP1 predicted poor prognosis of a0patientsThe expression and function of PDP1 in PDAC have not been reported A recent study showed that the expression of PDP1 was elevated in prostate cancer in which ectopic expression of PDP1 promoted cancer growth and progression [] Another study however observed that phosphorylation of PDP1 at Tyr94 suppressed its activity and reduced tumor growth [] These divergent observations indicated that PDP1 may have different functions in particular types of cancers To determine the role of PDP1 in PDAC we first examined its expression in human PDAC samples Data retrieved from two published datasets of PDAC GSE15471 and GSE28735 revealed that PDP1 was overexpressed in human PDAC tissues compared with the nonmalignant normal pancreatic tissues Fig a01a b Immunohistochemical examination of the tissue blocks confirmed that the protein expression of PDP1 was substantially elevated in the PDAC tissues Fig a01c To determine the clinical significance of this elevation we retrieved patient survival data from TCGA database We found that the expression of PDP1 predicted poor overall survival and diseasefree survival of the PDAC patients p and p respectively which indicates that PDP1 may be a poor prognostic factor in PDAC Fig a0 1d e Furthermore PDP1 may be a marker for patient mortality as its expression was significantly higher in patients who died than those who survived p Fig a01f These findings suggested that PDP1 was overexpressed in PDAC tissues and its overexpression can predict a poor prognosis for patientsPDP1 regulates the a0in a0vitro proliferation and a0invasion of a0PDAC cellsTo further elucidate the functional role of PDP1 in PDAC cells1 we first identified its in a0 vitro function in a set of PDAC cells The expression of PDP1 in different PDAC cells including MiaPaca2 Panc1 Panc2 KP3 and Capan1 was screened to select suitable cell lines for functional studies data not shown Panc2 and KP3 were selected due to their moderate level of PDP1 expression among the cell lines We then overexpressed PDP1 by transfecting a plasmid encoding the ORF of the genes while knocking down its expression by shRNA against PDP1 and established stably transfected cell lines by selection Fig a0 2a A proliferation assay was performed to examine whether PDP1 expression could alter cell growth in PDAC We found that in both cell lines PDP1 overexpression significantly promoted the growth rate of the cells while PDP1 knockdown had the opposite effects Fig a0 2b suggesting that PDP1 expression may promote tumor cell proliferation in PDAC This finding was further confirmed by colony formation assays which showed that PDP1 overexpression potently increased the number of colonies formed by PDAC cells while suppression of PDP1 reduced the ability to form colonies Fig a02c In addition as the in a0vitro invasiveness of tumor cells indicates the in a0vivo locoregional and distant spread of cancer we examined the invasion of PDAC cell lines with a chamber assay Overexpression of PDP1 significantly increased the number of cells invading through the extracellular matrix Fig a0 2d These observations suggested that PDP1 expression could promote the in a0vitro proliferation and invasion of PDAC cellsPDP1 overexpression accelerated the a0in a0vivo tumor growth of a0PDACTo further elucidate the in a0vivo functional role of PDP1 we established an orthotopic model of PDAC by injecting Panc2 cells into the pancreas of C57BL6J mice For noninvasive monitoring of tumor growth Panc2 cells with or without PDP1 overexpression were stably infected with a luciferase reporter and luciferin could be catalyzed to 0cLi a0et a0al Cell Biosci Page of Fig PDP1 was overexpressed in PDAC Data were retrieved from the GEO database and PDP1 was found to be overexpressed in PDAC tissues compared with normal pancreatic tissues in a GSE15471 and b GSE28735 c Immunohistochemical staining of PDP1 was retrieved from the Human Protein Atlas which suggested that PDP1 protein expression was upregulated in PDAC tissues Survival of PDAC patients was retrieved from TGCA database and patients with PDP1 expression above the median level had poor d overall survival and e diseasefree survival f Surviving PDAC patients had lower expression of PDP1 than the censored patientsFig PDP1 regulated PDAC cell proliferation and migration in vitro a Transfection of the PDP1 ORF clone or shRNA regulated endogenous expression of PDP1 in PDAC cells b Overexpression of PDP1 promoted PDAC cell proliferation while PDP1 suppressed PDAC cell proliferation c Overexpression of PDP1 promoted colony formation of PDAC cells while PDP1 suppressed PDAC cell colony number d Overexpression of PDP1 promoted PDAC cell migration while PDP1 suppressed PDAC cell migration p 0cLi a0et a0al Cell Biosci Page of emit bioluminescent signals Under an in a0vivo imager we observed that overexpression of PDP1 could significantly increase the signal intensity of tumor cellderived luciferase indicating a larger tumor formed by PDP1overexpressing PDAC cells than the control cells Fig a0 3a Endpoint measurement of the dissected tumors showed that tumor growth was accelerated by PDP1 overexpression Fig a03b Histological analysis by HE staining showed that the PDP1overexpressing PDAC cells had a more aggressive phenotype eg an unclear border between pancreatic tissues and the tumor tissues than the control cells Fig a03c This result was further proven by the immunoblotting of PCNA acellular marker of proliferation in PDAC tissues the expression was significantly higher in the PDP1overexpressing PDAC tumors than the control tumors Fig a03d These findings suggested that PDP1 overexpression could substantially accelerate the in a0vivo tumor growth of PDACPDP1 overexpression accelerated ATPassociated tumor cell growth in a0PDACMitochondrial PDP1 is an important regulator in energy production and metabolism in mammalian cells PDP1 catalyzes the dephosphorylation and concomitant reactivation of the α subunit of the E1 component of the pyruvate dehydrogenase complex PDC whose activity is critical for energy metabolism through the TCA cycle and oxidative phosphorylation [] A previous study suggested that suppression of PDP1 expression in embryonic stem cells is associated with reduced production of ATP the product of energy metabolism that is essential for many cellular processes [] Therefore we examined the cellular content of ATP in PDAC cells Overexpression of PDP1 could significantly increase the cellular ATP content in PDAC cells while knockdown of the protein showed the opposite effects Fig a04a To determine whether cellular ATP content plays an important role in mediating PDP1induced cell proliferation we supplied acetate as a substrate for ATP production by alternative mechanisms [] in PDAC cells with PDP1 knockdown The reconstitution of cellular ATP by acetate supplementation significantly reversed tumor cell proliferation in the cells with PDP1 knockdown Fig a0 4b Colony formation and cell invasion assays proved that the suppressive effect of PDP1 knockdown was attenuated by ATP supplementation in PDAC cells Fig a0 4c d These observations suggested that PDP1regulated cell proliferation and invasion in PDAC cells may be associated with the alteration of cellular ATP content and its related cellular activitiesmTOR is a0a a0downstream signaling pathway that a0mediates PDP1driven tumor cell proliferation in a0PDACmTOR signaling is often aberrantly hyperactivated in various types of cancer cells in response to the abundant cellular ATP to promote tumor cell proliferation [] In our study we examined the expression and activity of the mTOR pathway in PDAC cells with PDP1 knockdown Fig PDP1 overexpression promoted PDAC growth in vivo a PDP1 overexpression significantly increased the luciferase signal in the orthotopic tumors of PDAC in mice b PDP1 overexpression increased the tumor size of the orthotopic PDAC model c HE staining suggested that more cells were undergoing mitotic proliferation in the PDP1overexpressing tumors than the control tumors d PDP1overexpressing tumors expressed a higher level of PCNA than the control tumors p 0cLi a0et a0al Cell Biosci Page of Fig PDP1 induced PDAC cell proliferation and migration by inducing intracellular ATP a PDP1 overexpression induced intracellular ATP content while its knockdown reduced ATP levels Supplementation of an alternative substrate of ATP production acetate restored cell proliferation b colony formation c and migration of PDP1 knockdown PDAC cells d p Upon knockdown of PDP1 mTOR phosphorylation was significantly downregulated without notable changes in total protein expression Fig a0 5a To identify whether mTOR activity is crucial for PDP1mediated tumor cell proliferation we transfected a plasmid encoding the protein with a mutation that leads to constitutive activation of mTOR in PDAC cells with PDP1 knockdown Fig a05b Constitutive activation of mTOR restored the proliferation rate in the PDAC cells with PDP1 knockdown Fig a05c suggesting that mTOR activation is essential for PDP1driven tumor cell growth in PDAC In addition reactivation of mTOR in PDAC cells with PDP1 knockdown reconstituted the capacity of PDAC cells to form colonies and pass through the ECM Fig a0 5d e These findings indicate that mTOR activation mediates PDP1driven cell growth and invasion in PDAC as the downstream effectorAMPKα inhibition mediates PDP1induced mTOR activation in a0PDAC cellsWhen the cellular content of ATP is insufficient cells tend to activate AMPKα as a stress response pathway to limit growthrelated signaling [] Activation of AMPKα in PDAC was found to restrict cancer cell proliferation and aggressiveness in previous studies [ ] As PDP1 inhibition reduced ATP content as we observed in the present study we investigated whether AMPKα activity could be altered by PDP1 Overexpression of PDP1 potently suppressed the basal level of phosphorylated AMPKα without changing its total protein level while repressing PDP1 could activate AMPKα Fig a0 6a Inhibition of AMPKα in PDAC cells by its pharmacological inhibitor compound C CC restored mTOR activity Fig a0 6b suggesting that AMPKα acts as the upstream regulator of mTOR in the PDAC cells with PDP1 knockdown Furthermore inhibition of AMPKα significantly reactivated the proliferation colony formation and invasion of PDAC cells Fig a06ce A previous study showed that an increased AMPATP ratio would activate AMPK activity [] We then added AMP to the PDP1overexpressing cells and found that an increased AMPATP ratio led to the restoration of AMPK in the these cells Fig a0 6f An opposite trend in mTOR activity was observed as mTOR is a downstream effector of AMPK These observations together with the other findings in this study indicated that PDP1 regulates PDAC growth by modulating the ATPAMPKαmTOR pathwayDiscussionIn this study we observed that PDP1 was overexpressed in human PDAC Overexpression of PDP1 in PDAC cells promoted cell proliferation and migration in a0 vitro and stimulated tumor growth in a murine model of PDAC which could be related to the increase in intracellular energy production Fig a0 Expression of PDP1 was reported to be associated with ATP generation in previous studies for instance PDP1 expression and activity were shown to contribute to a higher aerobic capacity 0cLi a0et a0al Cell Biosci Page of Fig mTOR was responsible for PDP1mediated PDAC cell proliferation and migration a The phosphorylation of mTOR in PDAC cells representing its activity was measured by immunoblotting PDP1 overexpression activated mTOR signaling while its knockdown suppressed mTOR phosphorylation b Transfection of constitutively activated mTORC reactivated mTOR signaling in PDP1 knockdown PDAC cells mTORC represents a plasmid encoding a mutated mTOR with a change from arginine to proline which leads to constitutive activation of mTOR regardless of intracellular signaling Reactivation of mTOR restored cell proliferation c colony formation d and migration of PDP1 knockdown PDAC cells e p of the muscle suggesting a sustainable level of oxidative phosphorylationrelated ATP production [] Another study also suggested that activation of PDP1 in cancer cells could induce a switch from cellular glycolysis to oxidative phosphorylation the more efficient method of ATP production [] The depletion of cellular ATP abolished PDP1induced PDAC cell proliferation migration and invasion suggesting that ATP generation was the essential step by which PDP1 triggers these cellular processes PDP1 expression was also found to be critical in some other cellular processes such as recovery from injury and cell differentiation [ ] Given the important regulatory role of PDP1 in the physiological function of PDC our observations may suggest that overexpression of PDP1 in PDAC cells functions to hyperactivate the PDC system overloading the cellular substrate of the tricarboxylic acid cycle which in turn accelerates the oxidative phosphorylation process that can generate more ATP essential for cellular processesWe used acetate as an alternative carbon source in tumor cells with PDP1 knockdown PDP1 is a critical enzyme involved in the pathway of ATP production by consuming glucose PDP1 is involved the reaction of pyruvate to acetylCoA the direct source of the TCA cycle This facilitates the production of reducing equivalents in the form of NADH FADH2 etc by TCA cycles which can be used by oxidative phosphorylation for the production of ATP Acetate has been reported as an additional source for ATP production in cancer cells especially when glucoseglutamine is depleted [] The direct ligation of acetate to CoA by acetylCoA synthetases can yield acetylCoA in the TCA cycle [] The TCA cycle can therefore produce reducing equivalents in the form of NADH FADH2 etc which were used for ATP production by oxidative phosphorylation in PDP1 knockdown cells As we assumed that suppression of ATP production in PDP1 knockdown cells is responsible for inhibition of tumor growth we used acetate as an alternative source of ATP production in these cells and our observations proved that ATP restoration in PDP1 knockdown cells recovered tumor cell proliferation and growth supporting our claims We also observed that the addition of acetate had a minimal effect on tumor growth in mock tumor cells Mock cells can consume glucose to 0cLi a0et a0al Cell Biosci Page of Fig AMPK inactivation was responsible for PDP1mediated PDAC cell proliferation and migration a PDP1 overexpression suppressed AMPK signaling while its knockdown induced activation via phosphorylation b The presence of compound C mM inactivated AMPK signaling in PDP1 knockdown PDAC cells which in turn reactivated mTOR signaling Reactivation of mTOR restored cell proliferation c colony formation d and migration of PDP1 knockdown PDAC cells e f AMP was supplemented in the PDP1overexpressing cells to adjust the intracellular AMPATP ratio Increased AMPATP ratio in the PDPoverexpressing cells resulted in the restoration of AMPK activity but mTOR repression p Fig Schematic regulation of PDP1 in PDAC Overexpression of PDP1 may activate PDH which in turn accelerates the TCA cycle which provides reducing equivalents in the form of NADH FADH2 etc for the ATP production by oxidative phosphorylation The abundance of cellular ATP inhibits AMPK activation restoring the mTOR signaling that can promote proliferation and invasion of PDAC cellsproduce acetate which is then directly ligated by acetylCoA synthetases in the TCA cycle The reason for these results after addition of acetate to cells with an intact pathway of glucose metabolism is not fully understood however as the TCA cycle has a strict regulatory mechanism that involves production of reducing equivalents as 0cLi a0et a0al Cell Biosci Page of sources of ATP production this complicated process has ratelimiting steps even when the acetylCoA amount is overwhelming In this regard the addition of acetate may not further accelerate cell proliferation and growth in mock cells which can normally consume glucose to produce ATPWe observed that AMPKmTOR signaling was involved in PDP1 regulation of PDAC progression As an energy sensor AMPK is activated when the intracellular ATP level is insufficient and triggers a series of downstream responses that inhibit rapid cell proliferation hence AMPK has been frequently identified as a potential target in anticancer treatment [] PDP1 overexpression could induce activation of PDC which in turn generates more substrate for the production of ATP In this case overexpression of PDP1 in our study resulted in sustained repression of AMPK signaling due to the abundance of intracellular ATP that led to suppression of multiple downstream proliferationassociated mechanisms mTOR is a key molecule that mediates rapid protein synthesis during cancer progression [] Phosphorylation of mTOR signaling activates translational control by initiating the formation of the ribosome complex as well as the association of mRNA at its cap [] This process was halted under nutrientdeprived conditions in which mTOR was dephosphorylated and could not initiate capdependent translation [] The activation of AMPK as a nutrient sensor in some previous studies has been found to primarily downregulate mTOR activity in cancers [ ] PDP1mediated AMPK repression therefore could be a possible mechanism to facilitate mTORassociated PDAC progression which was confirmed by our observation that suppression of AMPK in PDP1 knockdown cells revoked mTOR signalingAMPK signaling which in turn suppressed mTOR activity Repression of AMPK in PDP1 knockdown cells restored the proliferation and migration of PDAC Our study suggested that PDP1 may be an attractive target for the diagnosis and treatment of PDACAcknowledgementsNot applicableAuthors contributionsLC and JZ conceived the idea designed the experiments analyzed the data and drafted the manuscript YL and JS performed the experiments and analyzed the data CC performed the experiment HG revised the manuscript All authors read and approved the final manuscriptFundingThis work was financially supported by the National Natural Science Foundation of China and Wu JiePing Medical Foundation Availability of data and materialsThe datasets used andor analysed during the current study are available from the corresponding author on reasonable requestEthics approval and consent to participateThe animal study was performed in accordance with the approved protocol by Fudan University Ref No FUSCCJSConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Integrated Oncology Fudan University Shanghai Cancer Center Shanghai China Department of Oncology Shanghai Medical College Fudan University Shanghai China Department of Oncology First Peoples Hospital of Pinghu Zhejiang China Department of Oncology Shaoxing Central Hospital Zhejiang China Received April Accepted July ConclusionIn summary in this study we identified the expression pattern and function of PDP1 in PDAC PDP1 was overexpressed in PDAC samples compared with normal pancreatic samples and high expression of PDP1 was correlated with a poor prognosis of PDAC patients Overexpression of PDP1 promoted the in a0 vitro proliferation colony formation and migration of PDAC cells while knockdown of PDP1 had the opposite effects Overexpression of PDP1 stimulated the in a0 vivo growth of orthotopic PDAC tumors in a murine model PDP1 regulated the production of intracellular ATP and supplementation with ATP recovered tumor cell proliferation and migration in PDP1 knockdown PDAC cells This result could be related to the alteration of mTOR activity in PDAC cells and recovery of mTOR activity in PDP1 knockdown PDAC cells restored cell proliferation and migration Mechanistically PDP1 knockdown activated References Collaborators GBDPC The global regional and national burden of pancreatic cancer and its attributable risk factors in countries and territories a systematic analysis for the Global Burden of Disease Study Lancet Gastroenterol Hepatol Rahib L Smith BD Aizenberg R Rosenzweig AB Fleshman JM Matrisian LM Projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the United States Cancer Res Kim J Bamlet WR Oberg AL Chaffee KG Donahue G Cao XJ Chari S Garcia BA Petersen GM Zaret KS Detection of early pancreatic ductal adenocarcinoma with thrombospondin and CA19 blood markers Sci Transl Med Hackert T Surgery for Pancreatic Cancer after neoadjuvant treatment Ann Gastroenterol Surg Satoi S Yamamoto T Yamaki S Sakaguchi T Sekimoto M Surgical indication for and desirable outcomes of conversion surgery in patients with initially unresectable pancreatic ductal adenocarcinoma Ann Gastroenterol Surg Chandana S Babiker HM Mahadevan D Therapeutic trends in pancreatic ductal adenocarcinoma PDAC Expert Opin Investig Drugs 0cLi a0et a0al Cell Biosci | Thyroid_Cancer |
IFNBased Biotherapeutics toHarness the Host AgainstFootAndMouth DiseaseGisselle N Medina Teresa de los Santos and Fayna DiazSan Segundo Plum Island Animal Disease Center PIADC ARS USDA Orient Point NY United States Kansas State University Collegeof Veterinary Medicine Manhattan KS United StatesFootandmouth disease FMD is a highly contagious vesicular disease of clovenhoofedanimals that severely constrains international trade of livestock and animal productsCurrently disease control measures include broad surveillance enforcement of sanitarypolicy and use of an inactivated vaccine While use of these measures has contributedto eliminating footandmouth disease virus FMDV from a vast area of the worldthe disease remains endemic in three continents and outbreaks occasionally appearin previously declared FMDfree zones causing economic and social devastationAmong others a very fast rate of viral replication and the need for days to achievevaccineinduced protection are the main limitations in controlling the disease Newfastacting antiviral strategies targeted to boost the innate immunity of the host to blockviral replication are needed Here we review the knowledge on the multiple strategiesFMDV has evolved to block the host innate immunity with particularly focus on the pastand current research toward the development of interferon IFNbased biotherapeuticsin relevant livestock speciesKeywords footandmouth disease virus FMDVIFNλ IFNÏinterferon IFN antivirals biotherapeutics IFNα IFNÎINTRODUCTIONThe Disease FootAndMouth DiseaseFootandmouth disease FMD is one the most serious livestock diseases that aï¬ects clovenhoofedanimals including cattle swine sheep and goats as well as numerous species of wild species The disease displays high morbidity but is usually not lethal except when it aï¬ects young animalsthat may develop myocarditis Infected animals secrete copious amounts of virus ps beforethe onset of the clinical phase of the disease Typical FMD clinical signs include fever and theappearance of vesicular lesions on the tongue mouth feet and teats Among ruminants thatrecovered from the disease a relatively large number become asymptomatic virus carriers although it is not clear what is the contribution of these carrier animals to disease transmissionin nature The World anization for Animal Health OIE lists FMD as a reportable diseaseand therefore by law participating nations are required to inform the anization about all FMDoutbreaks OIE member nations with reported cases of FMD are forbidden to engage in tradingof FMDsusceptible animals or their products Thus the presence of FMD in a country can havesevere economic consequencesDiï¬erent interventions to control an FMD outbreak include restriction of susceptible animalmovement slaughter of infectedcontact animals decontamination of infected and surroundingEdited byMariano PrezFilgueiraNational Agricultural TechnologyInstitute ArgentinaReviewed byMargarita S¡izSevero Ochoa Molecular BiologyCenter CSICUAM SpainKenneth James GenoveseAgricultural Research ServiceUnited States Department ofAgriculture United StatesCorrespondenceGisselle N MedinagissellemedinausdagovTeresa de los SantosteresadelossantosusdagovFayna DiazSan SegundofaynadiazsansegundousdagovSpecialty sectionThis was submitted toVeterinary Infectious Diseasesa section of the journalFrontiers in Veterinary ScienceReceived April Accepted June Published August CitationMedina GN de los Santos T andDiazSan Segundo F Use ofIFNBased Biotherapeutics to Harnessthe Host Against FootAndMouthDisease Front Vet Sci 103389fvets202000465Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVpremises and vaccination Vaccination is an option used mostlyin countries in which FMD is endemic but diseasefree nationsprefer to abstain from such practice In general FMDfreecountries that occasionally opted to vaccinate to better containthe outbreak did slaughter all vaccinated animals to regaincommerce rights faster as occurred in the outbreak inthe UK and the Netherlands The current approvedFMD vaccine consists of puriï¬ed chemically inactivated virus[binary ethylenimine BEItreated] formulated with oilbased oraluminum adjuvants that induces serotypespeciï¬c protection inapproximately days and it is applied with a boosting protocolfor ensuring longterm protection While this vaccine hasbeen successfully used for many decades leading to diseaseeradication of a vast area of our planet challenges remainFMD is endemic in most of Africa and Asia and occasionallyepizootics appear in South America or in nations that havebeen diseasefree for many years as it happened in the UKthe Netherlands South Korea Taiwan and Japan Novelvaccine technologies have been developed but to this end noneof them has fully addressed the limitations of the commerciallyavailable vaccine or is currently approved for massive use Alternatives or additional therapeutics that could complementor in some instances substitute for vaccination protocols includethe use of antivirals and biotherapeutics that act quickly priorto induction of vaccineinduced immunity The development ofsuch molecules requires a thorough understanding of the biologyof the virus and its intricate interactions particularly with theinnate immune molecular and cellular mechanisms evolved bythe hostThe AgentFootandmouth disease virus FMDV is a member of theAphthovirus genus within the Picornaviridae family and it is theetiologic agent of FMD The virus contains a singlestrandedRNA of positive polarity Its genome of ¼ nucleotidesconsists of a long reading frame ORF ï¬anked by a ² anda ²untranslated region UTR The ORF encodes a polyproteinof about amino acids which is processed by virusencodedproteases Processing results in the generation of precursors andmature protein products including four structural [1A VP4 1BVP2 1C VP3 1D VP1] and ten nonstructural NS proteins[Lpro 2A 2B 2C 3A three distinct copies of 3B VPg 3Cproand 3Dpol] Due to high genetic variability FMDV is categorizedin seven distinct serotypes A Asia1 C O and Southern AfricanTerritories SAT and numerous subtypes or topotypesUpon infection the virus spreads very rapidly usually achieving morbidity Depending on the route of entry less than tissue culture infectious doses are required to infect andcause disease in animals In fact FMDV is one of thefastest replicating RNA viruses in nature taking as little as h to induce cytopathic eï¬ects in susceptible tissue culture cellsOne could envisage that during FMDV replication almost everycomponent of the virus must play a role in dampening interferingcellular responses to allow such rapid virus replicationInnate Immunity and Interferon ActivationEarly protection against viralfundamentallymediated by the action of interferons IFNs the pillar moleculesof the innate immune system Expression of IFN isinfection istriggered by the recognition of molecular signatures collectivelynamed pathogenassociated molecular patterns PAMPs viacellular receptors pattern recognition receptors PRRs that candistinguish self from nonself molecules Figure Bindingof PAMPs to PRRs triggers a series of signal transduction eventsand posttranslational modiï¬cations PTMs phosphorylationubiquitination ISGylation etc that ultimately activate latenttranscription factors to induce IFN transcription Subsequentlysecreted IFN proteins bind to speciï¬c receptors on the plasmamembrane to activate in an autocrine and paracrine mannerdiscrete and overlapping cellular signal transduction pathwaysDepending on the cell type and aï¬ected tissue over speciï¬cIFNstimulated genes ISGs may be induced many of whichdisplay antiviral activity to control the viral infection There are three families of IFNs based on the speciï¬c receptorusage types I II and III Table Type I IFNsie IFNα and IFN signal through a heterodimeric receptorcomplex formed by IFNAR1IFNAR2 type II IFN IFNÎsignals through the complex IFNÎR1IFNÎR2 and type IIIIFNs bind the receptor complex IL28RαIL10R Despitethe receptor diï¬erencesthe three IFN families transducesignals through the Janus kinase JAKsignal transducer andactivator of transcription STAT pathway and type I and typeIII IFNs induce redundant responses Figure Overall therapid production of IFN helps to limit viral replication whilemodulating other immune functionsFOOTANDMOUTH DISEASE VIRUSIMPAIRS INNATE IMMUNITY MOLECULARINTERACTIONSRecognition of FMDV RNA by the host cell results in theestablishment of a rapid antiviral state to limit and controlinfection This selective pressure has allowed FMDV to evolvemany strategiesto ensure enhanced virulence and rapidinfectivity In general RNA viruses can bypass the IFN responseby blocking i global cellular transcription and translationii IFN induction and iii IFN signaling Similarly to otherRNA viruses FMDV can also target IFNindependent antiviralresponses mostly associated with cellular metabolic functionsie autophagy apoptosis stress granule formation etc thathave been extensively described elsewhere In thissection we will summarize the current literature on studiesconducted in vitro that explain how FMDV counteracts the hostinnate immune response at the molecular level including RNAsensing activation of adaptoreï¬ector proteins and regulation ofsignaling pathways by speciï¬c PTMsBlock on Cellular Transcription andTranslationFMDV inhibition of cellular gene expression and proteinsynthesis during infection is mainly driven by the viralencoded proteases Leader Lpro and 3C FMDV Lpro isa papainlike protease PLP thatthetranslation initiation factor eIF4G including eIF4GI and eIF4GII to disable capdependent protein synthesis AlsoFMDV Lpro causes degradation of the transcription factorinduces cleavage ofFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Antiviral signaling pathways induced during viral infection Cellular detection of microbial molecules known as pathogenassociated molecular patternsPAMPs ie viral RNA is mediated by pattern recognition receptors PRRs including cytosolic RNA sensors ie RIGI MDA5 or LGP2 andor membraneboundTLRs PAMPPRR interaction activates signal transduction cascades black arrows that result in the production of IFN and inï¬ammatory cytokines RIGI and MDA5contain two caspase recruitment domains CARD and an RNA helicase domain In the case of RIGI ubiquitination green circles is required for its effective activationActivated signals from either RIGI or MDA5 are transmitted downstream via the mitochondrial adaptor MAVS resulting in the formation of MAVS ï¬laments At thisstage different PTMs such as ubiquitination or ISGylation black circles can regulate their functions Endosomal RNAs are detected by TLR3 or TLR78 which signalthrough adaptor proteins TRIF and MyD88 respectively MyD88 uses other adaptors IRAK14 to allow for interaction with TRAF proteins In addition to their role asadaptor proteins TRAFs also serve as E3 ubiquitin Ub ligases to regulate signaling TRAFmediated induction of polyUb is sensed by NEMO thus recruitingdownstream effector kinases such as TBK1 or IKK These proteins form different signaling complexes ie NEMOTBK1 and NEMOIKK leading to phosphorylationblue arrows of transcription factors IRF37 to a lesser extent IRF1 and IRF5 are also phosphorylated IRF phosphorylation triggers dimerization and translocationContinuedFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE orange arrows to the nucleus where they bind mainly to IFN promotersenhancers Alongside with this pathway TRAF6E3 ligases can activate MAPK3and other kinases including ERK12 and JNK which phosphorylate the components of the AP1 heterodimer allowing for translocation to the nucleus and binding tothe IFN promoterenhancer to activate transcription Activated IKK also phosphorylates IκB releasing NFκB which then translocates to the nucleus and binds at theIFN promoter AP1 activating protein CARD caspase activation and recruitment domain DUB deubiquitinase ER endoplasmic reticulum IκB inhibitor of KBkinases IKK IκB kinase IL interleukin IRAK interleukin1 receptorassociated kinase IRF IFN regulatory factor LGP2 laboratory of genetics protein MAPKmitogenactivated protein kinase MAVS mitochondrial antiviral signaling protein MDA5 melanoma differentiationassociated gene MyD88 myeloid differentiationprimary response protein 88d NEMO NFκB essential modulator NFκB nuclear factorκB PKR protein kinase R PTM posttranslational modiï¬cation RIGIretinoic acidinducible gene I TANK TRAF family memberassociated NFκB activator TBK TANK binding kinase TLR Tolllike receptor TRAF TNF receptorassociated factor TRIF TIRdomaincontaining adapterinducing interferonTABLE Use of IFNbased therapies against FMDVTypeReceptSignalSubtypeSpeciesMilestoneType IIFNAR1IFNAR2JAK1 TYK2IFNαPorcinebovine ¢ Recombinant bacterial expressed IFNα is a potent biotherapeutic againstIFNαPorcine¢ Ad5 delivered poIFNα protects swine against different serotypes of FMDVFMDV in vitro IFNIFNδIFNÏ7IFNαÏIFNÏType IIIFNÎ R1 IFNÎ R2JAK1 JAK2IFNÎType IIIIFNλR1IL10R2JAK2 TYK2IFNλ1IFN΢ poIFNαprotection correlates with enhanced tissuespeciï¬c innate immune cellinï¬ltration in swine ¢ poIFNα protection correlates with upregulation of essential ISGs in vitro ¢ Ad5 delivered porcine poIFN protects swine against FMDV ¢ Bacterially expressed poIFNδ8 signiï¬cantly inhibits FMDV replication in vitro ¢ E coli produced poIFNÏ7 protects cells against FMDV ¢ Bacterially expressed IFNÎ±Ï added prior to infection resulted in a signiï¬cantreduction in FMDV replication in vitro ¢ Ovine IFNÏ has antiviral effect against FMDV in vitro ¢ Recombinant bovine IFNÎ reduced FMDV replication in BTY cell culture ¢ High dose of Ad5poIFNÎ protects swine against FMD ¢ Replication of FMDV in IBRS2 cells is inhibited by treatment with the puriï¬edrecombinant poIFNλ1 PorcinePorcinePorcinePorcineOvineBovinePorcinePorcineIFN CombosOtherIFNvaccinecombosIFNλ3Bovine¢ Inoculation with Ad5boIFNλ3 resulted in the induction of several ISGs in tissuesof the upper respiratory tract and protected cattle against challenge withFMDV PorcinePorcineIFNαIFN΢ Ad5poIFNλ3 protects swine against challenge with FMDV ¢ Use of a combination of Ad5poIFNÎ and Ad5poIFNα or Ad5poIFNαΠshowed an enhancement of the antiviral activity against FMDV in swinePoly ICPorcine¢ Double stranded ds RNA poly ICLC in combination with Ad5poIFNαprotected swine against FMDV siRNAPorcine¢ Combination of Ad5poIFNαΠwith Ad3siRNA targeting FMDV NS codingregions blocked replication of all serotypes of FMDV in vitro IRF73Porcine¢ Inoculation with Ad5IRF735D resulted in induction of IFNα and fully protectedmice and swine challenged with FMDV day after treatment IRESPorcine¢ Use of synthetic IRES in combination with adjuvanted typeO FMD improvedimmune response and protection against FMDV challenge IFNαPorcine¢ Use of a combination of Ad5poIFNα and Ad5A24 in swine resulted incomplete protection after challenge IFNαÎPorcine¢ Ad5poIFNαΠcoadministered with Ad5siRNA targeting NS regions of FMDVand a commercial inactivated FMD vaccine partially protected swine IFNλ3Bovine¢ Use of a combination of Ad5bovIFNÎ3 and AdtO1M in cattle resulted incomplete protection after aerosol challenge nuclear factor NFκB and results in blockage of speciï¬cdownstream signaling eï¬ectors Studies in porcine cellsdemonstrated that FMDV Lpro can promote its selfbindingto the transcription factor activitydependent neuroprotectiveprotein ADNP and negatively regulate the activity of the IFNα promoter In contrast chromatin changes that favor theupregulation of IFN and ISGs can inhibit FMDV replication Interestingly deletion or mutations in diï¬erent domainsof Lpro result in viral attenuation in vitro and in vivo Furthermore these studies have shown a strong type I IFNactivity upon infection with diï¬erent versions of FMDV Lpromutants Frontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVFIGURE Type I II and III interferon IFNmediated signaling All type I and type III IFN subtypes bind to respective receptors IFNAR1IFNAR2 and IFNLR1IL10R2These interactions trigger the phosphorylation of JAK1 and TYK2 kinases which in turn phosphorylate STAT1 and STAT2 JAK2 mediates type III IFNdependent STATphosphorylation Phosphorylated heterodimers of STAT1STAT2 bind to IRF9 forming the ISGF3G complex which then translocates to the nucleus and binds toIFNresponsive elements ISREs present in the promoters of over ISGs Type II IFN binds to the heterodimeric IFNÎR1IFNÎR2 receptor also inducingphosphorylation of JAK1JAK2 kinases In turn mostly STAT1 is phosphorylated Phosphorylated homodimers of STAT1 translocate to the nucleus and induce theexpression of genes controlled by gammaactivated sequence GASdependent promoter sequences IFNAR12 IFN alpha receptor12 IFNÎR12 IFNgammareceptor12 IFNALR1 IFNlambda receptor IL10R2 IL10 receptor ISGs IFNstimulated genes ISGF3G ISG factor gamma JAK12 Janus kinase STATsignal transducer and activator of transcriptionInterruption of cellular translation during infection can alsobe mediated by FMDV 3Cpro a chymotrypsinlike cysteineprotease that similarly to Lpro targets eIF4G and the capbindingcomplex eIF4A for cleavage although these events occur later inthe infection 3Cpro can also participate in the inhibitionof hostcell transcription by cleaving histone H3 upon FMDVinfection Block on Interferon InductionDuring infection the initial event that leads to the productionof IFN and proinï¬ammatory cytokines is the recognition ofviral RNA Figure Sensing of FMDVRNA is mediated byMDA5 a protein that belongs to a family of helicasesknown as retinoic acidinducible geneI RIGIlike receptorsRLRs Recent studies have shown that the interaction betweenRLRs RIGI and LGP2 and the FMDV proteins Lpro 2Band 3A interferes with the induction of type I IFN Indeed overexpression of either FMDV 2B or 3A resulted in thedownregulation of RIGI and MDA5 mRNA expression In contrast upregulation of LGP2 transcripts has been observedduring FMDV infection in porcine cells despite a detectablereduction of LGP2 protein levels presumably due to FMDVFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVLproinduced cleavage The apparent inconsistencybetween the levels of LGP2 mRNA and protein during FMDVinfection may be explained by LGP2s ability to serve as a positiveand negative regulator of RIGI and MDA5 signaling presumablyaï¬ecting multiple steps of the IFN induction pathway Inaddition to RLRs nucleotidebinding oligomerization domainNODlike receptors NLRs NOD1 and NOD2 also participatein the recognition of RNA A study by Liu et al describedthe association of NOD2 with FMDV 2B 2C and 3Cpro to blockinnate immunity activation Protein kinase R PKR is anotherrecognized PRR that acts as an RNA sensor Binding ofRNA to PKR induces a conformational change that leads toautophosphorylation and activation The primary target ofactivated PKR is the eukaryotic initiation factor α subuniteIF2α whose phosphorylation results in the blockage of cellularprotein synthesis a relatively common process during viralinfection Although no direct interaction between FMDVRNA and PKR has been demonstrated it has been reportedthat PKR activity modulates FMDV infectivity In fact in tissueculture experiments depletion of endogenous levels of PKR usingsiRNA resulted in increased FMDV titers Furthermoreit has been recently shown that overexpression of autophagyrelated ATG5ATG12 proteins induces transcription of PKR andsubsequent reduction of FMDV replication These resultssuggest that PKR has a complex role as an RNA sensor but also asan antiviral agent during FMDV infectionIt has been demonstrated that FMDV also targets DExDHbox RNA helicases formally accepted as PRRs and modulatorsof the antiviral signaling pathway In vitro experimentsintending to analyze proteinprotein interactions revealed theassociation between the RNA helicase DDX1 and FMDV 3D Interestingly these studies indicated that during FMDVinfection in porcine cells cleavage of DDX1 was detected whileoverexpression of DDX1 resulted in the upregulation of IFNand other ISG mRNAs which correlated with virus inhibition Other DExDHbox RNA helicases such as RNA helicaseH RHA are hijacked during FMDV infection and interact withFMDV UTR 2C and 3A to facilitate virus replication Signaling pathways downstream from RNA sensing involvethe activation of diï¬erent adaptor and eï¬ector proteins Oneof the pathways that lead to signal activation requires theformation of speciï¬c complexes such as NFκB essentialmodulator NEMO and the kinase IKK which bridges theactivation of NFκB and IFN regulatory factor IRF signalingpathways It has been demonstrated that FMDV 3Cpro interactswith NEMO and induces its cleavage resulting in impairedinnate immune signaling IRFmediated signals driven byIRF3 and IRF7 can also be targeted by FMDV proteinsSpeciï¬cally overexpression of Lpro in PK15 cells resulted inthe downregulation of IRF3 and IRF7 protein levels andinactivation of IFN and IFNλ1 promoter Other factors involved in the activation of IFN includeconventional PTMs such as phosphorylation and ubiquitinationwhich ensure eï¬ective regulation of these signaling pathways Also diï¬erent cellular deubiquitinases DUBs can reverseubiquitination to control the intensity of the immune signalingresponse Interestingly it has been shown that FMDV Lprocan remove ubiquitin Ub molecules from several proteinsrequired for IFN mRNA expression and those involved in theactivationrepression of the IFN loop This role became moreevident by the observation that during infection FMDV Lprocan cleave cellular substrates modiï¬ed with the Ublike moleculeISG15 Furthermore mutation of Lpro thatimpairsdeISGylaseDUB function results in viral attenuation Inthis regard identiï¬cation of FMDV targets for deubiquitinationand deISGylation may contribute to elucidate the role ofthose factors in counteracting the innate response and developnovel countermeasuresBlock on Interferon SignalingThe ligandmediated association of the speciï¬c IFN receptorspromotes a signaling cascade that results in the phosphorylationof the receptor by the action of JAKs These events result inthe generation of docking sites for downstream adaptor andeï¬ector proteins including signaltransducer and activatoroftranscription STAT proteins that associate with otherfactors and translocate to the nucleus inducing transcriptionof a plethora of ISGs described above and in Figure Although blockage of the JAKSTAT signaling pathway hasnot been reported during FMDV infection overexpressionof either FMDV 3Cpro or VP3 can inhibit this response Forinstance IFNtreated HeLa cells overexpressing FMDV 3Cprosuppressed IFNstimulated promoter activities and inducedproteasome and caspaseindependent protein degradationof karyopherin α1 KPNA1 the nuclear localization signalreceptor Thisinteraction inhibited the nuclear translocation of STAT1STAT2impeding maximal ISG promoter activity In another studyin HEK293T cells overexpression of VP3 followed by coimmunoprecipitation revealed the association between VP3 andJAK1 FMDV VP3 also inhibited virustriggered activation of theIFN promoter leading to the decrease in transcription of ISGspresumably due to lysosomalinduced degradation of JAK1 A yeast twohybrid screen identiï¬ed FMDV 2C in complex withNmyc and STAT interactor Nmi a protein known to augmentimmune function dependent on STATmediated transcriptionInterestingly such interaction resulted in the recruitment ofNmi to vesicular compartments followed by the induction ofapoptosis in BHK21 cells tyrosinephosphorylated STAT1forEvidently FMDV proteins can also target crosstalk pathwaysinduced by JAKSTAT signaling and due to this versatilityunderstanding of these signaling events during FMDV infectionis challengingFOOTANDMOUTH DISEASE VIRUSIMPAIRS INTERFERONMEDIATEDCELLULAR INNATE IMMUNE RESPONSESSimilarly to what happens in vitro FMDV manipulates theearly innate immune response in vivo to ensure a windowof opportunity that favors viral replication and spread beforeFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVthe onset of eï¬ective adaptive immunity required for virusclearance During infection FMDV interacts with a range of hostcells including natural killer NK cells dendritic cells DCsmonocytesMÏ and Îδ T cells All these cells play an importantrole in innate immune responses that trigger the productionof large quantities of IFN and other cytokines which serve asautocrine agents Shortly after FMDV infection in swinethe number ofcirculating NK cells transiently decreases and the remaining NKcells show a dysfunctional lytic activity against target cells and areduction of IFNÎ production In parallel FMDV blocks theability of porcine DCs to mature into conventional DCs cDCs dampening their response against Tolllike receptor TLRligands Another subset of porcine DCs plasmacytoid DCspDCs also referred to as the major professional systemic IFNαproducers are also aï¬ected by FMDV During infectionpartial depletion of pDCs in the peripheral blood has beendetected and the remaining pDCs are less capable of producingIFNα in response to ex vivo stimulation by TLR ligands or virus Similar to pDCs FMDV infection reduces the productionof IFNα on Langerhans cells LCs a distinct subset oftissueresident DCs of the skin It has also been suggestedthat porcine Îδ T cells and MÏ can serve as targets for FMDVinfection in swine although the interplay betweenthese cells and FMDV remains unclearComparably to swine FMDV infection in cattle triggersseveral early events in the innate immune system althoughthe eï¬ects are not exactly the same For instance bovine NKcells originated from FMDVinfected cows have an elevatedcytotoxic function against bovine target cells in vitro In addition some subsets of cDCs are signiï¬cantly decreasedduring the peak of viremia while the expression of majorhistocompatibility complex MHC class II molecules on allbovine cDCs is reduced and the processing of exogenous antigenis impaired Furthermore during FMDV infection thenumber of systemic mature bovine pDCs characterized bythe expression of CD4 and MHC class II is increasedpresumably to intensify a humoral response and T cell activationwhile levels ofimmature CD4 MHC class IIpDCs aredeclined Examination of bovine Îδ T cells revealed thatthese cells with the surface expression marker WC1 showa transient activated phenotype and increased expression ofIFNÎ FMDV also aï¬ects the innate immune response at the cytokinelevel in the natural host In vivo cytokine proï¬le analysis duringthe clinical phase of disease shows a systemic decrease of proinï¬ammatory cytokines [IL1 IL6 and tumor necrosis factorTNFα] and an increase of the antiinï¬ammatory cytokine IL and IFNα Most likely these changesare related to the early T cell unresponsiveness and lymph iadescribed in swine and cattle during FMDV infection Interestingly a signiï¬cant induction of inï¬ammatoryand antiviral factors at the local level is detected in cattle in sitesof abundant viral ampliï¬cation such as the nasaloropharynx orvesicular lesions A consistent upregulation of IFNα Î and λ mRNA in distinct microanatomical compartmentsof the nasopharyngeal mucosa concurrent with occurrence ofviremia has also been detected in cattle In contrast studiesin swine demonstrated that IFN expression in infected swineskin is inhibited These diï¬erences may be due to theanalysis of follicleassociated epithelium of the nasopharyngealmucosa in cattle vs skin in swine or to the speciï¬c samplingtechnique used in each experiment While in the cattle studylasercapture microscopy was used to focus only in areas of highFMDV replication in the swine study RNA was extracted froma piece of skin without discriminating between microanatomicalcompartments Evidently more studies are needed to elucidatethe intricate interactions between FMDV and the innate immunesystem of speciï¬c animal hostsEFFECTIVE USE OF INTERFERONAGAINST FOOTANDMOUTH DISEASEVIRUS IN VITROType I InterferonThe role of IFN in controlling FMDV replication was ï¬rstproposed in when Dinter and Philipson demonstrated thatcalf kidney cells exposed to FMDV could become persistentlyinfected and proposed this was a consequence of the inductionof an IFNlike inhibitor present in the supernatant of infectedcells Later studies also suggested that swine leukocytestreated with phytohemagglutinin produced an inhibitor ofFMDV replication with properties similar to IFN It wasnot until that new studies demonstrated that the abilityof FMDV to form plaques in cell culture correlated with thesuppression of type I IFN α protein expression Theseresults were further supported by detection of IFN protein andantiviral activity in the supernatants of primary porcine ovineand bovine kidney cells infected with an attenuated FMDVmutant leaderless as compared to the supernatants of cellsinfected with wildtype WT virus Later studies by the samegroup provided proof of concept on the use of recombinantbacterial expressed IFNα as a potent biotherapeutic againstFMDV This approach was further developed by deliveringrecombinant porcine IFNα using a replicationdefectivehuman Adenovirus vector Ad5poIFNα Infection ofIBRS2 cells with Ad5poIFNα resulted in secreted poIFNα IFN protein detected as early as h postinfection hpiand lasting for at least h Most important expressed IFNprotein displayed strong biological antiviral activity againstFMDV Followup studies by the same group showed that allFMDV serotypes are very sensitive to Ad5delivered poIFNαand sterile protection could be achieved in vivo highlightingthe potential of this approach for the development into abroad biotherapeutic strategy to control FMDV replicationIn the last years advancements is genomics have ledto the characterization of almost all type I IFN subtypes inthe porcine and bovine genome which are morenumerous than those identiï¬ed in primates and mice This hasrevealed diï¬erent functional genes and pseudogenes with diverseexpression proï¬les and antiviralfunctions against diï¬erentviruses mostly in swine In fact a recent studyFrontiers in Veterinary Science wwwfrontiersinAugust Volume 0cMedina et alIFN Against FMDVdemonstrated that poIFNÏ7 known for its ability to inducethe highest levels of antiviral activity when compared to otherpoIFNÏ subtypes elicits an antiviral state against FMDV inIBRS2 cells treated with the recombinant form of poIFNÏ7produced in Escherichia coli Other subclasses of type IIFN known to be produced in swine and cattle include IFNalphaomega IFNÎ±Ï also known as IFNµ and IFN delta IFNδ Signiï¬cant reduction in FMDV replication has been observedupon treatment of porcine cells with bacterially expressed IFNÎ±Ï or IFNδ8 prior to viral infection Recently another member of type I IFN family IFNÏ whichis only produced in ruminants has been evaluated as an antiviralagainst FMDV IFNÏ is a paracrine reproductive hormonesecreted constitutively by trophoblasts and endometrial cells toincrease the life span of the corpus luteum however productionis not induced upon viral infection While its secretionis restricted to ruminantsit has a broadspectrum activityagainst various crossspecies viruses Interestingly IFNÏ has homology with the amino acids of IFNα which allows forbinding to type I IFN receptors The property of IFNÏ thatmakes it an interesting therapeutic candidate for the treatmentof various viral diseases is its signiï¬cantly lower toxicity ascompared to other type I IFNsType II InterferonIn contrast to type I IFN the type II IFN family is composedof only one member IFNÎ which exerts its actions througha speciï¬c receptor IFNGR1IFNGR2 IFNÎ is weakly resistantto heat and acid and it is able to activate leukocytes suchas macrophages and granulocytes also exerting regulatoryfunctions on T and B lymphocytes Indeed productionof IFNÎ is used as a tool to measure cellmediated immuneresponses against FMDV in vaccinated cattle andin swine Interestingly IFNÎ responses as measured b | Thyroid_Cancer |
radiation induces epithelialmesenchymaltransition in human bronchial epithelial cellsBo Tang123 Yue Xi121State Key Laboratory of Radiation Medicine and Protection School of Radiation Medicine and Protection Soochow University Suzhou China 2Collaborative InnovationCenter of Radiation Medicine of Jiangsu Higher Education Institutions Suzhou China 3Department of Radiation Protection Safety Shandong Center for Disease Controland Prevention Jinan ChinaFengmei Cui12 Jin Gao12 Huiqin Chen12 Wentao Yu12 andYu Tu12Correspondence Yu Tu tuyusudaeducnObjective The present study aimed to analyze the mechanism by which longterm occupational exposure of workers to lowdose ionizing irradiation induces epithelialmesenchymaltransition EMT of the human bronchial epithelial cells using transcriptome profilingMethods RNAseq transcriptomics was used to determine gene expression in blood samples from radiationexposed workers followed by bioinformatics analysis Normal bronchialepithelial cells 16HBE were irradiated for different durations and subjected to immunofluorescence Western blotting scratch healing and adhesion assays to detect the progressionof EMT and its underlying molecular mechanismsResults Transcriptomics revealed that exposure to ionizing radiation led to changes in theexpression of genes related to EMT immune response and migration At increased cumulative doses ionizing radiationinduced significant EMT as evidenced by a gradual decrease inthe expression of Ecadherin increased vimentin elevated migration ability and decreasedadhesion capability of 16HBE cells The expression of fibronectin FN1 showed a gradualincrease with the progression of EMT and may be involved in EMTConclusion Ionizing radiation induces EMT FN1 may be involved in the progression of EMTand could serve as a potential biomarker for this processIntroductionPeople are inevitably exposed to ionizing irradiation in their daily lives and at work The uncertaintiesabout the health risks associated with longterm exposure to radiation at a lowdose in occupationallyexposed workers have been the focus of research for many years In their International Nuclear WorkersStudy INWORKS published in the International Journal of Epidemiology in Hamra specifically evaluated the radiation hazards and risk in a cohort of nuclear workers in countries []Abbott A explored the risk of lowdose irradiation in [] Longterm general and occupational exposure to radiation has been the focus of research in the past Keil conducted a cohort mortalitystudies of underground miners from the Colorado Plateau [] Kamiya conducted a cohort study ofnuclear workers in USA and reported the ERR values of cancers and cardiovascular diseases in []A recent epidemiological study emphasized the harmful effects of lowdose radiation exposure on humanhealth and reported elevated cancer mortality among nuclear workers exposed to radiation at a cumulative dose mSv and dose rate mSvyear [] However the biomarkers and molecular mechanismthat predict the longterm and lowdose effect of radiation are not clearThe carcinogenic effects of ionizing radiation rank first among other potential hazards of occupational exposure to radiation It is of great clinical significance to detect and evaluate the carcinogenicrole of radiation In the early stages of exposure to radiation several biological changes occur of whichepithelialmesenchymal transition EMT is an important indicator of malignant change EMT refers toThese authors contributedequally to this workReceived February Revised June 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 BSR20200453101042BSR20200453Table General information pertaining to the participantsAge yearWorking age yearMarital statusEducationIncome per month RMBGroup¥MarriedUnmarriedOtherElementary or lessJuniorSeniorUndergraduate or more¥Casesthe biological process in which epithelial cells are transformed into cells with interstitial phenotype characterized byloss of cell adhesion and enhanced ability to migrate [] Based on the specific biological environment in which MToccurs there are three subtypes of which type EMT is associated with epithelial malignancies Whether bronchialepithelial cells undergo EMT following longterm and lowdose radiation exposure and its mechanism are the focusof the present studyThe present study aimed to analyze the biological changes after exposure to longterm and lowdose ionizing radiation Highthroughput sequencing was used to analyze the mRNA expression profiles of workers with or withoutradiation exposure Normal bronchial epithelial cells underwent EMT following multiple rounds radiation and thedifferentially expressed mRNAs were validated using a cellular model These findings provide an experimental basisfor radiationinduced carcinogenesisSubjects and methodsSubjectsThe present study was approved by the ethic committee of the Soochow University and informed consent was obtainedfrom all participants The study included a total of participants as shown in Table which included workerswith no radiation exposure and workers exposed to radiation at a total dose of mSv for an average of yearsReagent and cell lineHuman normal bronchial epithelial cells 16HBE culture medium bronchial epithelial growth factor and penicillinstreptomycin were purchased from Shanghai Zhongqiao Xinzhou Biological Company Antibodies against FN1ab32419 vimentin VIM ab92547 Ecadherin Ecad ab40772 and the goat antirabbit IgG HL Alexa secondary antibody ab150080 were purchased from AbcamTranscriptome sequencingRNA library construction and sequencingThe ribosomal RNA rRNA was removed using the NEBNext rRNA Depletion Kit New England Biolabs Inc Massachusetts USA The RNA library was constructed using the NEBNext® Ultra¢ II Directional RNA Library PrepKit New England Biolabs Inc Massachusetts USA Quality control and quantification of the library were performed using a BioAnalyzer system Agilent Technologies USA and sequenced using the bp pairedendmethod in an Illumina Hiseq The reads were subject to quality control Q30 and checked using the Cutadaptprogram v193 The highquality reads were mapped to the human reference genome UCSC HG19 using theHisat2 program v204 Guided by the annotation from the TGF file the Cuffdiff program was used to generate thetranscript abundance of LncRNA and mRNA known as FPKM Fragments per kilobase of exon per million fragments mapped The foldchanges between two groups were calculated and the differentially expressed mRNAs wereidentified based on Pvalue for analysis of GO and KEGG pathway 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 BSR20200453101042BSR20200453Table Primers used in the present studyGene IDGene nameReferenceLAMB1KRT6BFN1ADAM9RNF182GAPDHPrimerForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseForwardReverseSequence 5cid23cid2AAATCTTGTGCTTGCAATCCTCCGCAAAGCAACTGTTGTTTAAGGTACCAGACAAAGTACGAGGAGGCTTGTTCTTAGCATCCTTGAGAATAGATGCAACGATCAGGACAGCAGGTTTCCTCGATTATCCTTAGGCTGAAGGAAAAGAGCATATAAGTCCCTTCCTTGTTGTAAGTCCGTAGAGACAAAGCCGCCTAAAGGCCACATGAAGGGTCTGGCCTCCAAGGAGTAAGACCAGGGGAGATTCAGTGTGGTGGO and KEGG analysisThe Gene Ontology GO project httpwwwgeneontology provides a set of structured controlled vocabulariesfor community use in annotating genes gene products and sequences which is divided by molecular function MFbiological process BP and cell component CC GO terms with Pvalue are considered to be statistically significant By mapping multiple types of omics data such as genomics transcriptomics proteomics and metabolomicsinto KEGG pathway the biological function of these genes can be interpreted Pvalue was considered to bestatistically significantRealtime PCRThe plasma sample was thawed out from ¦C and centrifuged at g for min at ¦C Samples μlwere transferred to another centrifuge tube ml and TRIzol LS Reagent μl and acetic acid μl wereadded The homogenized samples were incubated with chloroform ml After shaking the tube manually for s and incubating at to ¦C for min the sample was centrifuged at g for min at ¦C Followingcentrifugation the solution separated into a red phenol chloroform phase in the lower layer and a colorless aqueousphase in the upper layer All the RNA were distributed in the aqueous phase The volume of the aqueous phase wasapproximately of the TRIzol LS Reagent added during homogenization The aqueous phase was transferred to anew centrifuge tube and mixed with isopropanol for min to precipitate the RNA After centrifugation at g for min at ¦C the RNA became visible on the bottom and walls of the tube The supernatant was removed andthe RNA was washed with at least ml of ethanol After centrifugation at g for min at ¦C the RNAwas airdried for min The concentration and purity of RNA were determined using a NanoDrop® ND1000RNA was reverse transcribed into cDNA using the SuperScript¢ III Reverse Transcriptase Invitrogen RealtimePCR was performed using a qPCR SYBR Green master mix kit CloudSeq using the following condition ¦C min cycles ¦C s ¦C s The melting curve was then obtained with the condition ¦C s ¦C s ¦C s gradually from ¦C to ¦C ¦Cs The relative expression of the genes were calculated using theääCT method The primers used are shown in Table Cell culture and radiationHuman normal bronchial epithelial cells were purchased from Shanghai Zhongqiao Xinzhou Biological Companyand cultured in medium supplemented with bronchial epithelial growth factor and penicillinstreptomycin SciencellUSA When the cells were in logarithmic growth period the culture dish was placed in a biological Xray irradiatorRS2000Pro for radiation dose of Gy at a dose rate of Gymin After passage on the other day the cells wereexposed to radiation at the accumulative dose of Gy The radiationexposed cells were divided into and Gy groupsImmunoï¬uorescenceCells in the five groups were fixed with paraformaldehyde Meilunbio China cleared in Triton X100Meilunbio China for min and then blocked in the serum for min The cells were incubated with the primary antibody Abcam USA at ¦C overnight and then incubated with the secondary antibody Abcam 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 BSR20200453101042BSR20200453Figure Differentially expressed mRNAs in workers with or without radiation exposureA Cluster analysis of differentially expressed mRNA based on FPKM values via heatmap2 In the picture A is the control groupand B is the exposed group B Scatter plot based on the expression of the two groups Red dots upregulated genes greendots downregulated genes purple dots no significant change Foldchange was C Volcano plot based on foldchange andPvalue The red rectangle represents the differentially expressed mRNA P foldchange ¥ at room temperature for h in the dark The nuclei were counterstained using DAPI and examined by fluorescence microscopy Images were acquired using a scanning laser confocal microscope FV1200 and the results weresemiquantitatively analyzed using the ImageJ softwareWestern blottingCells were lysed in lysis buffer containing PMSF μl on ice for min transferred to a centrifuge tube ml and centrifuged at rpm for min at ¦C Protein concentration was determined using the BCA methodSolarbio China and the lysates were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresisSDSPAGE The proteins were transferred to polyvinylidene difluoride PVDF membrane and the membranes wereblocked with skim milk Oxoid UK for h The membranes were then incubated with the primary antibodyAbcam USA at ¦C overnight and the secondary antibody Abcam USA at room temperature for h Theproteins were detected using enhanced chemiluminescence ECL immunoassay Beyotime China and visualizedin an AI680 ultrasensitive multifunctional analyzer 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 BSR20200453101042BSR20200453 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 BSR20200453101042BSR20200453Figure GO analysis of differentially expressed mRNA in workers with or without radiation exposureA The top GO terms with the largest number of genes B The ratio of the number of differentially expressed genes in the top enriched GO terms with high fold enrichment to the total number of differentially expressed genes The left column pertains toupregulated mRNAs the right column pertains to downregulated mRNAs PTable Baseline characteristics of participantsAgeSmokeAlcoholTeaRiceVegetableOilSugarChiliSaltedFruitSaltMeatControl Radiation PScratch assayCells in the five groups were plated in a sixwell plate and cultured at ¦C When the cells grew to confluencea uniform scratch was made in the cell monolayer using a ml pipette tip Then the cells were washed with PBS andcultured with growth factorfree medium Images were collected at and h using a microscope IX73 andwere analyzed quantitatively using the ImageJ softwareAdhesion assayCell adhesion was tested using a cell adhesion detection kit BestBio China according to the manufactures protocolCells in the five groups were plated in 96well plates at the density of cellswell in triplicates for each groupAfter h of incubation at ¦C the medium was changed and μl of the staining solution B was added to the wellsThe OD was measured at nm after h using a microplate reader Synergy The formula used for calculatingthe cell adhesion rate was cell adhesion rate OD of test cells OD of the blank OD of the control ODof the blank CCK8 assayCells in the five groups were plated in a 96well plate at the density of per well in triplicates On the next day μl of CCK8 solution Meilunbio China was added to each well and the plates were incubated at ¦C After h a microplate reader Synergy was used to determine the OD at nm The formula used for calculating cellviability was cell viability OD of test cells OD of cellfree medium OD of control cell OD of cellfreemedium Statistical analysisThe general information of the participants was subjected to a normality test and expressed as mean standard deviation SD The data were analyzed using SPSS For the quantitative data with normal distribution and homogeneity of variance analysis of variance was used For the qualitative data chisquare tests were used The significancelevel was set to α and P005 was considered statistically significantResultsBaseline characteristics of participantsAll the participants were male Except for salt intake there were no significant differences among the other parameters including age smoking history and eating habits P005 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 BSR20200453101042BSR20200453Figure KEGG analysis of differentially expressed mRNA in workers with or without radiation exposureA The top pathways with high enrichment scores associated with the upregulated mRNA B The top pathways with highenrichment scores associated with the downregulated mRNADifferentially expressed mRNA in radiationexposed workersThe differentially expressed mRNA between the two groups were calculated using the Cuffdiff software with thefoldchange ¥ ie log FC ¥ Pvalue and FPKM value ¥ in at least one sample as the thresholdFinally we screened a total of differentially expressed genes including upregulated and downregulatedgenes Figure and Supplementary Material S1GO and KEGG analysis of differentially expressed mRNAWe then performed GO analysis on the differentially expressed mRNAs For the upregulated genes there were subclasses in BP in CC and in MF For the downregulated genes there are subclasses in BPs inCCs and in MFs Figure In BPs the upregulated mRNAs were involved in intracellular protein transportcellular protein localization ectoderm development etc In CCs the upregulated mRNAs were involved basal laminabasement membrane laminin complex etc In the MFs the upregulated mRNAs were involved in cell adhesionmolecule binding and integral binding For the downregulated mRNA the BPs mainly included immune responseimmune system process immune effector process and defense response among others the CCs mainly includedmembranebounded anelle cytoplasm and extracellular anelle and the MFs mainly included protein bindingidentification of protein binding and cofactor binding among othersKEGG analysis Figure revealed that pathways were related to the upregulated mRNA mainly including pro 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 BSR20200453101042BSR20200453Figure Validation of RNAseq by RTPCRP005 vs controltein export bacterial invasion of epithelial cells small cell lung cancer and ECMreceptor interaction among othersThere were pathways associated with the downregulated mRNA mainly including pertussis and transcriptionalmisregulation in cancerThrough the analysis of GO and KEGG we found that the GO terms with higher enrichment and KEGG pathwaywere closely related to EMT migration and ECMreceptor interaction Basement membrane laminin complex andcell adhesion molecule binding have been reported to participate in changes in cell morphology or cell migration Insummary we believe that exposure to a certain dose of radiation may cause EMT or EMTrelated regulatory processesRTPCR analysis of differentially expressed genesGO and KEGG pathway analysis revealed that EMTrelated regulatory processes cell migration and ECMreceptorinteraction were closely associated with these differential expressed mRNAs Based on these results five candidategenes were selected including LAMB1 KRT6B FN1 ADAM9 and RNF182 RTPCR analysis was performed toanalyze the expression of these genes in the blood samples which were also used for RNAseq The results showedthat data from the RTPCR assay were consistent with those from RNAseq Figure Establishment of EMT model in 16HBE cellsTo further explore the function of FN1 we first observed the changes of EMT in human bronchial epithelial cellsinduced by longterm and lowdose radiation As shown in Figure 5AB exposure to the cumulative dose of radiationresulted in a gradual decrease in the protein expression of Ecad and increase in VIM levels indicating that these cellsacquired EMT The migration capacity and proliferation activity were also examined Scratch healing assay revealedthat the irradiated cells had a significantly higher migration capacity than normal 16HBE cells Figure 5CD Celladhesion assay showed that the adhesion rates of the irradiated cells were significantly lower than those withoutradiation exposure which indirectly supported the observation that their migration ability was improved Figure5E Moreover CCK8 assay show that the activity of the irradiated cells increased to a certain extent Figure 5FFN1 expression in 16HBE cells during EMTWestern blotting was performed to analyze the expression of FN1 in 16HBE cells at different cumulative doses ofradiation As shown in Figure the expression of FN1 gradually increased with increasing cumulative doses of radiation The expression of FN1 was significantly increased at the radiation dose Gy showing that FN1 was involvedin the EMT of 16HBE cells and may be used as a potential biomarker of the cumulative doses of radiation 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 BSR20200453101042BSR20200453Figure Changes in VIM Ecad migration and proliferation of 16HBE cells following irradiationImages of Ecad A and VIM B expression at different cumulative doses of radiation observed under a confocal microscope Cand D Wound healing assay was performed to determine the cell migration capability E and F Cell migration and activity weredetermined by adhesion and CCK8 assays respectively P005 vs Gy 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 BSR20200453101042BSR20200453Figure Expression of FN1 in 16HBE cells following different cumulative doses of radiationP005 vs 0vGyDiscussionLung cancer has always been an area of intensive research in the biological effects caused by ionizing radiation andnot just the epidemiological study of radiation [] Owing to the occupational specificity and small sample size ofthe present study for example in some s the work content of many radiation workers involves confidentialityissues which brings great difficulties to the preinvestigation work and the information of the personnel cannot befully grasped so the sample size is generally small Studies using larger cohorts are still necessary to obtain moreinformation such as Mayak cohorts The conclusions from these studies need further corroboration due to the differences of the research applied and statistical methods used in the study [] In addition genderspecific analysiswas also limited due to the low number of female workers and the low exposure dose []To identify radiationrelated factors highthroughput sequencing was performed using peripheral blood of healthyradiationexposed workers RNAseq results revealed differently expressed genes between participants with orwithout radiation Among these genes the upregulated genes were mainly located on chromosome and while thedownregulated genes were in chromosome and However we did not identify any correlation between thedistribution of chromosomal positions of these differential genes and ionizing radiation Then bioinformatics analyses GO and KEGG were conducted to identify the potential functions of these differential mRNAs We found thatthe differential genes induced by radiation were mainly involved in the ECMreceptor interaction laminin complex 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 BSR20200453101042BSR20200453cell adhesion molecule binding and immune reaction Based on these results five differential genes were selectedand validated using the same samples Although there was no statistical difference in the smoking history betweenthe two groups smoking is a known highrisk factor for lung cancer and cannot be eliminated as a factor Studieshave shown that smokers and nonsmokers have different molecular mechanisms of lung cancer development suchas hypermethylation of gene promoters [] Therefore the abnormal expression of some genes needs to be furtherstudied to determine their correlation with ionizing radiationAmong the upregulated genes SEC62 which is reported to be overexpressed in lung cancer prostate cancer andthyroid cancer [] is involved in endoplasmic reticulum stress tolerance and cell migration and is identified as aprognostic marker for nonsmall cell lung cancer [] Moreover overexpression of SEC62 is also reported in the atypical fibrous xanthomas AFX [] CDC9 a member of tetraspanin superfamily is involved in the regulation of manydisease and physiological processes including cell migration and adhesion [] PLD1 is the mediummembraneprotein that is overexpressed in various cancers such as lung cancer breast cancer and kidney cancer [] It alsoplays a key role in LAinduced breast cancer cell migration and invasion [] Thus most of the upregulated genesidentified in our study are related to cancerSeveral prior studies used A549 as an in vitro model to study lung cancer [] However we used normalbronchial epithelial cells 16HBE in our study EMT in 16HBE cells can be induced by silicon dioxide [] smokingextract CSE [] and transforming growth factor β1 TGFβ1 [] The novelty of the present study was inthe use of the 16HBE cells and the induction of EMT using ionizing radiation Among the upregulated genes FN1was found to be closely associated with EMT Subsequently RTPCR and Western blotting further demonstrated thatthe expression of FN1 was increased in 16HBE cells following EMT FN1 a member of the glycoprotein family isinvolved in cell migration and adhesion [] Many studies have reported that FN1 has an indispensable role in EMT[] FN1 together with Ecad and VIM serves as a marker for EMT in nonsmall cell lung cancer MoreoverFN1 may play an important role in the pathogenesis of nasopharyngeal carcinoma [] and the drug resistance oflung cancer []In the present study the mRNA expression of workers with or without radiation exposure was profiled usinghighthroughput sequencing and bioinformatics analysis An EMT model of the normal bronchial epithelial cellswas established after repeated irradiation of the cells The differentially expressed mRNAs were selected based on thebioinformatics analyses and validated in the cellular model We found that FN1 was involved in EMT and could beused as a potential biomarker Our research provides evidence for the possible mechanism through which longtermexposure to ionizing radiation induces EMTCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThe work was supported by the Nuclear Energy Development Project [grant number ]Author ContributionConceptualization FMCand YT Investigation BT YX HQC and WTY Formal analysis BT YX and JG Funding acquisition YT Project administration YT Writing original draft BT YX and FMC Writing review editing BT YX FMCand YX All authors have read and agreed to published version of the manuscriptAbbreviations16HBE human normal bronchial epithelial cells EMT epithelialmesenchymal transition FN1 fibronectin rRNA ribosomalRNAReferences Hamra GB Richardson DB Cardis E Daniels RD Gillies M OHagan JA Cohort Proï¬le The International Nuclear Workers StudyINWORKS Int J Epidemiol 101093ijedyv122 Abbott A Researchers pin down risks of lowdose radiation Nature 101038523017a Keil AP Richardson DB and Troester MA Healthy worker survivor bias in the Colorado Plateau uranium miners cohort Am J Epidemiol 101093ajekwu348 Kamiya K Ozasa K Akiba S Niwa O Kodama K Takamura N Longterm effects of radiation exposure on health Lancet 101016S0140673615611679 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 BSR20200453101042BSR20200453 Hall J Jeggo PA West C Gomolka M Quintens R Badie C Ionizing radiation biomarkers in epidemiological studies An updateMutat Res 101016jmrrev201701001 MorenoBueno G Portillo F and Cano A Transcriptional regulation of cell polarity in EMT and cancer Oncogene 101038onc2008346 Edwards JK McGrath LJ Buckley JP SchubauerBerigan MK Cole SR and Richardson DB Occupational radon exposure and lungcancer mortality estimating intervention effects using the parametric gformula Epidemiology 101097EDE0000000000000164 Brown SC Schonbeck MF McClure D Baron AE Navidi WC Byers T Lung cancer and internal lung doses among plutoniumworkers at the Rocky Flats Plant a casecontrol study Am J Epidemiol 101093ajekwh192 Sokolnikov M Preston D Gilbert E Schonfeld S and Koshurnikova N Radiation effects on mortality from solid cancers other than lungliver and bone cancer in the Mayak worker cohort PLoS ONE e0117784 101371journalpone0117784 Wilson DA Mohr LC Frey GD Lackland D and Hoel DG Lung liver and bone cancer mortality after plutonium exposure in beagle dogsand nuclear workers Health Phys 101097HP0b013e3181b97318 Gilbert ES Sokolnikov ME Preston DL Schonfeld SJ Schadilov AE Vasilenko EK Lung cancer risks from plutonium an updatedanalysis of data from the Mayak worker cohort Radiat Res 101667RR30541 Gillies M Kuznetsova I Sokolnikov M Haylock R OHagan J Tsareva Y Lung Cancer Risk from Plutonium A Pooled Analysis of theMayak and Sellaï¬eld Worker Cohorts Radiat Res 101667RR147191 Boice Jr JD Ellis ED Golden AP Zablotska LB Mumma MT and Cohen SS Sexspeciï¬c lung cancer risk among radiation workers in themillionperson study and patients TBFluoroscopy Int J Radiat Biol Wu XM Chen Y Shao Y Zhou XL and Tang WR Association between cigarette smoking and RASSF1A gene promoter hypermethylation inlung cancer patients a meta analysis Asian Pac J Cancer Prev 107314APJCP201415198451 Korbel C Linxweiler M Bochen F Wemmert S Schick B Meyer M Treatment of SEC62 overexpressing tumors by Thapsigarginand Triï¬uoperazine Biomol Concepts 101515bmc20180006 Wemmert S Lindner Y Linxweiler J Wagenpfeil S Bohle R Niewald M Initial evidence for Sec62 as a prognostic marker inadvanced head and neck squamous cell carcinoma Oncol Lett 103892ol20164135 Muller CSL Kreie L Bochen F Pfuhl T Smola S Graber S Expression of 3q oncogene SEC62 in atypicalï¬broxanthomaimmunohistochemical analysis of cases and correlation with clinical viral and histopathologic features Oncol Lett Brosseau C Colas L Magnan A and Brouard S CD9 Tetraspanin A New Pathway for the Regulation of Inï¬ammation Front Immunol 103389ï¬mmu201802316 Blake DJ Martiszus JD Lone TH and Fenster SD Ablation of the CD9 receptor in human lung cancer cells using CRISPRCas altersmigration to chemoattractants including IL16 Cytokine 101016jcyto201805038 GomezCambronero J Phosphatidic acid phospholipase D and tumorigenesis Adv Biol Regul 101016jjbior201308006 DiazAragon R RamirezRicardo J CortesReynosa P SimoniNieves A GomezQuiroz LE and Perez Salazar E Role of phospholipase Din migration and invasion induced by linoleic acid in breast cancer cells Mol Cell Biochem 101007s11010019035178 Hu C Jiang R Cheng Z Lu Y Gu L Li H OphiopogoninB Suppresses Epithelialmesenchymal Transition in Human LungAdenocarcinoma Cells via the Linc00668miR4325pEMT Axis J Cancer 107150jca31338 Chen J Jiao D Li Y Jiang C Tang X Song J Mogroside V Inhibits Hyperglycemiainduced Lung Cancer Cells Metastasis throughReversing EMT and Damaging Cytoskeleton Curr Cancer Drug Targets 1021741568009619666190619154240 Camerlingo R Miceli R Marra L Rea G DAgnano I Nardella M Conditioned medium of primary lung cancer cells induces EMT inA549 lung cancer cell line by TGFss1 and miRNA21 cooperation PLoS ONE e0219597 101371journalpone0219597 Li X Yan X Wang Y Wang J Zhou F Wang H NLRP3 inï¬ammasome inhibition attenuates silicainduced epithelial to mesenchymaltransition EMT in human bronchial epithelial cells Exp Cell Res 101016jyexcr201712013 Liang X He X Li Y Wang J Wu D Yuan X Lyn regulates epithelialmesenchymal transition in CSexposed model through Smad23signaling Respir Res 101186s129310191166z Heijink IH Postma DS Noordhoek JA Broekema M and Kapus A House dust mitepromoted epithelialtomesenchymal transition inhuman bronchial epithelium Am J Respir Cell Mol Biol 101165rcmb20080449OC Xu L Xiang X Ji X Wang W Luo M Luo S Effects and mechanism of dehydroepiandrosterone on epithelialmesenchymal transitionin bronchial epithelial cells Exp Lung Res 1031090190 | Thyroid_Cancer |
The role of serum inflammatory cytokines andberberine in the insulin signaling pathwayamong women with polycystic ovarysyndromeHongying Kuang12¯ Yuwei Duan34¯ Dan LiID5a Yanwen Xu34b Wenxia Ai2 Wei Li1Ying Wang1 Sha Liu2 Mushan Li2 Xiaoqiu Liu2 Manqi Shao2 The First Affiliated Hospital of Heilongjiang University of Chinese Medicine Harbin China HeilongjiangUniversity of Chinese Medicine Harbin China The First Affiliated Hospital of Sun Yatsen UniversityGuangzhou China Guangdong Provincial Key Laboratory of Reproductive Medicine Guangzhou China Department of Acupuncture the Third Affiliated Hospital Beijing University Of Chinese Medicine BeijingChina¯ These authors contributed equally to this worka Current address The First Affiliated Hospital of Sun Yatsen University Guangzhou Chinab Current address Reproductive Medicine Center The First Affiliated Hospital Sun Yatsen UniversityGuangzhou Guangdong China 3509437qqcom DL Xuyanwenlivecn YXAbstractObjectiveTo study the role of selected serum inflammatory cytokines and berberine in the insulin signaling pathway among women with polycystic ovary syndrome PCOSMethodsSelected serum inflammatory cytokines were analyzed in the p cells which were interfered by berberine from infertile women who were to be treated with In Vitro FertilizationIVF Intracytoplasmic Sperm InjectionEmbryo Transfer icsiet Among them patientshad PCOS infertility and were nonPCOS patients whose infertility resulted from fallopian tube and male factors The elisa method was used to detect the changes in the expression levels of inflammatory factors in the cells The correlations between the seruminflammatory cytokine expression levels and the corresponding clinical hormones were analyzed The changes in the expression mRNA and protein levels of the serum inflammatorycytokines were studied by realtime quantitative PCR and protein printing Fluorescencemicroscope and flow cytometry were used to detect the glucose uptake capacity of ovariangranulosa cells in PCOS patients under the action of insulin after berberineResultsIn the PCOS group IL17a P IL1Ra P00001 and IL6 P were significantly higher than those in the nonPCOS group In the nonPCOS group AMH level wasnegatively correlated with inflammatory cytokines IL17a r 0819P IL1a r a1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Kuang H Duan Y Li D Xu Y Ai W Li W The role of serum inflammatorycytokines and berberine in the insulin signalingpathway among women with polycystic ovarysyndrome e0235404 101371journalpone0235404Editor Meijia Zhang China Agricultural UniversityCHINAReceived November Accepted June Published August Copyright Kuang 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 relevant data arewithin the manuscript and its SupportingInformation filesFunding This work was supported by theOutstanding talents funding project of HeilongjiangUniversity of Chinese Medicine China [grantnumber 2018JC05] the Foundation for youngacademic leaders of Heilongjiang University ofChinese Medicine China [grant number2018RCD08] and the Foundation for HeilongjiangProvince Postdoctoral Research Startup ChinaPLOS ONE 101371journalpone0235404 August PLOS ONE 0c[grant number LBHQ18119] the Project ofUndergraduate Colleges and Universities YouthInnovation Talents by Education Department ofHeilongjiang Province China [grant numberUNPYST2018227] the Natural ScienceFoundation of Heilongjiang Province China [grantnumber H2016083]Competing interests The authors have declaredthat no competing interests existThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway0716P IL1b r 0678P IL2 r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantly correlated with the levels of the examined inflammatory cytokines Berberine significantlyreduced the expression level of mTOR mRNA P and increased the expressionlevel of IRS1 mRNA P in the PCOS granule cellsConclusionIn this study we find that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 cause women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors alter their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroyingthe normal ovulation and fertilization system of women leading to polycystic ovary syndrome characterized by menstrual thinning and abnormal ovulation Berberine can improvethe sensitivity of insulin by regulating the signal pathway of insulin receptor substrate1IRS1 and mammalian target of rapamycin mTOR in PCOS patients and achieve a therapeutic effect of treating PCOS IntroductionPolycystic ovary syndrome PCOS is the most common endocrinopathy affecting reproductive aged women It affects reproduction infertility irregular menstruation hirsutism etcmetabolism insulin resistance impaired glucose tolerance etc and psychological characteristics anxiety depression and deterioration in quality of life [] Berberine BBR as a quaternary ammonium salt extracted from plants such as Coptis chinensis and Phellodendronchinensis is currently used in the treatment of diabetes hyperlipidemia and PCOS [] Recentstudies have found that berberine has good hypoglycemic and hypolipidemic effects and is aneffective insulin sensitizer Berberine reduces the synthesis of steroid hormones and theexpression of ovarian aromatase through the action on the hypothalamuspituitaryovarianaxis HPOA improves the insulin resistance status of PCOS patients reduces body weightinduces ovulation and regulates menstruation thereby increasing pregnancy rate and livebirth rate [] Clinical observations have demonstrated that even with longterm use of berberine its side effects are transient and mild suggesting that BBR is safe to use in PCOSpatients and a very promising plantbased compound for treating PCOS patients []Patients with PCOS have been found to be under a chronic lowgrade inflammation statusincluding high levels of leukocytes and disorder of the proinflammatory cytokines [] Interleukin IL6 is a multipotent cytokine that mediates inflammatory response by controllingcell differentiation migration proliferation and apoptosis thus playing a role in the development of insulin resistance [] IL17a is the signature cytokine secreted by the Th17 CD4ve T cell subset Activation of Th17type responses is important not only for host immunecontrol of extracellular bacterial and fungal infections but also associated with chronic inflammation and autoimmunity [] The IL1RA protein is a naturally occurring antagonist of proinflammatory cytokines These proinflammatory cytokines are involved in the underlyingmechanism of various chronic inflammatory conditions [] Therefore we hypothesize thatinflammatory factors are one of the important factors influencing the formation of PCOS andberberine may be an important drug that regulates PCOS inflammatory factorsPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayAntiMu¨llerian hormone AMH is an indicator of a patients ability to respond quicklyand efficiently to ovarian reserve In women AMH is produced by the granulosa cells of theovarian follicles and its secretion begins at puberty and lasts until menopause [] PCOS ischaracterized by hyperandrogenism and follicular blockade These two characteristics may bedue to an imbalance between AMH and follicle stimulating hormone [] Circulating insulinlevels in patients with PCOS increase thereby inducing follicular developmental disorderswhich in turn lead to ovarian polycystic ovary formation and higher than normal AMH HighAMH is one of the indicators of stubborn anovulation [] Research has shown that serumAMH level is relatively stable throughout and between menstrual cycles [] More and morestudies have used AMH as a biomarker for PCOS []In the present study we examined the effect and mechanism of serum inflammatory cytokines including IL6 IL17a IL1RA etc on insulin sensitivity of PCOS In addition weassessed whether berberine can improve insulin sensitivity of PCOS by antagonizing the proinflammatory effect of serum inflammatory cytokines Materials and methods SampleSerum samples and granulosa cells were collected from infertile women who were to betreated with In Vitro Fertilization IVF Intracytoplasmic Sperm InjectionEmbryo Transfericsiet Among them patients had PCOS infertility and were nonPCOS patientswhose infertility resulted from fallopian tube and male factors The nonPCOS women wererequired to have regular menstrual cycles The essays were analyzed by Guangdong ProvincialKey Laboratory of Reproductive Medicine at the first affiliated hospital of Sun YatSen University Data collection time ParticipantsInclusion criteria age Diagnosis of PCOS according to Rotterdam StandardRotterdam Diagnostic Criteria for PCOS Ovarian Ovulation Disorder Manifests Oligomenorrhea or Amenorrhea Clinically or biochemically determined androgen level increasesmore than 2nmolL or clinically manifested hirsute acne excluding Kaohsiung caused byother diseases Ovarian morphology showed polycystic changes Only of the above items can be met IVFET treatmentExclusion criteria Having orally taken drugs in the past three months that affect the results such as contraceptives or other hormone drugs insulin sensitizers and lipidlowering drugs Suffering from other androgen excess related diseases including congenital adrenal hyperplasia with hydroxylase deficiency androgen secreting tumor excessive use of androgenproducing drugs Cushing syndrome severe insulin resistance thyroid dysfunction andhyperprolactinemia Having a history of anic diseases of heart lung liver kidney and other important ansor patients with mental diseases and other reasons that may interfere the present studyoutcomesPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathway Ethics statement The study was approved by the ICE for Clinical Research andAnimal Trials of the First Affiliated Hospital of Sun Yatsen University reference [] No and was conducted in accordance with the ethical standards of the Declaration ofHelsinki All participants have signed written informed consent and there were no minors ReagentBerberine was provided by Yuanye BioTechnology Co Ltd Shanghai China2NBDG N13195 Invitrogen DMEMF12 Gibco and Fetal bovine serumSV3008702 Hyclone were purchased from Thermo Fisher Scientific Waltham USA Thefollowing is a list of used reagents and their manufacturers informationRealtime quantitative PCR kits ROCHE CAT Reverse transcriptase PROMEGA CAT M1701RIPA pyrolysis fluid yantian Bi CAT P0013BBcl2 abcam CAT ab196495BAD abcam CAT ab90435BAX abcam CAT ab182733ACTINβ abcam CAT sc70319 Methods Granulosa cell acquisition and grouping The follicular fluid was centrifuged at rpm for minutes at room temperature The supernatant was discarded and the precipitate after centrifugation was resuspended by phosphate buffer saline PBS The cell suspension was slowly centrifuged to percoll level at room temperature with 1800rpm for 10minAfter centrifugation the white floc was sucked out and washed with PBS three times at1200rpm for 5min at room temperature We discarded the supernatant and added typeIV collagenase and blew it evenly Digestion was performed at ËC for 1520min We addedthe same amount of culture solution to stop digestion and blew it evenly The filtrate was filtered by micron cell filter and centrifuged at room temperature with 1000rpm for 4minThe supernatant was discarded the cells were suspended again with PBS 3ml erythrocytelysate was added to mix well and the mixture was allowed to stand at ËC for 10min It wasthen centrifuged at 1000rpm for 4min at room temperature Some white precipitate whichwas verified as granulocytes were observed after centrifugation After the seed plate of PCOSwomen granulosa cells was seeded the fluid was changed once hours After hours solvent or berberine was added for continuous intervention for hours which led to the PCOScontrol group and PCOS berberine intervention group Detection of serum inflammatory cytokines The Merck luminex testing platformwas used to detect serum cytokines according to the manufacturers instructions Realtime quantitative PCR At the end of berberine treatment Trizol method wasused to extract total RNA in the cells which was then used to reverse transcription of mRNATotal RNA was isolated from cells using Trizol regent Takara BioInc Tokyo Japan and I mgmRNA was reverse transcribed to cDNA using a reverse transcription Takara Bio Inc TokyoJapan and subjected to quantitative PCR which was performed with the BioRad CFX96Touch TM RealTime PCR Detection System BioRad CA using iQ TM SYBR Green Supermix BioRad CA and threshold cycle numbers were obtained using BioRad CFX ManagerSoftware Real time PCR was carried out using the following conditions min at ËC min at ËC and cycles of s at ËC min at ËC using 1μl of cDNA reverse transcribed as mentioned above Quantifast SYBR green PCR kit Qiagen Cat No and500nM of forward and reverse primers The following primers were usedPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayβactin GCCGTTCCGAAAGTTGCCTT GAGCGCGGCGATATCATCAAMPK ACAGCCGAGAAGCAGAAACA TTGCCAACCTTCACTTTGCCmTOR CTTAGAGGACAGCGGGGAAG TCCAAGCATCTTGCCCTGAGSTAT3 CTGAAACGGGCTTCAGGTCA TCCAGGGAGAAAGGGAGTCASOCS3 TCTGTCGGAAGACCGTCAAC CCTTAAAGCGGGGCATCGTAIRS1 TCTCTTCCCACGGCGATCTA TGACACTGCGGAAGGAACTCWe used the 2ÎÎCT method to calculate the relative expression level of target genes Western blot experiment After the end of each cell culture the cells were lysedusing RIPA lysate in an ice bath and the protein lysate was collected After the total proteinconcentration was determined by BCA method polyacrylamide gel electrophoresis was carried out according to the standard of μg of total protein per group After the electrophoresisthe protein in the gel was transferred to the polyvinylidene fluoride PVDF membraneblocked with skim milk powder and then incubated with the corresponding dilutedprimary antibody at ËC overnight and finally labeled with the corresponding horseradish peroxidase After incubation for one hour at room temperature the ECL luminescentsolution was added and detected by autoradiography using a Biored gel imaging systemImmunoreactive protein bands were visualized with enhanced chemiluminescence ECL on aChemiDoc MP Imaging System Blots were scanned and quantified with the Image analysissoftware BioRad Image Lab All specific protein band densities were normalized to βactin amounts Detection of glucose uptake capacity in granulosa cells Granulosa cells of bothnonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granulosacells were randomly divided into two groups one group was cultured normally and the othergroup was added with berberine with the final concentration of 100μM After 24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of100nM were replaced at ËC After 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and the difference in fluorescence density between different groups was compared by using flowjov10 Statistical methodsData were represented by mean±standard deviation SD and SPSS statistical softwarewas used for analysis Analysis of variance ANOVA was used to test betweengroup differences and the pvalues were corrected by Bonferroni p value less than was considered statistically significant Results Analysis of clinical baseline indicators of PCOS group and nonPCOSgroupAs shown in Table compared with the nonPCOS group the PCOS group had significantlyhigher BMIP LHP00001 LHFSHP00001 FPGP AMHP00001 the number of follicles obtainedP00001 ICSI mature eggsP and thenumber of normal fertilization P FSH levels were significantly lower in the PCOSgroup than those in the nonPCOS group P Detection of serum inflammatory cytokinesAs shown in Table IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group IL10 P IL13PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Comparison of clinical indicators between PCOS and nonPCOS groupsnonPCOS n PCOS n PvalueAge years oldMenarche ageSBP mmHgDBPmmHgPulse sminBMI kgm2FSH mIUmlLH mIUmlLHFSHE2 pgmlPRL ngmlT nmolLFPG mmollAMHngmleggs obtainedICSI mature eggsNormal fertilized eggsMeanSDMedianMeanSDMedian101371journalpone0235404t001Table Comparison of serum inflammatory cytokines between the PCOS and nonPCOS groupsIL10 pgmlIL13 pgmlIL17a pgmlIL1Ra pgmlIL1α pgmlIL1β pgmlIL2 pgmlIL6 pgmlIL8 pgmlTNFα pgml101371journalpone0235404t002nonPCOSn ±±±±±±±±±±±PCOSn ±±±±±±±±±PvalueP IL1α P IL1β P IL8 P and TNFα P inthe two groups was not statistically significantly different Correlation analysis between serum inflammatory cytokines anddiagnostic clinical indicatorsWe examined the correlation between serum inflammatory cytokines and clinical serum hormones and other levels in PCOS and nonPCOS patients In the nonPCOS group AMH levelwas negatively correlated with inflammatory cytokines IL17ar 0819P IL1ar 0716P IL1br 0678P IL2r 0765P and IL8r 0705P However in the PCOS group AMH levels were not significantlyPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaycorrelated with the levels of these inflammatory cytokines Among the diagnostic clinical indicators except AMH level there was no significant correlation between other clinical indicatorsand the expression level of inflammatory cytokines in the nonPCOS group and the PCOSgroup Tables Table Correlation analysis of BMI FSH and other inflammatory cytokines in the nonPCOS groupnonPCOSBMIIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarP101371journalpone0235404t003FSHPrLHLHFSHrPrTable Correlation analysis of E2 PRL and other inflammatory cytokines in the nonPCOS groupnonPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarPrPrPr101371journalpone0235404t004Table Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the nonPCOS groupnonPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFarAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t005PPPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayTable Correlation analysis of BMI FSH and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαBMIFSHLHLHFSHrPrPrPr101371journalpone0235404t006Table Correlation analysis of E2 PRL and other inflammatory cytokines in the PCOS groupPCOSE2PRLTFPGIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrPrPrPr101371journalpone0235404t007PPTable Correlation analysis of AMH the number of follicles obtained and other inflammatory cytokines in the PCOS groupPCOSIL10IL13IL17aIL1raIL1aIL1bIL2IL6IL8TNFαrAMHPeggs obtainedICSI mature eggsNormal fertilized eggsrPrPrP101371journalpone0235404t008 Detection of protein imprinting in granulosa cellsIt can be seen from Fig 1A that there was no significant difference in the expression levels ofAMPK P PAMPK P and ACC P protein in PCOS groupPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Granule cell protein expression level B Gray expression analysis of protein expression A Representativewestern blot images and B Summary of expression changes among relative genes Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than wasconsidered statistically significant101371journalpone0235404g001PLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayFig A Fluorescence density map of sugar uptake capacity of granular cells B Fluorescence density analysis Granulosa cells ofboth nonPCOS and PCOS patients were obtained in vitro for in vitro culture PCOS granule cells were randomly divided into twogroups one group was cultured normally and the other group was added with berberine with final concentration of 100μM After24h the serumfree culture solution containing 2nbdg with a final concentration of 50μM and insulin of 100nM was replaced at ËCAfter 1h incubation the difference in green fluorescence intensity of 530nm was compared under fluorescence microscope and thedifference in fluorescence density between different groups was compared by using flowjov10 Data were represented by mean±standard deviation SD and SPSS statistical software was used for analysis Pvalue less than was considered statisticallysignificant101371journalpone0235404g002compared with the nonPCOS group while the expression levels of mTORP00001 PmTORP00001 and STAT3P00001 were significantly increased At the same time theprotein expression level of IRS1P was significantly decreased Detection of granulated cell sugar uptake capacityAs shown in Fig 2A and 2B compared with granulosa cells obtained from the nonPCOSgroup the fluorescence intensity of granulosa cells in PCOS group was significantly reducedP00001 and the fluorescence density in the cells increased significantly after berberineP00001 Detection of expression of key factors of glucose metabolism ingranulosa cellsAs shown in Fig 1B the increased expression level of AMPK mRNA after the addition of berberine was significantly different from that in the PCOS group P Compared with thenonPCOS group the level of mTOR in PCOS granule cells was significantly increasedP and the expression level of IRS1 mRNA was significantly reduced P Berberine significantly reduced the expression level of mTOR mRNA in PCOS granule cellsP and increased the expression level of IRS1 mRNA in PCOS granule cellsP Discussion The correlation between selected serum inflammatory cytokines andPCOSThis study finds that IL17a P IL1Ra P00001 and IL6 P in the PCOSgroup were significantly higher than those in the nonPCOS group Studies have shown thatPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayPCOS associated with lowgrade chronic inflammation interleukin17A IL17A is one of themajor members of proinflammatory cytokines and is mainly involved in the development ofinflammatory and autoimmune diseases Within the immune process the genetic factors ofIL17A play a major role in the susceptibility of PCOS [] The level of IL1Ra is significantlyincreased in patients with PCOS which can lead to decreased insulin resistance and blood glucose metabolism causing obesity and metabolic syndrome in PCOS patients [] Tarkun [] studied the serum levels of IL6 in patients with PCOS higher than the normal control group and IL6 was significantly associated with insulin resistance and fasting blood glucose The increase of IL6 level is an important inflammatory factor inducing the occurrenceof PCOS AMH levels and inflammatory cytokines in PCOS patientsThis study finds that in the nonPCOS group AMH level was negatively correlated withinflammatory cytokines such asIL17a r 0819P 0004IL1a r 0716P 0002IL1b r 0678P 0031IL2 r 0765P 001IL8 r 0705P However in thePCOS group AMH levels were not significantly correlated with the levels of various inflammatory cytokineStudies have found that AMH plays a key role in the anovulatory mechanism of PCOS []AMH can promote the growth of preantral follicles to the small sinus stage in vitro whileincreasing the production of steroid hormones and paracrine factors as well as oocyte maturation AMH is a key follicular paracrineautocrine factor that has a positive effect on preantralfollicle survival and growth [] The alienation of AMH function affects the regulation of follicles and promotes the increase of small follicles in the body Due to the abnormal expressionof the number and function of follicles the regulation of the ovary on the matrix is weakenedand the follicular irregular growth is induced without follicular atresia The expression of normal levels of inflammatory cytokines may promote normal cell apoptosis Elevated inflammatory cytokines IL17aIL1aIL1bIL2IL8 may disrupt the ovarian follicle atresiaweakening the ability of apoptosis and inhibiting the maturation of oocytes In turn the AMHfunction is out of control and the correlation between AMH and inflammatory factor levels isinducedIt can be concluded that AMH levels under normal conditions can effectively regulate thelevel of inflammatory factors and promote the bodys own metabolism and reproductive function The expression of high AMH levels in PCOS patients causes a loss of correlation withinflammatory factors The abnormal level of AMH increases the level of inflammatory factorsresulting in a continuous low concentration of systemic inflammatory state in the humanbody leading to metabolic diseases such as insulin resistance and glycolipid metabolism andreproduction disfunction The effects of Berberine on granulosa cells insulin resistanceOur study finds that berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients Berberine can increase insulin sensitivity in patients reduce blood sugar improveinsulin resistance and achieve the therapeutic effect of treating PCOS Berberine is the mainactive ingredient of Chinese herbal medicine Coptis Cork and Mink and has been used totreat diarrhea metabolic disorders and infertility []Berberine regulates glucose metabolism through a variety of mechanisms and signalingpathways such as increased insulin sensitivity activation of the adenosine monophosphateactivated protein kinase AMPK pathway regulation of the intestinal microbiota andPLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwayinhibition of liver sugar Exogenous stimulates peripheral cell glycolysis promotes intestinalglucagon like protein1 GLP1 secretion upregulates liver lowdensity lipoprotein receptormRNA expression and increases glucose transporter []PCOS is a multifactorial endocrine disease that affects reproduction and metabolism Studies in dheainduced PCOS mouse models have found that mTOR and pmTOR serine2448are highly expressed in the ovary In normal mice in another study mTOR protein levels inthe corpus luteum of PCOS patients were the same as in healthy women However comparedwith healthy patients receiving insulin stimulation the expression of mTOR protein in the corpus luteum of PCOS patients is less so another link between PCOS and mTOR is metabolicdisorders during PCOS [] The combination of activation of the mTOR pathway and oxidative damage to DNA that causes replication stress is a particularly effective factor in promoting aging and aging [] Berberine inhibits the level of DNA damage signals [] It canreduce the expression and activation of γH2AX including tumor A and TK6 cells as wellas normal WI38 cells or mitogenic human lymphocytes [] It can also reduce intracellularreactive oxygen species and mitochondrial transmembrane potential which is a sign of mitochondrial activation [] However these drugs also significantly reduce phosphorylationlevels of Ser235 of ribosomal S6 protein RpS6 Ser2448 of mTOR and Ser65 of 4EBP1These data indicate that the reduction of mTOR S6K signal which in turn reduces the translation rate and is accompanied by a reduction in oxidative phosphorylation which leads to areduction in ROS and a reduction in oxidative DNA damage [] The mechanism by whichBRB exerts these effects may be by targeting mitochondriaThe protective effect of berberine on islet function may involve two pathways On the onehand berberine can improve insulin sensitivity in patients with PCOS with IR as the core []On the other hand berberine can promote insulin secretion from islet cells of PCOS patientsand protect islet cells through antioxidant activity [] Kong [] found that themolecular mechanism of berberine on insulin resistance by upregulating the expression ofinsulin receptors confirmed that berberine can reduce fasting blood glucose and fasting seruminsulin At the same time they also found that berberine activated its promoter through protein kinase CPKCdependent increasing insulin receptor mRNA and protein expressionIn fat and muscle cells berberine may stimulate cells by upregulating glucose transporter type GLUT1 expression and inhibiting retinol binding protein4 RBP4 At the same timeberberine also has a certain effect on the phosphorylation of IRS1 which can finally alleviateinsulin resistance []In other words berberine can inhibit inflammatory factor levels increase AMPK mRNAand IRS1 mRNA levels and reduce the level of mTOR mRNA in granulosa cells of PCOSpatients The mechanism by which berberine regulates the metabolism of glycolipids is not tostimulate the patient to increase insulin secretion but to increase the glucose consumption ofthe patients cells and improve glucose tolerance At the same time by increasing insulin sensitivity in patients further lowering blood sugar and improving insulin resistance and achievingthe effect of treating PCOS ConclusionIn this study we found that the elevated levels of serum inflammatory factors IL17a IL1Raand IL6 caused women to be in a subclinical inflammatory state for a long time Abnormalchanges in inflammatory factors altered their original negative correlations with AMH levelsthereby weakening the metabolism of glycolipids promoting insulin resistance destroying thenormal ovulation and fertilization system of women and leading to polycystic ovary syndromecharacterized by menstrual thinning and abnormal ovulation Berberine can improve thePLOS ONE 101371journalpone0235404 August PLOS ONE 0cThe role of serum inflammatory cytokines and berberine in the insulin signaling pathwaysensitivity of insulin by regulating the signal pathway of mTOR mRNA and IRS1 mRNA inPCOS patients and achieve the therapeutic effect of treating PCOSSupporting informationS1 Raw ImagesPDFAuthor ContributionsConceptualization Yuwei Duan Wei LiData curation Wei LiFormal analysis Wei LiFunding acquisition Hongying KuangSoftware Manqi ShaoWriting original draft Hongying Kuang Yuwei Duan Wenxia AiWriting review editing Dan Li Yanwen Xu Wenxia Ai Wei Li Ying Wang Sha LiuMushan Li Xiaoqiu Liu Manqi ShaoReferencesTeede H Deeks A Moran L Polycystic Ovary Syndrome A complex Condition with PsychologicalReproductive and Metabolic Manifestations That Impacts on Health Across the Lifespan BMC Med ZHANG F MA T CUI P Diversity of the Gut Microbiota in DihydrotestosteroneInduced PCOSRats and the Pharmacologic Effects of Diane35 Probiotics and Berberine [J]Frontiers in microbiology IMENSHAHIDI M HOSSEINZADEH H Berberine and barberry Berberis vulgaris A clinical review [J] LI M F ZHOU X M The Effect of Berberine on Polycystic Ovary Syndrome Patients with Insulin Resistance PCOSIR A MetaAnalysis and Systematic Review [J] LI W LI D KUANG H Berberine increases glucose uptake and intracellular ROS levels by promoting Sirtuin ubiquitination [J] Biomedicine pharmacotherapy Biomedecine pharmacotherapie RONDANELLI M INFANTINO V Polycystic ovary syndrome management a review of the possibleamazing role of berberine [J] Benson S Obesity Depression and Chronic Lowgrade Inflammation in Women with PolycysticOvary Syndrome Brain Behav Immun 101016jbbi200707003PMID Rehman K Akash MSH Liaqat A Role of Interleukin6 in Development of Insulin Resistance andType Diabetes | 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 |
"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 |
"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 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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 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Inflammation plays a leading role in the pathogenesis of nephrolithiasis The association of the dietary inflammatory index DII with urinary lithogenic factors is unclear This study aimed to evaluate the relation of DII to urinary risk factors of kidney stones formationResults Of participants n n n n and n had hyperoxaluria hypercreatininuria hypercalciuria hyperuricosuria hypocitraturia respectively There was a significant increasing trajectory in urinary calcium uric acid and creatinine as well as a decreasing trend in urinary citrate across tertiles of DII score all P After multivariate adjustment for energy intake age physical activity and body mass index high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI Ptrend hypercalciuria OR 95CI Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend No association was identified between DII and hyperoxaluriaKeywords Dietary inflammatory index Kidney stones Hypercalciuria Hypocitraturia Hyperoxaluria Hyperuricosuria HypercreatinuriaIntroductionNephrolithiasis is one of the most prevalent urologic disorders and impose a substantial burden on human health globally [] The high recurrence rate of kidney stones is yet unsolved [ ]Thus there is an urgent need to target modifiable risk factors to prevent the development and recurrence of renal stonesHigher urinary excretions of oxalate calcium creatinine and uric acid as well as lower excretions of citrate are potential modifiable a0 urinary lithogenic risk factors Correspondence mina_mirzaei101yahoocom Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box IranFull list of author information is available at the end of the involved in the formation of kidney stones [] Inflammation is also another mechanism which plays a leading role in the pathogenesis of nephrolithiasis [] Dietary modifications toward decreasing inflammation may have a potential to prevent kidney stones or their recurrence Several micronutrients or foods such as magnesium vitamin E vitamin C carotenoids fruits and fish had an antiinflammatory impact [] In contrast simple sugars red meats highfat dairy products and refined grains are associated with elevated inflammatory markers [] Nevertheless nutrients or foods are not consumed separately but as part of the whole diet [] The Dietary Inflammatory Index DII is developed to measures the overall inflammatory potential of diets [] which has been recognized to be related to the biomarkers of inflammation [] A proinflammatory diet has 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 0cMaddahi a0et a0al BMC Res Notes Page of been found to be related to the reduced kidney function [] However there is no study investigating the relation of DII to urinary lithogenic factors Therefore this study aimed to assess the association of DII with hypercalciuria hypocitraturia hyperoxaluria hyperuricosuria and hypercreatinuria in patients with nephrolithiasisMain textMethodsSubjectsThis crosssectional study was performed on a total of stone former men aged a0years in Tehran Iran in Participants were recruited from the Urology Research Center of Sina Hospital Tehran Iran Inclusion criteria for this study were having a history of kind stone formation and age ¥ a0years People with a history of thyroid disease fatty liver disease malignancy stroke diabetes cardiovascular disease and hypertension were excluded Participants who were on medications such as corticosteroids diuretics anticancer drugs multivitamins potassium citrate calcium and vitamin D or C supplements were not eligible for this study Furthermore all alcohol drinkers and drugs abusers were excluded Patients were included in the study after signing written informed consentsDietary assessmentUsual food intake of patients during the previous year was measured by a validated semiquantitative 168item food frequency questionnaire FFQ[] DII was calculated using the method reported by Shivappa et a0al [] The DII is based on scientific papers scoring food parameters based on whether they elevated reduced or had no impact on six inflammatory biomarkers [Creactive protein interleukin IL1 beta IL10 IL4 IL6 and tumor necrosis factoralpha As mentioned Shivappa et a0 al calculated DII according to the food parameters As dietary patterns of different populations are different with each other some food parameters used in the study by Shivappa may not be available in different FFQs Hence researchers calculate DII according to available foods in the FFQ by modification of the method used by Shivappa et a0al [] In the current study the DII score was calculated using the corresponding food parameters available from the FFQ used in our study This approach has been used broadly in the previous studies [] The DII score was calculated with the use of the corresponding nutrients or food parameters available from the FFQ including energy protein total fat carbohydrate dietary fiber monounsaturated fatty acids n3 fatty acids n6 fatty acids polyunsaturated fatty acids saturated fatty acids cholesterol trans fatty acids vitamin A thiamin niacin riboflavin Vitamin B6 folate vitamin B12 vitamin E vitamin C Vitamin D bcarotene iron magnesium zinc selenium as well as caffeine onion greenblack tea paper and garlic The inflammatory effect scores for dietary components used for calculation of DII in this study are reported in Additional file a0 Table a0S1 To calculate the DII score for each participant the mean intake of each nutrient or food parameter was standardized by subtracting mean global intake of food items from the actual individuals intake and dividing it by the global SD to create a zscore Zscore is used to express an individuals exposure relative to the standard global exposure This approach both anchors the individuals exposure to a robust range of dietary patterns in a variety of cultural traditions and obviates completely the problem of noncomparability of units because the a0Zscores is independent of the units of measurement These zscores then were converted to proportions and centered by multiplying values by and subtracting to normalize the scoring system and to avoid skewness The centered percentile values for food items were then multiplied by the corresponding food itemspecific inflammatory effect scores to obtain the food itemspecific DII scores Calculation of DII for carbohydrate intake in a participant in our study as an example for DII calculation is presented in Additional file a0 Table a0S2 similar approach was followed for the calculation of DII for other nutrients Information about global daily mean intake standard deviation for global intake and overall inflammatory effect score of all nutrientsfood items used for DII calculation is reported in the study by Shivappa et a0al [] The overall DII score for each individual was calculated by summing food itemspecific DII scores [] Higher DII scores indicate a more proinflammatory diet while lower DII scores indicate a more antiinflammatory dietMeasurements of a0study outcomesThe 24h urine samples were collected from all participants and urine was analyzed using an AutoAnalyzer as described previously [] Hyperoxaluria a0 was a0 defined a0 as a0 the urinary oxalate Ë a0 mgday hypocitraturia as a0urinary citrate of a0mgday hyperuricosuria as urinary uric acid over a0 gday hypercreatininuria as urinary creatinine of Ë a0 mgday and hypercalciuria as a0a urinary calcium ¥ a0mgday []Measurement of a0other variablesGeneral Information was obtained using interview Physical activity was measured using of a0 the a0 International a0Physical a0Activity a0Questionnaires a0IPAQ [] Body weight was measured in minimal clothing after removal of shoes by a digital scale Seca Germany with a precision about a0kg Height of individuals was assessed in 0cMaddahi a0et a0al BMC Res Notes Page of standing position without shoes using a calibrated stadiometer Seca Germany to the nearest a0cm BMI was calculated as weight divided by the square of height kgm2Statistical analysesDII was categorized into tertiles T1 to T2 to T3 to Analysis of variance ANOVA and Chi square tests were used to compare continuous and nominalordinal variables across tertiles of DII respectively Continuous variables are reported as mean ± SE and nominalordinal variables as frequency Odds ratio OR and confidence interval CI for the relation of DII to study outcomes was calculated using the logistic regression analysis Statistical significance was set at p for all tests All analyses were undertaken using the statistical Package for Social Science Version SPSS Inc Chicago IL USAResultsParticipants in the highest tertile of the DII had significantly higher total daily energy intake P and lower physical activity P than those in the other tertiles There was a significant increasing trajectory in urinary calcium P uric acid P and creatinine P and a decreasing trend in urinary citrate P across tertiles of DII score Table a0DII score and a0urinary lithogenic factorsIn the crude model it was found that higher adherence to the DII was significantly related to the increased odds of hypercreatininuria OR 95CI Ptrend hypercalciuria OR 95CI Table Characteristics of a0the a0study participants across a0tertiles of a0the a0DII scoreAge yearHeight cmWeight kgBMI kgm2Physical activity scoreEnergy intake kcaldayUrinary creatininekg weight mgdaykgTotalN ± ± ± ± ± ± ± ± ± ± ± Dietary inflammatory index scoreTertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± P value Urinary citrate mgdayUrinary oxalate mgdayUrinary uric acid gdayUrinary calcium mgdayJob status Engineerphysician Clerk Student Teacher Selfemployed Retired Worker UnemployedMarital status Married SingleEducation level Illiterate Diploma University degreeCategorical variables are presented as frequency n and continuous variables as mean ± SE Oneway ANOVA was used for continuous variables and persons Chi square test for categorical variables 0cMaddahi a0et a0al BMC Res Notes Page of Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend After multivariate adjustment for energy intake age physical activity and BMI high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI hypercalciuria OR 95CI Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend The relation of DII to hypercreatininuria hyperoxaluria and hyperuricosuria was not significant after adjustment for carbohydrate fiber and protein intake Table a0 Ptrend adjustment for covariates dietary intakes of protein and fiber were slightly related to the decreased odds of hypocitraturia Protein OR 95CI fiber OR 95CI and hypercalciuria Protein OR 95CI fiber OR 95CI while the intake of protein and fiber was not to associated with hypercreatininuria hyperoxaluria and hyperuricosuriaDiscussionWe revealed that in stone former men a diet with a high DII is significantly related to the increased odds of having hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia but not to hyperoxaluriaIt has been confirmed that kidney stone formers could be susceptible to recurrence in stones formation We also evaluated the association of dietary protein and fiber intake on urinary risk factors Table a0 After Table Univariate and a0 multivariate logistic regression models for a0 the a0 relation of a0 DII score to a0 urinary risk factors of a0kidney stone formationDietary inflammatory index scoreModel Crude modelModel Model Model Odds ratio CIOdds ratio CIOdds ratio CIOdds ratio CIHypercreatininuria T1 T2 T3 P value for trendHypocitraturia T1 T2 T3 P value for trendHyperoxaluria T1 T2 T3 P value for trendHyperuricosuria T1 T2 T3 P value for trendHypercalciuria T1 T2 T3 P value for trend Model adjusted for energy intakeModel additionally adjusted for age BMI and physical activityModel adjusted for carbohydrate fiber and protein intake 0cMaddahi a0et a0al BMC Res Notes Page of Table Multivariate logistic regression models for a0the a0relation of a0dietary fiber and a0protein intake as a0continues variable to a0urinary risk factors of a0kidney stone formationFiberOdds ratio CIProteinOdds ratio CIModel Model Model Model HypercreatininuriaHypocitraturiaHyperoxaluriaHyperuricosuriaHypercalciuriaModel adjusted for energy intakeModel additionally adjusted for age BMI and physical activitybecause of unhealthy dietary patterns [] Inconsistent with our finding a study did not report any significant difference in creatinine across tertiles of DII in subjects with chronic kidney disease [] A randomized controlled trial a0 study by Noori et a0 al [] on recurrent stone formers showed that a a0DASH diet which in contrast to a diet with a high DII is featured by a high intake of whole grains fruits lowfat a0 dairy products and vegetables and a low intake of total fat cholesterol saturated fat meat and refined grains is significantly associated with a decrease in calcium oxalate supersaturation and an increase in citrate excretion Moreover another study reported that greater adherence to the Mediterranean a0dietary pattern a0is related to the reduced risk for incident kidney stones [] The relationship between systemic inflammation and nephrolithiasis has been identified previously [] Since both DASH and Mediterranean diets attenuate a0 inflammation [ ] the protective effects of these dietary patterns on kidney stones formation may be mediated at least partly by reducing systemic inflammation A crosssectional study conducted on diabetic patients also reported that higher intake of vegetable and fish dietary pattern is related to a lower creatinine rates [] Vegetables and fish as components of DII are identified to have antiinflammatory effects [ ] The DII is a tool to assess the overall impact of a diet on inflammatory potential [] and is associated with markers of systemic inflammation including such as IL6 [] and CRP [] [] IL6 and CRP are two of the inflammatory biomarkers considered in the calculation of DII [] It has been revealed that the DII score is inversely related to the Dietary Approaches to Stop Hypertension Score DASH Mediterranean Diet Score and Healthy Eating Index2010 [ ] Taken together these findings support that a likely mechanism for the relation of DII scores to hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia could be explained by the higher systemic inflammation level among people following a proinflammatory dietSince DII positively depends on protein intake that is also a metabolic risk factor for hypercalciuria hyperuricosuria and hypocitraturia and on contrary dietary fiber has a negative impact on DII and is a factor that can reduce calcium absorption we also adjusted the analysis for protein carbohydrate and fiber intake to differentiate the metabolic impact of DII from its proinflammatory impact It was found that DII is related to hypercalciuria and hypocitraturia independent of dietary intake of protein carbohydrate and fiber however the relationship between DII and hyperuricosuria disappeared showing that this association may be resulted from metabolic effects of DII Nevertheless protein and fiber intake was inversely associated with hypercalciuria and hypercalciuriaConclusionIn conclusion this study found that a diet with high inflammatory property might be unfavorably associated with urinary risk factors of kidney stone formation in men with a history of nephrolithiasisLimitationFirst since the participants of the current study were limited to men our findings may not be generalizable to women therefore it is essential to conduct such a study on women too Third causation cannot be inferred the crosssectional design of the present investigation Finally the calculation of DII by FFQ has a potential recall bias for the evaluation of dietary intake 0cMaddahi a0et a0al BMC Res Notes Page of Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 yAdditional file a0 Table a0S1 Inflammatory effect scores for dietary components used for calculation of DII Table a0S2 calculation of DII for carbohydrate intake in a participant in our study as an example for total DII calculationAbbreviationsIPAQ International physical activity questionnaires DII Dietary inflammatory index PUFAs Polyunsaturated fatty acids CRP C reactive protein FFQ Food frequency questionnaire ANOVA Analysis of variance OR Odds ratio CI Confidence interval BMI Body mass indexAcknowledgementsWe would like to thank the Tehran University of Medical Sciences This work was supported by Tehran University of Medical Sciences Grant ID13951046Authors contributionsSMKA designed the research and collected the samples NSM and SHA wrote the paper HY and MSY analyzed data KhM conducted research and had primary responsibility for final content All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsThe data are not publicly available due to containing information that could compromise the privacy of research participantsEthics approval and consent to participateEthics approval for the study protocol was granted by The Human Ethics Committee of Tehran University of Medical Sciences Grant ID IRTUMSVCRREC13951046 All participants signed written informed consent formsConsent for publicationNot ApplicableCompeting interestsAll authors declared that they have no competing interestsAuthor details Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box Iran Department of Urology Sina Hospital Tehran University of Medical Sciences Tehran Iran Department of Epidemiology and Biostatistics School of Public Health Tehran University of Medical Sciences Tehran Iran Received March Accepted July References Li Y Zhang J Liu H et al Curcumin ameliorates glyoxylateinduced calcium oxalate deposition and renal injuries in mice Phytomedicine Fink HA Akornor JW Garimella PS et al Diet fluid or supplements for secondary prevention of nephrolithiasis a systematic review and metaanalysis of randomized trials Eur Urol Littlejohns TJ Neal NL Bradbury KE Heers H Allen NE Turney BW Fluid intake and dietary factors and the risk of incident kidney stones in UK Biobank a populationbased prospective cohort study European urology focus Ong CN Minerals from drinking water Bioavailability for various world populations and health implications Nutrients in Drinking Water Yagisawa T Chandhoke PS Fan J Metabolic risk factors in patients with firsttime and recurrent stone formations as determined by comprehensive metabolic evaluation Urology Grases F Melero G CostaBauza A Prieto R March J Urolithiasis and phytotherapy Int Urol Nephrol Khan SR Reactive oxygen species inflammation and calcium oxalate nephrolithiasis Translational Androl Urol Bo S Durazzo M Guidi S et al Dietary magnesium and fiber intakes and inflammatory and metabolic indicators in middleaged subjects from a populationbased cohort Am J Clin Nutri Chrysohoou C Panagiotakos DB Pitsavos C Das UN Stefanadis C Adherence to the Mediterranean diet attenuates inflammation and coagulation process in healthy adults the ATTICA Study J Am Coll Cardiol Upritchard JE Sutherland W Mann JI Effect of supplementation with tomato juice vitamin E and vitamin C on LDL oxidation and products of inflammatory activity in type diabetes Diab Care Neale E Batterham M Tapsell LC Consumption of a healthy dietary pattern results in significant reductions in Creactive protein levels in adults a metaanalysis Nutri Res Esmaillzadeh A Kimiagar M Mehrabi Y Azadbakht L Hu FB Willett WC Dietary patterns and markers of systemic inflammation among Iranian women J Nutri Mohseni R Mohseni F Alizadeh S Abbasi S The Association of Dietary Approaches to Stop Hypertension DASH Diet with the risk of colorectal cancer a metaanalysis of observational studies Nutrition and cancer Alizadeh S Djafarian K Alizadeh M ShabBidar S The relation of healthy and Western dietary patterns to the risk of endometrial and ovarian cancers a systematic review and metaanalysis Int J Vitamin Nutrition Res Alizadeh S ShabBidar S Mohtavinejad N Djafarian K A posteriori dietary patterns and risk of pancreatic and renal cancers Nutrition Food Science Mohseni R Abbasi S Mohseni F Rahimi F Alizadeh S Association between dietary inflammatory index and the risk of prostate cancer a metaanalysis Nutr Cancer Shivappa N Steck SE Hurley TG et al A populationbased dietary inflammatory index predicts levels of Creactive protein in the seasonal variation of blood cholesterol study SEASONS Public Health Nutrition Xu H Sjögren P Ãrnlöv J et al A proinflammatory diet is associated with systemic inflammation and reduced kidney function in elderly adults J Nutri Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relative validity of food group intake in a food frequency questionnaire developed for the Tehran lipid and glucose study J Epidemiol Shivappa N Steck SE Hurley TG Hussey JR Hébert JR Designing and developing a literaturederived populationbased dietary inflammatory index Public Health Nutri Bondonno NP Blekkenhorst LC Bird AL et al Dietary inflammatory index and the aging kidney in older women a year prospective cohort study Eur J Nutri Maddahi NS Mirzaei K Aghamir SMK Modaresi SS Yekaninejad MS Major Dietary Patterns and kidney stone formation among Iranian men J Nutri Sci Dietetics Moghaddam MB Aghdam FB Jafarabadi MA Allahverdipour H Nikookheslat SD Safarpour S The Iranian version of international physical activity questionnaire IPAQ in Iran content and construct validity factor structure internal consistency and stability World Appl Sci J Trinchieri A Mandressi A Luongo P Longo G Pisani E The influence of diet on urinary risk factors for stones in healthy subjects and idiopathic renal calcium stone formers Br J Urol Rouhani MH Najafabadi MM Surkan PJ Esmaillzadeh A Feizi A Azadbakht L Dietary inflammatory index and its association with renal function and progression of chronic kidney disease Clin Nutri ESPEN 0cMaddahi a0et a0al BMC Res Notes Page of Noori N Honarkar E Goldfarb DS et al Urinary lithogenic risk profile in sgÃ¥rd R Rytter E Basu S AbramssonZetterberg L Möller L Vessby B recurrent stone formers with hyperoxaluria a randomized controlled trial comparing DASH Dietary Approaches to Stop Hypertensionstyle and lowoxalate diets Am J Kidney Dis Leone A FernándezMontero A de la FuenteArrillaga C et al Adherence to the Mediterranean dietary pattern and incidence of nephrolithiasis in the Seguimiento Universidad de Navarra Followup SUN cohort Am J Kidney Dis Saneei P Hashemipour M Kelishadi R Esmaillzadeh A The Dietary Approaches to Stop Hypertension DASH diet affects inflammation in childhood metabolic syndrome a randomized crossover clinical trial Ann Nutr Metab Hsu CC Jhang HR Chang WT et al Associations between dietary patterns and kidney function indicators in type diabetes Clin Nutr Duda MK OShea KM Tintinu A et al Fish oil but not flaxseed oil decreases inflammation and prevents pressure overloadinduced cardiac dysfunction Cardiovasc Res High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type diabetes Scand J Food Nutri Wood LG Shivappa N Berthon BS Gibson PG Hebert JR Dietary inflammatory index is related to asthma risk lung function and systemic inflammation in asthma Clin Exp Allergy Hodge A Bassett J Shivappa N et al Dietary inflammatory index Mediterranean diet score and lung cancer a prospective study Cancer Causes Control Wirth MD Hébert JR Shivappa N et al Antiinflammatory dietary inflammatory INDEX scores are associated with healthier scores on other dietary indices Nutri Res Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims 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"deltalike protein DLL3 may be related with prognosis inpatients with small cell lung cancer SCLC However this finding remains controversial in small cell lung cancerThis metaanalysis was systematically performed to evaluate the prognostic value of DLL3 in SCLCMethods The PubMed EMBASE and Web of Science databases were retrieved to collect the eligible referencesThrough Stata software we pooled hazard ratios HR with confidence intervals CI by using random orfixedeffects models to evaluate the association between DLL3 and SCLC survival resultsResults A total of interrelated studies including patients were qualified After we removed study theremaining studies including patients were pooled to testify that high expression of DLL3 was an inferiorprognostic for patients with SCLC in Asian populations HR CI I2 p Thepooled results showed that DLL3 might be higher expression in advanced metastasis SCLC in Asian populations RR CI I2 p But the expression of DLL3 was not correlated with sex RR CI I2 p smoking history RR CI I2 p and tumour stage RR CI I2 p Conclusions Our metaanalysis confirms that in Asian populations high expression of DLL3 was a potential poorprognostic biomarker for SCLC and DLL3 highly expressed in advanced stage SCLC in Asian populationsKeywords Deltalike protein DLL3 Prognostic Small cell lung cancer SCLC MetaanalysisIntroductionLung cancer has the highest morbidity and mortality ofall malignant tumours which is one of the most common cancers worldwide [] The major histologic subtypes oflung cancerNSCLC and small cell lung cancer SCLC [] SCLC is an invasive highgrade malignancy withearly development and a bad prognosis Most patientswith SCLC are advanced with widespread metastasiswhen they are diagnosed and systemic chemotherapy islung cancer are nonsmall cell Correspondence ligaofenghl126comDepartment of Thoracic Surgery The Third Affiliated Hospital of KunmingMedical University Yunnan Cancer Hospital Kunming Yunnan Chinathe most effective therapy [] Few therapeutic methodsare available after SCLC relapse and the prognosis is especially poor Therapeutic treatments for patients withSCLC have essentially remained unchanged in recentyears [] Thus further research into the mechanism ofSCLC and the exploration of new therapeutic targets forSCLC is imperativeDeltalike protein DLL3 is a transmembrane protein that promotes the development of neuroendocrinetumours through its reciprocity with the Notch pathway[] It is expressed in cancer tissues in approximately of patients with SCLC and other neuroendocrineis not expressed in nonneuroendocrinecancer but 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 World Journal of Surgical Oncology Page of tumours or normal tissues [] Most studies have shownthat DLL3 is a latent treatment target point for SCLC[] Recently many researches initiate to focus on theprognostic value of DLL3 in SCLC Some of the researchers demonstrated that DLL3 had no correlationwith prognosis in SCLC patients [ ] but other investigators showed that DLL3 was associated with thesurvival of patients with SCLC [] So previous studiesof the prognostic value of DLL3 in SCLC remain controversial we performed this systematic metaanalysis forresolving this controversialPublished s discussing the prognostic value ofDLL3 in SCLC were systematically reviewed in ourmetaanalysis We aimed to include all correlationalstudies to assess the prognostic value of DLL3 andattempted to identify an accurate biomarker to guideprognosis and treatment for SCLC in the futureMethodsSearch strategyThe PRISMA guidelines were followed for our metaanalysis see Additional file [] The literatures werecollected through retrieving the PubMed EMBASE andWeb of Science databases from the initial date to February The search strategy was DLL3 OR deltalike protein all fields AND nonsmall cell lung cancer OR NSCLC OR small cell lung cancer OR SCLC ORLung Adenocarcinoma OR Lung Squamous cell carcinoma OR lung cancer OR lung tumour OR lung neoplasm OR lung carcinoma all fields There was nolimitation on the publication status All eligible studieswere retrieved and the headlines and s of all thereference lists of the reviews or studies were independently filtrated through three authors based on the criteria Differences between two authors were resolved bythe third authors opinionsInclusion criteriaResearches were included by following the standards studies reported the prognostic value of DLL3 in SCLC studies were published as original s studiesreported the data of HR and CI or provided survivalcurve and studies in which the prognostic value wasinvestigated by survival analysis with overall survivalOS diseasefree survival DFS progressionfree survivalRFS ordiseasespecific survival DSS We excluded the animalresearches and other review literaturesPFS relapserecurrencefree survivalData extractionTwo researchers independently extracted the data included the author country edition year sample capacitypatients sex smoking history distant metastasis tumourstage histologic subtype biomarkers scoring methodsboth univariateand multivariatecutoffs value and the data of HR and CI We extracted the multivariate analysis outcome if one study includedanalysisoutcomes When we could not extract the data of HRand CI directly in the but the survival curveswere reported we extracted and digitized the survivalcurves by operating the Engauge Digitizer softwarehttpdigitizersourcefenet and we estimated thedata of HR and CI by the Excel programme files asexploited by Tierney [] When the data of HRand survival curves were not reported we contacted thecorresponding authors of eligible s by email to obtain the original data these s were excluded ifthere was no responseAssessment of study qualityTwo researchers independently used the Quality InPrognosis Studies QUIPS tool to assess risk of bias ofall the publications [] According to the criteria every was evaluated as low risk moderate risk or highrisk by following six different areas study participationstudy attrition prognostic factor measurement outcomemeasurement study confounding and statistical analysisand reporting [] Differences were settled throughdiscussionStatistical analysisTwo authors independently extracted the HR and CI from the original s The HR was used to describe the high DLL3 expression versus low DLL3 expression We took the reciprocal of the HR if the sshowed the low DLL3 expression vs high DLL3 expression We observed that high expression of DLL3 portended a worse prognosis when HR and that HR indicated preferable prognosis For the analysis results p was considered statistically significant Statisticalheterogeneity was assessed by calculating the I2 statistic[] The presence of heterogeneity was indicated whenI2 and then a randomeffects model was appliedto poolthe results [] a fixedeffects model wasemployed to pool the results when I2 [] Weperformed further subgroup or sensitivity analysis toanalyse the heterogeneity The publication bias was estimated by Beggs and Eggers tests when p indicates no publication bias [] Through Stata software we performed this metaanalysis and acquiredthe forest plotsResultsSearch results and study characteristicsUsing the searching strategy described above a total of original documents were identified from the databases with approximately studies remaining after excluding duplicates Afterandscreening thetitles 0cChen World Journal of Surgical Oncology Page of s of the publications publications werenot related to evaluating the prognosis role of DLL3in SCLC Finally we were left with eligible studiesafter screening the full text among which swere included in our final analysis Fig []Ten s were excluded for the following reasonsseven s were review researches and commentaries one did notreport hazard ratios andKaplanMeier curves and s were s anddid not report relevant outcomes The eligible s characteristics are shown in Table All theses came out from the initial date to February and the patients were diagnosed as SCLC byhistopathology Among these studies all studies investigated DLL3 byinparaffinembedded tissueimmunohistochemistryIHCQuality assessment of the included studiesWe performed quality evaluations of the s following the QUIPS tool and two authors independently evaluated allthe literature Differences were resolved bydiscussion After screening all included s we foundthat there were no researches that reported study attritionand there were no standardization of cutoffs value forevaluating DLL3 expression And then studies were evaluated as low risk were evaluated as moderate risk and study was evaluated as high risk Table This outcomeindicated that most of the studies we included were of amedium or high qualityPrognostic value of DLL3 in SCLCAs shown in Fig eligible s were pooled foranalysing the prognostic value of DLL3 in SCLC TheFig The flow diagram of studies selection 0cChen World Journal of Surgical Oncology Page of Table Study characteristics of the eligible sStudyYear Country No ofsamplesSexmalefemaleSmokinghistorynonyesYan [] ChinaDistantmetastasisnegativepositiveXie [] USATanaka [] JapanRegzedmaa [] ChinaHuang [] ChinaFuruta [] JapanNANANANANANANATumourstageIIIIIIIVHistologicsubtypeMethod Outcomes BiomarkersCutoffvalueNANASCLCSCLCSCLCSCLCSCLCSCLCIHCIHCIHCIHCIHCIHCOSOSOSOSDLL3TTF1DLL3DLL3DLL3CTLA4MSTNscore ¥ ¥ ¥ score ¥ OSPFSDLL3OSDLL3ASCL1score ¥ ¥ SCLC small cell lung cancer IHC immunohistochemistry OS overall survival PFS progressionfree survival DLL3 deltalike protein CTLA4 cytotoxic T lymphocyteassociated protein MSTN mesothelin ASCL1 achaetescute homologue1 NA not applicableresults showed that high expression of DLL3 indicatedno prognostic value in patients with SCLC HR CI I2 p Howeverprominent heterogeneity existed in the pooled results sowe conducted further subgroup analysis Among the sixstudies three s were from China two were fromJapan and one was from America We divided the studies into the Asian group and the American group forsubgroup analysis As shown in Additional file FigA1we found that the heterogeneity was in the Asiangroup and we believed that the main cause of the heterogeneity was the study from American So we decidedto eliminate the study from American and only analysedthe remained studies After we removed the Americanstudy the outcomes indicated that high DLL3 expressionwas a poor prognosis marker in SCLC HR CI I2 p Fig Then wedivided the studies into two groups according to thesize of the sample and the final outcomes indicated thathigh DLL3 expression was a poor prognosis marker inthe group with sample size greater than HR CI p Fig But there was nosignificant relationship between DLL3 expression andprognosis in the group with sample size less than HR CI I2 p Fig Beggs test p and Eggers test p there was nodemonstratedresultsthatpublication bias in our metaanalysis Fig Finallysensitivity analysis showed that allthe studies werestable see Additional file FigA6The clinical characteristics of patients with DLL3expression in SCLCThe pooled results showed that the expression of DLL3was not correlated with sex RR CI I2 p Table Additional file FigA2 smoking history RR CI I2 p Table Additional file FigA3 and tumour stage RR CI I2 p Table Additional file FigA4 But DLL3 was highly expressed in patients withdistant metastasis RR CI I2 p Table Additional file FigA5DiscussionMany studies have shown that DLL3 played a significantprognosis value in patients with cancer For examplehigh DLL3 expression was associated with shorter OSand PFS in small cell bladder cancer [] High DLL3expression was associated with bad OS and usuallyexpressed in older patients and advanced stage in endometrial cancer [] Xie reported that high DLL3expression predicted a better OS in patients with SCLC[] and other studiesforeboded that high DLL3Table Quality assessment of included studiesStudyStudyparticipationYan []Xie []Tanaka []Regzedmaa []Huang []Furuta [] indicates low risk of bias StudyattritionPrognostic factormeasurementOutcomemeasurementStudy confoundingStatistical analysisand reportingTotal riskof biasLowModerateLowLowHighModerate moderate risk of bias and high risk of bias 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in SCLC patients DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneityexpression was an inferior prognostic marker for SCLC[ ] Thus the results of different studies remainedcontroversial We included a total of studies with patients with SCLC to assess the prognostic value ofDLL3 in SCLC by pooling the data of HR and CIFirst we performed this metaanalysis using studiesBut heterogeneity was observed in our pooled resultsThrough further subgroup analysis we found that astudy from the American was the main cause of heterogeneity As we removed the American study we foundFig Forest plots of prognostic value of DLL3 in Asia patients with SCLCDLL3 deltalike protein USA the United States of America HR hazardratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneity 0cChen World Journal of Surgical Oncology Page of Fig Forest plots of prognostic value of DLL3 in different studies with different sample sizes DLL3 deltalike protein HR hazard ratios CI confidence intervals Isquared percentage heterogeneity between studies p test for heterogeneitythat high expression of DLL3 is a marker of poor prognosis in SCLC But we also noticed that the results ofthe American study were contrary to our conclusionsthey reported that high expression of DLL3 is a markerof good prognosis in SCLC One interpretation of thisresult was that DLL3 expression varies between differentpopulations The American study indicated that the highexpression of DLL3 was a marker of good prognosis[] and there was no significant correlation betweenDLL3 expression and prognosis in several Japan studies[ ] while the high expression of DLL3 is a markerof poor prognosis in China studies [ ] These results showed that the expression of DLL3 might be different in different populations However only one studyFig Funnel plot of DLL3 present on overall survival DLL3 deltalike protein HR hazard ratios 0cChen World Journal of Surgical Oncology Page of Table Summary of the correlation between DLL3 expression and the clinical characteristics of patients with SCLCClinical characteristicsSex malefemaleRR CI No of studiesI2 p Smoking historynonyesDistant metastasis negativepositive P valueTumour stage IIIIIIIVSCLC small cell lung cancer RR relative ratios CI confidence intervals DLL3 deltalike protein I2 percentage heterogeneity between studies p testfor heterogeneity researched the correlation with DLL3 expression andprognosis in SCLC outside of Asia so the results needto be treated with caution In addition more studieswere suggested for the future to further verify the existence of such differencesOur other explanation of this result was that the expression of DLL3 was the same in different populationsAmong the included studies the sample size of studieswas less than including the America and Japanstudies Therefore we speculated that insufficient sample size might cause bias in the results We conducted asubgroup analysis according to the sample size and theresults showed that the high expression of DLL3 in thestudies with large sample sizes N ¥ was associatedwith poor prognosis while the pooled results of thestudies with low sample sizes N showed no significant correlation between the expression of DLL3 andprognosis Therefore the sample size may be one of thereasons for the differences in DLL3 expression in eachstudy Moreover immunohistochemistry was used to detect DLL3 expression in all of the studies Immunohistochemical staining is a semiquantitative method and isevaluated with great subjectivity [] Different antibodies and different cutoff values for DLL3 expressionwere employed in all the included studies and thus couldalso be another cause of the differences in results TheAmerican study also explained their different results byclaiming that many of the other studies were performedusing mRNA expression instead of protein expression orused different cutoffs value of DLL3 expression []Researches havereported that DLL3 is highlyexpressed in SCLC [ ] which suggested that DLL3might promote the development of SCLC Therefore wealso discussed the correlation between DLL3 expressionand the clinical characteristics of patients with SCLCThe pooled results showed that DLL3 expression had nosignificant correlation with patients sex smoking statusand stage while DLL3 often highly expressed in metastasis patients of SCLC Our survival analysis outcomeswere consistent with this result which suggested thathigh expression of DLL3 might be one of the factorscontributing to poor prognosis in patients with advancedmetastatic SCLC in Asia However only a few studiesreported the correlation between the expression of DLL3and clinical characteristics and the methods and cutoffs values used to detect the DLL3 expression in eachstudy were not uniform Therefore more reliable studiesare needed to further verify our outcomes Our resultssuggest that it is valuable to further investigate the correlation between DLL3 and the clinical characteristics ofpatients with SCLCOur metaanalysis is the first to focus on the prognostic value of DLL3 in SCLC The significance of thismetaanalysis lies in providing a basic direction and evidence for further research into the mechanism of DLL3in SCLC For SCLC Notch1 over expression could induce G1 cell cycle arrest [] Previous studies reportedthat DLL3 downregulated the Notch receptor expression thereby the Notch signalling pathway was inhibitedwithin the cell [] Therefore high expression of DLL3can promote the development of SCLC by inhibiting theNotch signalling pathway Studies also have shown thatthe high expression of DLL3 may reduce the sensitivityof chemotherapy drugs [] These studies have demonstrated that DLL3 may be associated with the prognosisof SCLC and also consistent with our metaanalysis results Thus studies of the corresponding targeted drugsof DLL3 can effectively inhibit the expression of DLL3and thus improve the survival of SCLC Rovalpituzumabtesirine RovaT is a new antibodydrug conjugate directed against DLL3 in SCLC [] A phase I trial foundthat patients with high DLL3 expression in SCLCshowed a better response to RovaT than those with alow DLL3 expression [] However disappointingly thephase III TAHOE trial has been stopped because theRovaT group showed a worse OS compared to the control group [] But more clinical trials are recruitingparticipator to investigate RovaT as maintenance therapy in advanced stage SCLC The lack of progress withthis drug does not prevent us from making a breakthrough with other similar drugs Some researches foundthat the intratumoural and intertumoural distributionof DLL3 protein in SCLC is homogeneous [] supporting the conclusion that biopsy specimens are a reliablesource for DLL3 evaluation for targeted therapy Inaddition most studies have demonstrated that DLL3 is 0cChen World Journal of Surgical Oncology Page of highly expressed in SCLC while it is not or is lessexpressed in other types of lung cancer and normal tissues [] Therefore the expression of DLL3 can be detected by biopsy as an indicator for diagnosis predictingtherapeutic efficacy and monitoring recurrence or metastasis of SCLC in the futureAlthough our study fully explains the prognostic valueof DLL3 in SCLC our analysis still has several limitations Firstlarge heterogeneity was observed in thepooled results This is explained by the observation thatthe evaluation criteria for the expression of DLL3 areparticularly mixed and there are no international standards for cutoffs values to determine the expression ofDLL3 Thus the scoring methods and cutoffs values ofDLL3 should be unified to strengthen our conclusionsOtherwise the detection method of DLL3 in most studies is mainly immunohistochemistry at present which isa semiquantitative subjective and inaccurate detectionmethod Different studies show different prognosticvalues of DLL3 Therefore we need other more precisedetection methods to evaluate the expression of DLL3 inSCLC in the futureSecond the therapy method is also a key limitationThe current studies only focus on tissue specimens frompatients with SCLC after surgery or biopsy and fewstudies reported the treatment methods in their researches However the prognostic value of DLL3 may liein the therapeutic method Therefore every study shouldpay attention to the impact of patienttreatmentmethods on prognosis in the futureThird some of the original studies did not report thedata of HR and CI The HR and CI resultswere measured from survival curves an evaluationmethod with certain deviation and subjectivity whichmight influence the authenticity of the resultsConclusionIn summary our metaanalysis confirmed that high expression of DLL3 was a potential poor prognostic biomarker for SCLC in Asian populations moreover DLL3expression was correlated with advanced metastasisSCLC in Asian populations However the relationshipbetween DLL3 expression and the prognostic or clinicalcharacteristics of patients with SCLC in European andAmerican populations need to be further verified Thusdetecting the expression of DLL3 in tumour tissue willbe helpful to guide therapy in Asian patients of SCLCFor our research other highquality studies especiallyfrom European and American countries are required toconfirm our findings about the prognosis value of DLL3in SCLC in the future In view of the limitations of ouranalysisthe conclusions should be interpreted withcautionSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s12957020020045Additional file The PRISMA checklistAdditional file FigA1 Forest plots of prognostic value of DLL3 inSCLC SCLCsmall cell lung cancer HRhazard ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA2 Forestplots of the correlation between DLL3 expression and sex of patientswith SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA3 Forestplots of the correlation between DLL3 expression and smoking history ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA4 Forestplots of the correlation between DLL3 expression and tumour stage ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA5 Forestplots of the correlation between DLL3 expression and metastasis ofpatients with SCLC SCLCsmall cell lung cancer RRrelative ratios CI confidence intervals DLL3deltalike protein I2percentageheterogeneity between studies ptest for heterogeneity FigA6Sensitivity analysis of all the studiesAbbreviationsCI Confidence intervals DLL3 Deltalike protein DFS Diseasefree survivalDSS Diseasespecific survival HR Hazard ratios IHC ImmunohistochemistryOS Overall survival PFS Progressionfree survival QUIPS Quality In PrognosisStudies RFS Relapserecurrencefree survival RR Risk ratio SCLC Small celllung cancerAcknowledgementsNot applicableAuthors contributionsAll authors contributed to the study conception and design Materialpreparation data collection and analysis were performed by Benchao ChenHeng Li and Chao Liu The first draft of the manuscript was written byBenchao Chen and all authors commented on previous versions of themanuscript All authors read and approved the final manuscriptAuthors informationNot applicableFundingThis work was supported by the National Natural Science Foundation ofChina [No ]Availability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsReceived July Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwide 0cChen World Journal of Surgical Oncology Page of Regzedmaa O Li Y Li Y Zhang H Wang J Gong H Prevalence ofDLL3 CTLA4 and MSTN expression in patients with small cell lung cancerOnco Targets Ther httpsdoi102147OTTS216362 Huang J Cao D Sha J Zhu X Han S DLL3 is regulated by LIN28B and miR518d5p and regulates cell proliferation migration and chemotherapyresponse in advanced small cell lung cancer Biochem Biophys ResCommun 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"clinical development of immune checkpoint inhibitors ICIs therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit Therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of ICIs and more indepth and comprehensive understanding has beencontinuously explored in recent years Predictive markers of ICIs efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers With thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers Comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs and discuss their future directions in achievingprecision immunooncologyKeywords Neoplasm Immune checkpoint inhibitor Predictive biomarker Tumor mutation burden Programmeddeath ligand1BackgroundImmune checkpoint inhibitors ICIs therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors Antiprogrammed cell death1programmed cell deathligand PD1PDL1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma RCC head and neck squamous cell carcinoma HNSCC and gastroesophageal Correspondence cuijwjlueducnCancer Center the First Hospital of Jilin University Xinmin StreetChangchun Jilin Chinacancer [ ] However despite the breakthrough in clinical treatment with ICIs most patients do not benefitPembrolizumab or nivolumab has an objective responserate ORR of in firstline melanoma and insecondline nonsmall cell lung cancer NSCLC []Therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of ICIs and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid 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 0cBai Biomarker Research Page of technologyimmunohistochemicalmade many new advances in the corresponding fieldsWith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers The development of predictive biomarkers contributes to revealing the therapeutic mechanisms of ICIs and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs It should be pointed out here that when reading and collating we try to read and include all therelevant s In the process of selecting s we include the authoritative s published in highleveljournals or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedAdvances of multiple predictive biomarkers toICIs efficacyi Tumor genome and neoantigen biomarkersTumor mutation burdenSignificant correlations between high tumor mutationburden TMB and response to ICIs have been reported inseveral cancer types [] including urothelial carcinoma[] small cell lung cancer SCLC [] NSCLC []melanoma [] and human papilloma virus HPVnegative HNSCC [] A metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log TMB [] The National ComprehensiveCancer Network NCCN guidelines have adopted TMBas the recommended test for patients with NSCLC receiving immunotherapy Although the results in some clinicalstudies of RCC [] HPVpositive HNSCC [] and melanoma receiving antiPD1 after recurrence [] showedthat TMB alone also did not clearly distinguish respondersand predict OS it is still exciting that multiple studies inthe American Society of Clinical Oncology ASCOmeeting have confirmed the predictive value of TMB inimmunization or combination therapy KEYNOTE061study [ ] CONDOR study [] EAGLE study []EPOC1704 study [] etc consolidating its status ofTMB as an independent predictor And in April theUS Food and Drug Administration FDA prioritized theapproval of TMB as a companion diagnostic biomarkerfor pembrolizumabNonetheless the cutoff values of TMB were defineddifferently across studies and assay platforms such asatezolizumab mtMb in urothelial cancer pembrolizumab mtMb in NSCLC and atezolizumab¥ ¥ or ¥ mtMb in NSCLC [] andnivolumab plus ipilimumab ¥ mtMb in NSCLC [] which needs further study to confirm the optimalcutoff value in different tumors Moreover the NGSpanels have approved by the FDA that can be used to estimate TMB include the MSKIMPACT and FoundationOne CDx panel the detection results of which arehighly consistent with whole exome sequencing WES[ ] and other solutions are under development Astudy detecting TMB cutoff value at mtMb in NSCLC patients with the FoundationOne platformcontaining a gene panel found that compared withTMBL patients overall survival OS and DCR was significantly improved in TMBH patients treated withantiPD1L1 drug [] Both WES and targeted NGS a422cancergene panel performed in patients withNSCLC treated with antiPD1L1 demonstrated thatTMBH population has a significantly better durableclinical benefitDCB and progressionfree survivalPFS [] These findings demonstrate the feasibility ofcomprehensive genomic profiling CGP but the designof optimal next generation sequencing NGS panel thatis more accurate comprehensive and costeffective isstill not clear In addition given that bTMB was identified as a predictor of PFS but failed to differentiate patients with OS benefits researchers consider the need toexplore other more precise factors eg allele frequencyAF A study that developed a new bTMB algorithm intwo independent cohorts POPLAR and OAK showedthat modified bTMB low AF bTMB LAFbTMB mutation counts with an AF was significantly associated with favorable HR 95CI p PFS HR 95CI p andORR p after immunotherapy but required tobe prospectively validated [] Finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions A recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment TMB was only associated with OS in untreated patients while early 4week ontreatment changein TMB ÎTMB was strongly associated with antiPD1response and OS in the entire cohort [] The detectionof ÎTMB is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cBai Biomarker Research Page of In addition epigenetic changes are associated withTMB The latest study investigated the association between TMB and DNA methylation DNAm to explorepotential complimentary biomarkers for NSCLC immunotherapies The results showed that high TMBNSCLCs had more DNAm aberrance and copy numbervariations CNVs showing certain value in predictingefficacy such as HOX gene methylation status and TMB[] Thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction Exploration in Chinese NSCLC patientsshowed that NSCLCs with high TMB had DNAm aberrance and CNVs Some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens [] In the pancancer analysis of cancer types evaluated in The Cancer Genome AtlasTCGA RCC had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnsSNVs[] Somatic copy number alterations SCNAs are another feature of the genomic landscape of tumors andpancancer TCGA analysis revealed an inverse correlation between SCNAs atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested [] and this result was subsequently replicated in a larger study of TCGA []single nucleotide variantsDNA damage response pathwaysGenetic variation involved in DNA mismatch repairMMR pathway can lead to microsatellite instabilityMSI a specific type of high TMB tumors and increased numbers of CD8 tumor infiltrating lymphocytesTILs PD1TILs and indoleamine 23dioxygenaseIDO tumor cells have been shown in MMR deficiencydMMR colorectal cancer [] Recently five clinical trials Keynote016 including multipletumor types have shown that patients with dMMRMSIH can achieve durable responses to pembrolizmabBased on this pembrolizumab is approved by the USFDA for the treatment of any advanced solid tumor withdMMRMSIH and nivolumab in combination with ipilimumab has also shown promising response in dMMRMSIH colorectal cancer [] In addition dMMR canalso cause mutations in the DNA polymerase gene epsilondelta POLEPOLD1increasing the mutationload and neoantigen load Analysis of POLEPOLD1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher TMB and OS Therefore it may be an infordependentInidentifying patients who benefitaddition pathways of base excision repairBERand prognostic markerfrom ICIs []risk factorhomologous recombination repair HRR MMR in theDNA damage response DDR signaling network contribute more significantly to TMB or neoantigens whichhave the highest levels when comutated [] It hadbeen identified that comutations in the DDR pathwaysof HRR and MMR or HRR and BER defined as comutare associated with increased levels of TMB neoantigenload and immune gene expression signatures Comutpatients showed a higher ORR and longer PFS or OS indicating that comut can be used as predictors of response to ICIs and provide a potentially convenientmethod for future clinical practice []Specific mutated gene pathways in tumor cellsIt is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy Patients with mutations in the interferon IFNγpathway genes IFNGR12 JAK12 and IRF1 are poorlyresponsive to ICIs treatment and confer resistance []A study found that in patients receiving immunotherapytumor cells can downregulate or alter IFNγ signalingpathways such as lossoffunction alleles of genes encoding for JAK12 and changes in STAT1 to escape the influence of IFNγ [] resulting in poor efficacy andresistance Recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling SWISNF complex and unique genes of the PBAFcomplex Pbrm1 Arid2 and Brd7 lead to sensitivitiesto ICIs [ ] Loss of function of the PBAF complexincreased chromatin accessibility to transcription regulator elements of IFNγinducible genes within tumorcells and subsequently increased production of CXCL9CXCL10 chemokines leading to more efficient recruitment of effector T cells into tumors [] In human cancers expression of Arid2 and Pbrm1 are related toexpression of T cell cytotoxicity genes which confirmedin Pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic T cells and responsive to immunotherapy [ ]In addition doublestranded RNAdsRNA editing enzyme adenosine deaminase acting onRNA ADAR1 protein can block the IFNγ signalingpathway and lead to poor ICIs efficacy and resistanceLoss of function of ADAR1 in tumor cells can reduce AtoI editing of interferoninducible RNA species and leadto dsRNA ligand sensing by PKR and melanomadifferentiationassociated protein MDA5 This resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy [] Finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding PDL1 and IFN signaling pathway genes sensitizingto anticytotoxic Tlymphocyteassociatedprotein4 CTLA4 therapy []it 0cBai Biomarker Research Page of In addition to the IFNγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy Epidermal growthfactor receptor EGFR and anaplastic lymphoma kinaseALK mutations have been shown to be associated withreduced response rates to ICIs and low TMB and therefore the FDA does not recommend firstline ICIstreatment in patients with EGRF or ALK positive tumors[ ] certain types of mutations in MDM2MDM4and ARID1A can predict nonresponse to ICIs in highTMB tumors [] NSCLC with KRAS and STK11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antiPD1 therapy [] and STK11 deficiency was an independent indicator of poor antiPD1response in NSCLC with KRAS mutant however at the American Association for Cancer Research AACRmeeting of patients in the Keynote042 studyNCT02220894 update data were tested for STK11 andKEAP1 and the results showed that patients could benefit from pembrolizumab regardless of STK11 and KEAP1status but patients with STK11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted NGSpanels suggested that duration of ICIstreatment was associated with certain BRAF and MET alterations butnot TMB status [] NOTCH signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells Recent breakthrough findings have distinguished deleterious NOTCH mutation showing that itcan be used as a potential predictor of favorable ICI response in NSCLC potentially via greater transcription ofgenes related to DNA damage response and immune activation [] Another tumorspecific inheritance thatmay influence ICIs efficacy is the aberrant expression ofendogenous retroviruses ERVs Pancancer analysisidentified a positive correlation of transcript expressionof ERVs with Tcell activity in various tumors [] andpatient prognosis [] Furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones For example the analysis of studydata of secondline PD1L1 inhibitor therapy found thatthe mPFS of patients with KRAS G12C or G12V was significantly better than that of patients with KRAS mutations at other sites []In addition several pancancer biomarkers are recentlyapproved by the FDA For example given the effectiveORR of and a disease control rate DCR of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent FDA approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor FGFR2 fusion or rearrangement and the comprehensive genomicassay FoundationOne CDxdeveloped by Foundation Medicine as a companiondiagnostic Also exciting is the recent FDA approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic NSCLC with MET exon skippingMETex14 mutations including firstline patients andpreviously treated patients also using FoundationOneCDx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofNeoantigen loadNeoantigen load the number of mutations actually targeted by T cells may be directly related to the responseto ICIs [] A retrospective study showed thatclonal neoantigen burden was associated with the longerOS in primary lung adenocarcinomas p []Traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexMHC binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall TMB inpredicting ICIs efficacy or survival [] In recent practice this neoantigen can be assessed by the difference inpredicted MHCI binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index DAI reflectingclinically relevanttumor peptide[] A high DAI value indicates that the mutant peptidesignificantly increases binding affinity to MHC compared to the wildtype sequence and can generate moreimmune responses Studies on previously published cohorts treated with three ICIs have shown that DAI outperforms TMB and the traditionally defined neoantigenload in predicting survival [ ] In additionlowneoantigen intratumour heterogeneity might also be important for ICIs response Analysis of the lung adenocarcinoma TCGA database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with OSand longer lasting clinical benefit than either variablealone [] Anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the Immune Epitope Database IEDB [] butit does not account for all possible human leukocyteantigen HLA contexts In addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes A study developed the Neopepseealgorithm using a machine learning approach incorporating 0cBai Biomarker Research Page of integration of nine immunogenicity features and gene mutation expression levels [] and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction Recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion [] indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii Tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score IPS Herbst [] showed that response toatezolizumab treatment was significantly associated withhigh levels of PDL1 expression on the surface of TILs before treatment but not with PDL1 expression on tumorcells p Finally other inhibitory immune pathways may affect the response to ICIs therapy including Tcelllymphocyte activationgene3 LAG3 and Vdomain Ig suppressor of Tcell activation VISTA which can be used as potential biomarkers for ICIs responseimmunoglobulin3 TIM3PDL1 expressionGiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between PDL1 expression and response to ICIs or OS even in firstlinecombination therapy [] pembrolizumab is currently approved by the FDA for use in patients with PDL1 PDL1 ¥ of tumor cells in firstline treatmentand ¥ in secondline treatment NSCLC and PDL1immunohistochemistry IHC as a companion diagnosticfor antiPD1 therapy in NSCLC patients [ ] However some studies have not detected a significant correlation between PDL1 expression and response to ICIs[ ] and PDL1 negative patients can still benefitclinically with treatment with ICI or combination treatment with ICIs [] with ORRs ranging from to Therefore PDL1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing FirstlyPDL1 assay and antibody are not standardized []Secondly PDL1 expression is temporally and spatiallyheterogeneous [] A study of metastatic NSCLCtreated with ICIs showed that PDL1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases Andthe predictive value of PDL1 at different biopsy sites forthe benefit of ICIs in NSCLC may vary higher PDL1 inlung or distant metastasis specimens was significantly associated with higher response rate PFS and OS whilePDL1 in lymph node metastasis biopsy was not associated with either response or survival [] Thirdly positive score and cutoff value of PDL1 expression is notstandardized [] At present PDL1 positive scoremainly focuses on the PDL1 expression level of tumorcells that is tumor proportion score TPS But PDL1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scoreCPS which is the proportion score of the sum of PDL1 expressed by tumor cells and tumorassociated immune cells In addition PDL1 expression on immuneresponseto ICIsimmunetreatmentBiomarkers of tumorinfiltrating immune cellsOverall immune status of tumor microenvironmentThe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert [] Immuneinflamed is characterizedby the presence of CD8 and CD4 T cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules [] indicating a potential antitumor[]immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] Analysis of pretreatment samples forantiPD1PDL1 revealed a relatively high abundance ofCD8T cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of CD8T cells into tumor parenchyma []while immunedesert phenotype is characterized by theabsence of abundant T cells in the parenchyma orstroma of tumors and poor response to ICItreatment[] Recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment TME classifying tumors aswell as predicting treatment response and prognosis[] which involves the density of two lymphocyte populations CD8 and memory [CD45RO] T cells in thecenter and invading margin of tumor [] Mlecnik et al[] evaluated immunoscore in specimens of stageIIV colorectal tumor and confirmed that it was significantly associated with PFS DFS and OS and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival The valueof immunoscore to predicting ICIs efficacy is being validated internationally in clinical trials of melanoma andNSCLC []A wider assessment of active immune responses withinTME by immune gene expression profiling might effectively predict clinical benefit to ICIs strategies Analysisof total RNA and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cBai Biomarker Research Page of expression of immunerelated genes in clinically active patientsincluding cytotoxic T cell markers egCD8A perforin granzyme B Th1 cytokines or chemokines MHCII and other immunerelated genes egNKG7 IDO1 [] Ascierto [] screened morethan immunerelated genes in patients with recurrent breast cancer years after treatment and thosewithout recurrence more than years later and foundthat five genes IGK GBP1 STAT1 IGLL5 and OCLNwere highly overexpressed in patients with recurrencefree survival In addition IFNγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with ICIs The study developed IFNγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding IFNγ granzymes AB perforin IDO1 and other immunerelated genesBoth gene scores showed significant associations withbest overall response rate and PFS Optimized cutoffvalues for IFNγ scores based on receiver operatingcharacteristic curve ROC curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders []Immune cells with specific phenotypes in TMEThe phenotype of TILs also influences the efficacy ofICIs The study used singlecell mRNA sequencingscRNAseq data analysis to identify two major CD8Tcell phenotypes within melanoma memorylike andexhausted [] the proportion of which is strongly correlated with response to ICIs The research furtherfound that the transcription factor TCF7 is selectivelyexpressed in memorylike T cells so the ratio ofCD8TCF7 to CD8TCF7TILs is strongly correlatedwith improved response and survival in melanoma patients treated with antiPD1 [] Balatoni []found that of immune cells in TME were positivelyassociated with OS after treatment including CD4 andCD8 T cells FOXP3 T cells CD20 B cells CD134and CD137 cells and NKp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes Researchers found that only asmall proportion of CD8 TILsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential CD39 expression was the key molecule thatdistinguished the two populations [] Analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of CD39 on CD8 TILs indicating thatCD39CD8TIL may be a promising predictive biomarker [] The fact of very low level of CD39 expression on CD8TILs in of EGFRmutant NSCLC isconsistent with their low response rate to antiPD1immunotherapyIn addition a study showed that Fc domain glycan ofthe drug and Fcγ receptor FcγR expressed by the hostbone marrow cells could determine the ability of PD1tumorassociated macrophages TAMs to capture antiPD1 drugs from the surface of T cells which leads toPD1 inhibitor resistance [] and the association ofTAMs and poor antiPD1 response was reported inmelanoma cohorts [] antiPD1 response was associated with an increase in CD8T cells and natural killercells NK cells and a decrease in macrophages [] andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mPFS after antiPDL1 therapy in RCC emphasizing the inhibitory role of myeloidcells in response to ICIs [] In conclusion immunecells in TME show a great promise in the developmentof predictive biomarkers for ICIsimmunerepertoireDiversity of immune repertoires in TMEEffective T cell responses involve the activation and expansion of specific antigenreactive T cell clones so diversity ofin intratumoral orperipheral may correlate with ICIs responses and can bequantified as richness and clonality [] However theresults seem to be complex with some studies finding apositive correlation between TIL clonality and the response to ICIs before [] or after [] treatment whileothers showing that only an increase in TIL clonalityduring treatment is associated with the response to antiPD1 [ ] others show that intratumoral T cellclonality is not associated with survival while peripheralT cell clonality is inversely associated with PFS and OS[] Tumeh [] further investigated whetherbaseline TILs have a narrow T cell receptor TCR repertoire focusing on tumorspecific immune responsesand whether this narrow TCR repertoire correlates withpembrolizumab responses They found that respondingpatient had more restricted usage of the TCR beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients Notablybaseline TCR clonality was not highly correlated withTIL density suggesting that some patients with restricted TCR clonality specific for tumor antigens maystill benefit from antiPD1 therapy even though TILdensity is low Recently researchers have proposed theimmune repertoire IRIndex the average frequency ofshared TCR clones in T clones in TILs and peripheralPD1CD8 T cells They found that neoantigenstimulated TCR agreed with IRIndex and patients withhigh IRindex had better immune activation and highergene expression profiles GEPs score subsequently they 0cBai Biomarker Research Page of confirmed the predictive value of IRindex to ICIs efficacy DCRPFS But considering that it is difficult tosort out PD1CD8 T cells in tumor tissue based ontwo separate patient cohorts a research confirmed thatTCR repertoire diversity and clonality of peripheral PD1CD8T cells may serve as noninvasive predictors ofclinical outcomes after ICIs in patients with NSCLC[] The viewpoints of T cell diversity and TCR clonality as markers of ICIs efficacy need to be further validated in a large patient populationiiiLiquid biopsy biomarkersPeripheral blood cell biomarkersPeripheral blood is a noninvasive source to explore potential biomarkers for ICIs and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies Analysis of melanoma treated with ipilimumabshowed that improved OS and PFS were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio NLR low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of FoxP3 Treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased FoxP3Treg concentratreatmenttions and increased lymphocyte and eosinophil counts[] Reports in patients with melanoma treated withpembrolizumab and in patients with NSCLC treatedwith nivolumab have shown that NLR is associated withworse tumor response [ ] Multivariate analysis inmelanoma patients treated with antiPD1 antibodiesshowed that NLR was the only factor associated withworse ORR and shorter PFS indicating that NLR is astrong predictor of worse outcome in patients treatedwith ICI [] Low baseline lactate dehydrogenase LDHlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedOS in antiPD1 and CTLA4 treated melanoma [] Given that previous studies have proposed the importance of baseline derived NLR dNLR and LDHlevels as prognostic markers a recent study proposed acomposite prognost | Thyroid_Cancer |
"RNA molecules with a unique closed continuous loop structure CircRNAs areabundant in eukaryotic cells have unique stability and tissue specificity and can play a biological regulatory role atvarious levels such as transcriptional and posttranscriptional levels Numerous studies have indicated that circRNAsserve a crucial purpose in cancer biology CircRNAs regulate tumor behavioral phenotypes such as proliferation andmigration through various molecular mechanisms such as miRNA sponging transcriptional regulation and proteininteraction Recently several reports have demonstrated that they are also deeply involved in resistance toanticancer drugs from traditional chemotherapeutic drugs to targeted and immunotherapeutic drugs This review isthe first to summarize the latest research on circRNAs in anticancer drug resistance based on drug classification andto discuss their potential clinical applicationsKeywords Circular RNA Cancer Drug ResistanceIntroductionCancer has become the most serious public health problem worldwide [] As the early diagnosis of cancer urgently needs to be improved many patients are alreadysuffering from advanced cancer at the first clinical visit[] These patients miss the opportunity for surgery andanticancer drugs are their major treatment option Sincethe first clinical report of chemotherapy for advanced cancers multiple anticancer drugs have been developed andhave effectively improved the clinical outcomes of patientswith advanced cancers [] In addition targeted and immunotherapeutic drugs have recently ushered in a newera in medical therapy for cancer [ ] However anticancer drug resistance still cannot be avoided resulting incancer relapse The mechanisms underlying anticancerdrug resistance are multifaceted Different hallmarks such Correspondence wangzhaoxianjmueducn weichenchen1990126com Tianwei Xu Mengwei Wang Lihua Jiang and Li Ma contributed equally tothis work1Cancer Medical Center The Second Affiliated Hospital of Nanjing MedicalUniversity Jiangjiayuan road Nanjing Jiangsu PR ChinaFull list of author information is available at the end of the as tumor growth selective therapeutic pressure immunesystem characteristics and the tumor microenvironmentdetermine the biological behaviors of anticancer drug resistance [] These hallmarks are driven by complexunderlying molecular regulatory mechanisms [] Identifying the key molecules in these processes could help us tounderstand the occurrence of resistance and these molecules play considerable roles in the prediction and reversion of anticancer drug resistance []With the development of sequencing studies haveshown that approximately of the human genome istranscribed but only encodes proteins [] Theremaining RNA which does not encode proteins wasinitially thought to be transcriptional junk [] Accumulating evidence has revealed that these noncodingRNAs ncRNAs also exert a great influence on physiological activities and pathological changes especially incancer [ ] Importantly ncRNAs are classified bysize into small RNAs such as miRNAs nt and longnoncoding RNAs lncRNAs nt Dysregulation ofin the molecular and cellularncRNAs participates 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 0cXu Molecular Cancer Page of processes related to cancer The roles of ncRNAs are divided into oncogenes and tumor suppressors based ontheir effect on the cancer For example miR21 was reported to promote tumor growth and metastasis by targeting PTEN in lung cancer and other cancers such asmelanoma and B cell lymphoma [ ] As shown inour previous study high serum miR21 levels indicate apoor prognosis for patients with nonsmall cell lung cancer NSCLC [] In contrast miR34a functions as adirect downstream target of p53 to block cancerprogression [] HOTAIR a wellstudied oncogeniclncRNA is generally upregulated in cancers and epigenetically silences tumor suppressors such as p21 []However lncRNA MEG3 modulates the expression ofp53 and some other tumor suppressors to increases chemotherapeutic sensitivity [] Several ncRNAs may alsofunction as either tumor suppressors or oncogenesdepending on the context For example the lncRNANKILA reduces cancer metastasis by negatively regulating NFkB signaling while it also promotes tumor immune evasion [] Since ncRNAs such as miRNAs andlncRNAs have been confirmed to play an important rolein cancer studies of new ncRNAs will continue to improve our understanding of cancerIn recent years a novel class of lncRNAs designated circular RNAs circRNAs has also been identified and extensively studied in cancer biology [] CircRNAs are derivedfrom premRNAs through a noncanonical alternative splicing event called backsplicing This process endows circRNAs with a unique fundamental structural feature thatdiffers from common linear lncRNAs namely a covalentlyclosed continuous loop structure without a polyadenylatedtail CircRNAs are very stable because their unique structure is resistant to exonucleasemediated degradation []Fig Biogenesis of circRNAs a Classification of circRNAs CircRNAs can be divided into ecircRNAs EIciRNAs and ciRNAs by their composition bFunctions of circRNAs CircRNAs can regulate gene transcription as their parental genes do in the nucleus CircRNAs can act as miRNA spongesinteract with proteins as protein scaffolds or decoys and under certain circumstances be translated in the cytoplasm 0cXu Molecular Cancer Page of Although backsplicing is much less efficient than canonicalsplicing in linear RNAs circRNAs are still enriched in tissues serum and even urine [] Although circRNAs arewidely expressed the expression pattern of circRNAs displays tissue specificity and cell type specificity For exampleplatelets express more circRNAs than neutrophils whichare also blood cells vs circRNAs [ ]CircRNAs can be divided into subtypes exonic circRNAsEcircRNAs which contain only exon sequences intronic circRNAs ciRNAs which contain only intron sequences and exonintron circRNAs EIciRNAs whichcontain both exon and intron sequences [] Fig 1aStudies have demonstrated that circRNAs may exert biologicalfunctions by transcriptional regulation miRNAsponging protein interactions or under certain circumstances selftranslation [ ] Fig 1b For exampleciRS7 is a wellknown circRNA that is enriched in neurexhibitonalknockout miceCiRS7tissuesneuropsychiatric disorders and the underlying mechanismmay be attributed to the ciRS7miR7Fos axis []CircRNAs also participate in various pathological proincluding the development of cancer [] Accessescording to the classification ofthe mechanism intumors we present some representative circRNAs withclear mechanisms biological functions and clinical significance Table First circRNAs are differentiallyexpressed in almost all cancers including lung cancergastrointestinal cancers and urological cancers SecondcircRNAs are differentially expressed rather than simplyupregulated or downregulated in the same tumor Forexample circPVT1 and circFADS2 are upregulated [] and circNOL10 and circPTPRA are downregulatedin lung cancer [ ] Third the same circRNAs arealso differentially expressed in a variety of tumors butthe upregulated or downregulated expression patternsare not the same in different tumors CircHIPK3 isTable Representative circRNAs with clear mechanisms biological functions and clinical significance in cancersMechanismTranscriptionalregulationInhibit cell proliferation and promote cellapoptosisClinical significanceLung cancer differentiationCircRNAcircNOL10 DownCancerLung cancerExpression Biological functionsColorectalCancercircITGA7 DownInhibit cell growth and metastasisTumor sizeLymph metastasis Distant metastasisand TNM stageBreast CancercircFECR1 UpPromotes tumor metastasisMetastases Advanced stagesMiRNAspongingLung cancercircPVT1UpPromote cell proliferation and Inhibit cellapoptosisTumor size TNM stage Overall survivalcircFADS2 UpPromote proliferation and invasionTNM LNM Overall survivalcircPTPRA DownInhibit proliferation and migrationMetastasis Overall survivalGastric CancercircHIPK3 UpPromote proliferation and migrationOverall survival Infiltrative type GC cellAdvanced TNM stagecircPVT1UpPromote proliferationOverall survival Diseasefree survivalcircLARP4 DownInhibit proliferation and invasionOverall survivalColorectalcancercircHIPK3 UpPromote proliferation migration invasionmetastasis autophagy and inhibitapoptosisAdvanced TNM stage Lymph nodemetastasis Distant metastasis Advancedtumorcirc_DownInhibit proliferation migration andinvasionTNM stageOverall survivalHepatocellularcarcinomacircHIPK3 UpProliferation migrationcirc_DownInhibit proliferation and migrationTumor differentiation Advanced TNMstage HBVDNA copy numbers LivercirrhosisDifferentiation and tumorSatelliteBladder cancer circHIPK3 DownInhibit Migration invasion andangiogenesisAdvanced tumor Lymph nodemetastasisProteininteractionGastric cancerSelftranslation GlioblastomacircDONSONUpcircAGO2 UpcircSHPRHDowncircFBXW7 DownRef ReferenceFacilitate cancer growth and invasionTNM stage Lymph node metastasisOverall survival and Diseasefree survivalPromote growth invasion and metastasis Metastasis Overall survivalSuppress tumor progression andtumorigenesisInhibit proliferation and cell cycleaccelerationOverall survivalOverall survivalRef[][][][][][][][][][][][][][][][][][] 0cXu Molecular Cancer Page of upregulated in most types of cancers such as gastriccancer colorectal cancer and hepatocellular carcinoma[ ] However it is downregulated in bladdercancer [] Based on these findings the expression pattern of circRNAs in malignant tumors is very complexand thus a highthroughput detection method is neededto determine the differential expression profile of circRNAs to further understand the importance of the differential expression patterns of circRNAs in cancertumorssuggestsThe complex expression pattern of circRNAs also implies complex biological functions and clinical significance in cancer CircRNAs modulate multiple biologicalfunctions of cancer such as proliferation metastasis andangiogenesis [ ] For example circPVT1 promotescell proliferation and inhibits cell apoptosis in lung cancer [] and gastric cancer [] while circHIPK3 inhibitsangiogenesis in bladder cancer [] CircRNAs have important tumorrelated clinical significance and are associated with various clinical features of cancer and patientoutcomes As shown in Table the clinical features ofcancer mainly include cancer differentiation lymph nodemetastasis distant metastasis and the TNM stage [] The association between circRNAs and the clinicalcharacteristics ofthe potentialdiagnostic value of circRNAs For examplelowcircNOL10 expression may indicate a low level ofdifferentiation of[] High circDONSON expression suggests that clinicians shouldclosely monitor lymph node metastasis in patientswith gastric cancer [] The main circRNArelatedindicators of outcomes in patients with cancer areoverall survival and diseasefree survival [ ] Inpatients with gastric cancer high circPVT1 expression indicates poor overall survival and diseasefreesurvival [] while high circLARP4 expression is related to longer overall survival [] CircFBXW7 andcircSHPRH are good prognostic biomarkers for glioblastoma because their high expression results inlonger overall survivaltheimportant role of circRNAs in tumors must not beignored Moreover the crucial role of circRNAs inmediating anticancer drug resistance is emerging[] Here we are the first to summarize the role ofcircRNAs in anticancer drug resistance from the perspective of drug classification[ ] In conclusioncancerlungNonplatinum cytotoxic drugsTraditional anticancer drugs can be divided into cellcyclespecific anticancer drugs and cell cyclenonspecificanticancer drugs according to their cellular kinetics [] Classical cell cyclespecific drugs include topoisomerase inhibitors antimetabolite drugs and some drugs derived from plants [] Typical cell cyclenonspecificanticancer drugs are anticancer antibiotics []isindistributedabundantlyTopoisomerase inhibitorsTopoisomerasealleukaryotic nuclei and resolves topological problems during bioprocesses such as DNA replication and DNAtranscription [] In the 1970s camptothecin was foundto have anticancer activity and topoisomerase I wasidentified as its major target [] Irinotecan is an Sphasespecific camptothecin derivative that exerts efficient anticancer activity against metastatic colorectalcancer CRC [] However clinical resistance to irinotecan is common [] Jian [] found that circ_ increases migration and is involved in irinotecanresistance in CRC Circ_001680 acts as an oncogene bysponging miR340 to upregulate BMI1 in CRC BMI1has been recognized as a positive regulator that inducescancer stem celllike properties [ ] which aredeeply involved in irinotecan resistance [ ] Sphereformation assays showed that SW480 and HCT116 cellswith circ_001680 overexpression formed more stem cellspheres after treatment with irinotecan and had a morerobust cell growth ability than control cells In vivo thesizes and weights of tumors in the control group weremarkedly decreased after treatment with irinotecan butwere not significantly changed in the circ_001680overexpressing group These results indicate that the circ_001680miR340BMI1 axis may contribute to irinotecanresistance by regulating cancer stem celllike propertiesAntimetabolite drugsPemetrexed is a folate antagonist that disrupts folatedependent metabolic processes [ ] Itfunctionsmainly during Sphase Xu [] reported a novelcircRNA circMTHFD2 related to pemetrexed resistancein gastric cancer Overexpression of circMTHFD2 wasconfirmed in pemetrexedresistant gastric cancer cellsFurther investigation showed that circMTHFD2 couldbind with miR124 and promote the protein expressionof FDZ5 and MDR1 to induce pemetrexed resistance5Fluorouracil 5FU a fluoropyrimidine can inhibitthymidylate synthase to induce thymineless cell deathand functions mainly as an Sphase antimetabolite [] 5FU has been the standard treatment for manysolid tumors including CRC for decades [ ] Thebenefit of 5FU is always limited by the development ofresistance and the specific molecular mechanisms arecomplex [] Xiong [] identified differentiallyexpressed circRNAs in 5FUresistant CRC cells bymicroarray hybridization Fortyseven of these circRNAswere upregulated and were downregulated ThesecircRNAs were distributed on each chromosome butmost were located on chromosomes and and respectively suggesting that these chromosomes are more strongly correlated with 5FU resistancethan other chromosomes The top most strongly 0cXu Molecular Cancer Page of upregulated circRNAs were predicted to be capable ofregulating the Wnt signaling pathway which is deeplyinvolved in 5FU resistance [] In addition several FUresistancerelated miRNAs such as miR8853p []may be targets of these circRNAsGemcitabine is another widely used Sphase antimetabolite drug [] Yan [] reported that circ_0035483is significantly increased in TK10 and UO31 renal cancercell lines after treatment with gemcitabine The results ofan MTT assay showed that high expression of circ_ in gemcitabinetreated TK10 cells could enhancecell viability to induce gemcitabine resistance The resultsof RNA pulldown experiments revealed that circ_0035483can bind with miR335 as a sponge Further dualluciferase assay results identified CCNB1 as a target ofmiR335 CCNB1 is an important checkpoint molecule inthe cell cycle and is deeply involved in cell cyclespecificgemcitabine resistance [ ] Promotion of circ_ expression increased the expression of CCNB1and cell apoptosis was inhibited Thus circ_0035483 promotes gemcitabine resistance in human renal cancer cellsby sponging miR335 to upregulate CCNB1found to be differentiallyline and the parental cellGemcitabine resistance can also be developed by selection under exposure to an increasing gradient of gemcitabine in pancreatic cancer PC celllines [] Theline was develgemcitabineresistant SWl990GZ cellline and ciroped from the parental SWl990 cellexpressedcRNAs werebetween this cellline []Twentysix of these circRNAs were upregulated and were downregulated Four circRNAs were validated byqRTPCR Pathway analysis indicated that these dysregulated circRNAs are deeply involved in gemcitabine resistance in PC via modulation of MAPK and mTORsignaling pathways [ ]etShao[]alIn additionestablished theline and comgemcitabineresistant PANC1GR cellpared the differential circRNA profiles between thePANC1GR cell line and the parental PANC1 cell lineA total of circRNAs were identified by highthroughputtranscriptome sequencing as significantlydifferentially expressed in these two celllines ofwhich were upregulated and downregulated Moreover gene ontology GO term and pathway analysis results indicated the functions of these circRNAs in PCprogressionrelated signaling pathways such as the ErbBpathway [] The two most significantly upregulatedchr14101402109 and chr4circRNAs were further validatedandfunctional experiments showed that silencing these twocircRNAs restored gemcitabine sensitivity in PANC1GR cells while overexpression of these circRNAs promoted gemcitabine resistance in both the PANC1 andMIA PACA2 cellIdentification of potentiallinesbinding miRNAs of these two circRNAs by sequenceanalysis suggested that miR1455p a tumor suppressorinhibiting PC progression [] could bind both circRNAs and act as a downstream target Moreover theplasma expression levels of the two circRNAs and miR1455p in gemcitabinetreated PC patients were verifiedvia qRTPCR Consistent with previous results the twocircRNAs were upregulated but miR1455p was downregulated in the plasma of gemcitabineresistant patientsThis finding confirms their potentialfor monitoringgemcitabine resistance via liquid biopsyDrugs derived from plantsTaxol is an Mphase specific plant drug that was originally derived from the bark of the Pacific yew and has become one of the most widely used agents [] Unlikeantimetabolite drugs Taxol can bind tubulin to arrestmitosis and induce apoptosis resulting in cell death []The mechanism underlying Taxol resistance remainspoorly understood [] however ncRNA dysregulationplays an important role in this process [ ] Taxol isan effective chemotherapeutic drug in firstline therapyfor gastric cancer [] Liu [] reported thatcircPVT1 a circRNA derived from the oncogeniclncRNA PVT1 locus [] can mediate Taxol resistancein gastric cancer Overexpression of circPVT1 was confirmed both in Taxolresistant gastric cancer tissues andcells and circPVT1 knockdown promoted apoptosistriggered by Taxol ZEB1 is a wellknown promoter ofTaxol resistance [ ] Mechanistically circPVT1 canpromote ZEB1 expression by sponging miR1243p Inaddition Taxol resistance remains problematic in thetreatment of ovarian cancer [ ] Expression profilesof dysregulated circRNAs were identified in Taxolresistant ovarian cancer tissues A total of aberrantlyexpressed circRNAs were found were upregulatedand downregulated [] Among these circRNAscirc_0063809 was further confirmed by qPCR to be upregulated in both Taxolresistant tissues and cells Invitro and vivo experiments demonstrated that silencingcirc_0063809 promotes Taxolinduced cytotoxicity inTaxolresistant ovarian cancer cells Moreover the results of mechanistic experiments revealed that circ_ can upregulate FOXR2 expression by spongingmiR1252 to contribute to Taxol resistance in ovariancancer Taxol is also recommended for firstline treatment of HER2negative metastatic breast cancer BC[] CircAMOTL1 was found to contribute to Taxol resistance by activating the AKT pathway []Xu [] reported the differentially expressed circRNA profile in the Taxolresistant A549Taxol cell linecompared to the parental A549 cell line A total of circRNAs were identified among which circRNAs were upregulated and were downregulated 0cXu Molecular Cancer Page of GO analysis results showed that the most significantlyenriched terms were linked to Taxol resistancerelatedterms such as GTPase binding and alterations in cell adhesion [ ] In addition Cytoscape was used tovisualize the circRNAmiRNA interaction network Several miRNAs previously reported to drive Taxol resistance were predicted For example miR141 has beensuggested to induce Taxol resistance in ovarian cancer[] and miR34c5p can downregulate p53 to induceTaxol resistance in lung cancer [] Circ_0071799which was upregulated is related to miR141 Downregulated circ_0091931 can interact with miR34c5p Inaddition Li [] reported that circ_0002483 overexpression can enhance Taxol sensitivity in nonsmallcell lung cancer NSCLC Circ_0002483 has been confirmed by a dualluciferase reporter assay to spongemiR1825p and pathway analysis revealed that the circ_0002483miR1825p interaction may be deeply involvedin the FoxO signaling pathway Further analysis indicated that the ² untranslated regions ²UTRs ofGRB2 FOXO1 and FOXO2 contain miR1825p comcirc_plementary0002483miR1825pGRB2FOXO1FOXO3 axiscanreverse Taxol resistance in NSCLCsequences MechanisticallytheYu [] evaluated the overexpression of circ_ in the docetaxelresistant lung adenocarcinomaA549DTX and H1299DTX cell lines Knockdown ofcirc_0003998 in A549DTX and H1299DTX cells partially restored docetaxel sensitivity via promotion of cellapoptosis In addition miR326 was predicted by theCircular RNA Interactome CircInteractome database[] as a potential target of circ_0003998 MiR326 is awellrecognized tumor suppressor and its downregulation is deeply involved in multidrug resistance in tumors[] and a significant negative correlation betweencirc_0003998 expression and miR326 expression wasfound in lung adenocarcinoma tissues Further dualluciferase reporter assays confirmed the direct bindingbetween circ_0003998 and miR326 In addition miR inhibitors were found to significantly reverse theincreased sensitivity to docetaxel caused by circ_ siRNA In summary overexpression of circ_ induces docetaxel resistance in lung adenocarcinoma partially by sponging of miR326 Docetaxelcan also be used in firstline chemotherapy for patientswith metastatic nasopharyngeal carcinoma NPC []Hong [] reported that circCRIM1 binds tomiR422a to upregulate FOXQ1 in NPC FOXQ1 is intimately involved in chemotherapeutic resistance byregulating the epithelialmesenchymal transition EMT[] In vivo and in vitro functional experiments haveconfirmed that FOXQ1 overexpression induced by circCRIM1 also contributes to metastasis and docetaxelresistance in NPC []Anticancer antibioticsAs a cell cyclenonspecific anticancer drug doxorubicinAdriamycin is an anthracycline antibiotic antineoplastic drug widely used in the clinic [] Its underlyinganticancer mechanisms are inhibition of DNA synthesisinterference with topoisomerase II activity and inductionof free radical damage to cells [] CircRNAs have alsobeen reported to play a role in doxorubicin resistanceThe differential expression profile of circRNAs in adoxorubicinresistant acute myeloid leukemia AMLcell line THP1ADM was identified by analysis of ahuman circRNA array Fortynine circRNAs were foundto be differentially expressed with a fold change of inTHP1ADM cells compared to THP1 cells Thirtyfivewere upregulated and were downregulated Amongthese circRNAs circPAN3 was considered a candidatemediator of doxorubicin resistance due to its substantialdifferential expression and the important role of its hostgene PAN3 in AML [] In addition circPAN3 expression was higher in the bone marrow of patients with refractoryrecurrent AML than in the bone marrow ofdoxorubicinsensitive patients Moreover knockdown ofcircPAN3 reversed doxorubicin resistance in THP1ADM cells TargetScan showed that miR1535p andmiR1835p could be target genes of circPAN3 XIAP awellknown chemoresistancerelated gene in AML []was found to be a downstream target of both miR1535pand miR1835p Further mechanistic experiments alsoconfirmed the circPAN3miR1535pmiR1835pXIAPaxis which contributes to doxorubicin resistance in AMLDoxorubicin has also been approved by the FDA for clinical use in patients with BC [] The results of a microarray screen also indicated the important role of circRNAsin doxorubicin resistance in BC [] Eighteen circRNAswere identified as significantly differentially expressed betweenanddoxorubicinsensitive MCF7 cells were upregulatedwhile were downregulated Kyoto Encyclopedia of Genesand Genomes KEGG analysis revealed that the most relevant signaling pathways included the MAPK and PI3KAktsignaling pathways which are closely related to doxorubicinresistance [ ] In addition circ_0006528 expressionwas measured by qRTPCR The results confirmed circ_ overexpression in doxorubicinresistant cell linesand tissues Downregulation of circ_0006528 restored thesensitivity of MCF7ADM and MDAMB231ADM cellsto doxorubicin Moreover miR75p which has been reported to be involved in PI3KAkt signaling pathway activation and chemoresistance in BC [] was predicted to bea target of circ_0006528 while Raf1 a MAPK signalingpathway activator is a direct target of miR75p [] Further experimental results proved that the circ_0006528miR75pRaf1 axis may be responsible for doxorubicin resistance in BCdoxorubicinresistantMCF7ADM 0cXu Molecular Cancer Page of resistanceinreverseddoxorubicinCircKDM4C was reported by Liang to be atumor suppressor in BC [] In vitro experimentsidentified decreased expression of circKDM4C in MDAMB231DOX cells compared with the parental MDAMB231 cells CircKDM4C knockdown in MDAMB cells promoted resistance to doxorubicin whileoverexpression of circKDM4C in MDAMB231DOXcells inhibited cell proliferation enhanced apoptosis andfinallydosedependent and timedependent manners MiR548p waspredicted by CircInteractome database analysis to be atarget of circKDM4C and upregulation of miR548p wasfound to partially abolish circKDM4C overexpressioninduced inhibition of doxorubicin resistance FurthermiRNA target prediction indicated that the ²UTR ofphenazine biosynthesislike domaincontaining proteinPBLD is considered a putative target of miR548pPBLD is associated with various tumor progressionrelated signaling pathways such as the MAPK pathway[] Luciferase reporter assay results confirmed thecircKDM4CmiR548pPBLD axis and the contributionof this axis to doxorubicin resistance was proven bothin vitro and in vivoPlatinum drugsPlatinum drugs such as cisplatin and oxaliplatin arewidely used in the treatment of human cancers and haveachieved clinical success as standard therapies []Oxaliplatin is a platinum complex with oxalate and 1R2R12diaminocyclohexane DACH ligands Oxaliplatin uptake has been reported to be mediated by the anic cation transporters OCT1 and OCT2 which areoverexpressed in CRC cells [] This overexpressionpartially explains why oxaliplatin is an efficient component in the adjuvant FOLFOX treatment regimen inmetastatic CRC Oxaliplatin resistance has become anissue in CRC Drugresistant HCT116 HCT116R cellswere developed by exposure to FOLFOX and circRNAmicroarray analysis was performed to compare circRNAexpression profiles between HCT116R and parentalHCT116 cells [] A total of upregulated and downregulated circRNAs were identified The presenceof two upregulated circRNAs circ_32883 and circ_0338in extracellular vesicles was further validated Circ_ was found to be significantly upregulated in CRCtissues Several microRNAs such as miR5015p werepredicted to interact with circ_32883 to bind to theirtarget genes Hon [] isolated exosomes fromthe cell culture medium of HCT116R and parentalHCT116 cells and used by circRNA microarray analysisto identify significantly upregulated and downregulated circRNAs Consistent with a previous reportcirc_32883 and circ_0338 were upregulated in HCT116R cellderived exosomes In addition the resistance ofHCT116R cells to FOLFOX was partially reversed byknockdown of circ_0338 indicating a potential role forcirc_0338 as a biomarker for FOLFOX resistance inCRC Wang [] identified a novel circRNA circ_ in exosomes derived from oxaliplatinresistantCRC cells This circRNA could be transferred intooxaliplatinsensitive CRC cells and promote glycolysisand oxaliplatin resistance in the recipient cells Furtherexperiments showed that circ_0005963 can sponge miR to upregulate PKM2 In addition sicirc_0005963transferred by exosomes inhibited glycolysis and reversed resistance to oxaliplatin Thus this exosomallyinitiated circ_0005963miR122PKM2 signaling axis hasgreat therapeutic potential for oxaliplatinresistant CRCCisplatin was first synthesized by Michele Peyrone andwas approved by the US FDA for use in testicular andovarian cancer in [] Cisplatin often leads to aninitial therapeutic response but many originally sensitivetumors gradually develop resistance [] Huang et al[] screened circRNAs derived from EIF3a the largest subunit of the translation initiation factor EIF3 incisplatinresistant A549DDP cells Two circRNAs transcribed from EIF3a circ_0004350 and circ_0092857were validated to exhibit differential expression betweenA549DDP and parental A549 cells Moreover downregulation of circ_0004350 and circ_0092857 reversed cisplatin resistancecells Functionalenrichment analysis revealed that these two circEIF3asmay have a synergistic functional effect with their parental EIF3a gene on cisplatin resistancein lung cancerCirc_0076305 is another circRNA identified to promote cisplatin resistance in lung cancer Significantly elevated expression of circ_0076305 was proven incisplatinresistant tissues and cells [] Moreover cisplatin treatment was found to effectively upregulate theexpression of circ_0076305 in A549 and H1650 cellsFurther gain and lossoffunction experiments indicatedthat circ_0076305 can regulate cisplatin resistance inlung cancer cells Bioinformatic and mechanistic experiments such as circRNA immunoprecipitation circRIPand luciferase reporter assays were used to validate thebinding of circ_0076305 to miR2965p as well as tomiR2965p and STAT3 Reports have indicated thatmiR2965p acts as a tumor suppressor in lung cancerand that STAT3 can contribute to cisplatin resistance[ ] Pearson correlation analysis showed that theexpression levels of circ_0076305 are negatively correlated with those of miR2965p and positively correlatedwith those of STAT3 These results suggest that circ_ increases cisplatin resistance by acting as amiR2965p sponge to promote STAT3 expression inlung cancer Zheng [] reported that circPVT1contributes to cisplatin resistance in lung adenocarcinomaaxis Howeverthe miR1455pABCC1via 0cXu Molecular Cancer Page of circRNAs can also act as suppressors of cisplatin resistance in lung cancer Circ_0001946 expression was significantly lower in A549DDP cells than in parentalA549 cells [] The nt long circ_0001946 is derived from cerebellar degenerationrelated protein CDR1 Knockdown of this circRNA was found to increase cisplatin resistance in A549 cells and the resultsof a host cell reactivation HCR assay and Western blotanalysis demonstrated that silencing circ_0001946 activates the nucleotide excision repair NER signalingpathway which increases the ability for DNA damage repair [] to reduce cisplatin sensitivity in lung cancerTreatments for osteosarcoma OS the most commonmalignant bone tumor in teenagers are limited []Cisplatinbased neoadjuvant chemotherapy has greatly impro | Thyroid_Cancer |
"The principal function of iodine acts on thyroid function but in recent years the role of iodinedeficiency in metabolism has also been gradually revealed We aimed to investigate the current status of iodizedsalt consumption and urinary iodine concentration UIC in an urban Chinese population with type diabetes andto further explore whether UIC was associated with diabetic microvascular complicationsMethods Four thousand five hundred fiftynine subjects with diabetes from communities in downtownShanghai were enrolled in the crosssectional Metal Study in UIC was detected using an inductively coupledplasmamass spectrometer Diabetic kidney disease DKD was defined as urinary albumintocreatinine ratioUACR mgg or estimated glomerular filtration rate mLmin173 m2 Diabetic retinopathy DR wasevaluated by highquality fundus photographs and was remotely read by ophthalmologistResults The median UIC of subjects with diabetes was μgL in downtown Shanghai Among allthe subjects consumed noniodized salt and were iodine deficient Iodine deficiency UIC μgLwas associated with an increased odds of DKD OR 95CI after adjustment for age sex educationcurrent smokers BMI HbA1c duration of diabetes dyslipidemia thyroidstimulating hormone and free thyroxineNo association was observed between UIC and DR after multivariable adjustmentConclusions A concerning number of subjects with diabetes consumed noniodized salt and suffered from iodinedeficiency in coastal regions of China Low UIC might be a risk factor for DKD which should be further confirmedby longitudinal prospective studiesKeywords Iodized salt Type diabetes Diabetic kidney disease Urinary iodine concentration Epidemiology Correspondence wnj486126com luyingli2008126com Chi Chen and Yi Chen contributed equally to this workInstitute and Department of Endocrinology and Metabolism Shanghai NinthPeoples Hospital Shanghai JiaoTong University School of MedicineShanghai 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 Nutrition Metabolism Page of IntroductionType Diabetes Mellitus T2DM has become a seriousglobal health care burden causing microvascular complications which are associated with increased disabilityreduced quality of life and life expectancy [ ] It wasestimated that there were million cases of adult diabetes worldwide in and the number was projectedto increase to million by [] leading to highincidence and prevalence of microvascular complications Approximately one third with T2DM will developdiabetic retinopathy DR and one quarter will developdiabetic kidney disease DKD [] Thus it is critical toidentify and control some novel modifiable risk factorscontributing to microvascular complications in patientswith T2DMIodine is an indispensible micronutrient for the synthesis of thyroid hormones Iodine deficiency ID in early lifeimpairs neurodevelopment and also has many adverse effects throughout various life stages [] Over the past years substantial progress has been achieved in the worldwide effort to eliminate iodine deficiency disorders IDDby salt iodization program However this program is vulnerable and requires a longterm commitment from governments In several countries where ID had been onceeliminated salt iodization programs were discontinuedand ID has now reappeared []The principal function of iodine acts on thyroid function but in recent years the role of ID in metabolismhas also been gradually revealed Analysis of data fromthe National Health and Nutrition Survey NHANES found that in US adults low urinary iodineconcentration UIC was associated with dyslipidemia[] and coronary artery disease [] Moreover O S AlAttas [] reported that UIC is markedly decreasedin T2DM and urinary iodine was negatively associatedwith insulin resistance in patients with T2DM Most recently Mingyue Jin [] also found that at a lowerUIC μgL the prevalence of diabetes significantlyincreased relative to an UIC of μgL Animalstudies also showed that iodine supplementation couldreduce the blood glucose levels and improve the insulinsensitivity in goats [] However the role of iodine nutrition on diabetic microvascular complications has notbeen studiedThe aim of this study was to investigate the currentstatus of iodized salt consumption and UICs in an urbanChinese population with T2DMand additionallywhether UIC is associated with diabetic microvascularcomplications including DKD and DRMethodsStudy populationThe crosssectional METAL study Environmental Pollutant Exposure and Metabolic Diseases in Shanghaiwwwchictrcn ChiCTR1800017573 was launchedto investigate the association between iodine nutritionand microvascular complications in Chinese adults withdiabetes We recruited study participants from sevencommunities in Huangpu and Pudong new districtShanghai China Huangpu district located in downtownShanghai is the administrative economic and culturalcenter of the metropolitan coastal city [] Pudong newdistrict is the symbol of Chinas reform and ingup[] We randomly selected half of patients with diabetesfrom the registration platform in each communityhealthcare center Chinese citizens ¥ years old whohad lived in their current area for ¥ months were included In August a total of subjects withT2DM who were years of age received an examination Participants with missing UIC values n were excluded Finally participants were involvedin the present analysisThe study received ethical approval from the EthicsCommittee of Shanghai Ninth Peoples Hospital Shanghai Jiao Tong University School of Medicine All procedures followed were abided by the ethical guidelines ofthe Declaration of Helsinki as reflected in a prioriapproval by the appropriate institutional review committee Informed consent was received from all participantsincluded in the study prior to the data collectionMeasurementsinThe same welltrained and experienced personnelSPECTChina study [] used a questionnaire to collect information on sociodemographic characteristicseducation medical history family history and lifestylerisk factors Weight and height were measured using abalance beam and a vertical ruler in light clothing andwithout shoes Body mass index BMI was calculated asthe ratio of weight in kilograms divided by height in meters squared Current smoking was defined as havingsmoked at least cigarettes in ones lifetime and currently smoking cigarettes [] Especially For the pastthree years which type of salt was used in your familywas applied to collect information about type of saltThree options for this item were provided only iodized salt only noniodized salt bothBlood samples were drawn between am and am after an overnight fast Blood was refrigerated immediately after phlebotomy and in hours it was centrifugated and the serum was aliquoted and frozen in acentrallaboratory Glycated hemoglobin HbA1c wasmeasured by highperformance liquid chromatographyMQ2000PT Medconn Shanghai China Fastingplasma glucose serum creatininetotalcholesterol high HDL and lowdensity lipoproteinLDL were performed with a Beckman Coulter AU Brea USA Serum thyroidstimulating hormone TSHtriglycerides 0cChen Nutrition Metabolism Page of and free thyroxine FT4 were measured by electrochemiluminescence Roche E601 GermanyMorning fasting spot urine samples collected were refrigerated immediately and frozen at °C in hoursUIC was detected using an inductively coupled plasmamass spectrometer ICPMS No 7700x Agilent Technologies Inc USA The concentrations of urine albuminand creatinine were determined with a Beckman CoulterAU Brea USA using a turbidimetric immunoassayand an enzymatic method respectively Urinary albumintocreatinine ratio UACR was calculated as the urinaryalbumin concentrations divided by the urinary creatinineconcentrations and expressed in mggDR screening was evaluated by mydriatic binocular indirect ophthalmoscopy Topcon TRCNW400 NonMydriatic Retinal Camera Oakland USA Fundus photographs were read by an experienced ophthalmologistspecialized in retinaOutcome definitionDyslipidemia was defined as total cholesterol ¥mmolL mgdL triglycerides ¥ mmolL mgdL LDL ¥ mmolL mgdL HDL mmolL mgdL or selfreported previous diagnosisof hyperlipidemia by physicians according to the modified National Cholesterol Education ProgramAdultTreatment Panel IIIThe estimated glomerular filtration rate eGFR was calculated using the Chronic Kidney Disease EpidemiologyCollaboration CKDEPI equation for Asian origin []As suggested by American Diabetes Association ADAhigh UACR was defined as UACR ¥ mgg reducedeGFR as eGFR mlmin173m2 and DKD as UACR mgg or eGFR mLmin173 m2 []The internationally accepted DR classification by theGlobal Diabetic Retinopathy Project Group in was applied [] The classification was no retinopathynonproliferative DR intraretinal microaneurysmshemorrhages venous beading prominent microvascularabnormalities and proliferative DR neovascularizationor vitreouspreretinal hemorrhagesStatistical analysisStatistical analysis was run with SPSS IBM Corporation Armonk NY USA General characteristics arepresented as median with the interquartile range IQRfor continuous variables or as proportion for categoricalvariables MannWhitney U test and the KruskalWallistest were used for comparison of two or more groups ofnonnormally distributed data Pearsons Ï2 tests wereperformed to compare categorical variablesThe associations of UIC with elevation of UACR reduction of eGFR DKD and DR were analyzed with logistic regression analyses The results are presented as oddsratio OR and confidence intervals CIs Model was unadjusted Model was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Table General characteristics of study participants by UIC categoriesUrinary iodineAge yrAdequate Low Women UIC μgLFPG mmolLHbA1c BMI kgm2Duration of diabetes yrCurrent smokers Beyond high school education TSH mIULFT4 pmolLBlood lipidsTotal cholesterol mmolLLDLC mmolLHDLC mmolLTriglycerides mmolLMore than adequate Excessive P Data are summarized as median interquartile range for continuous variables or as number with proportion for categorical variablesUIC Urinary iodine concentration FPG Fasting plasma glucose HbA1c Glycated hemoglobin BMI Body mass index LDLC Low density lipoprotein cholesterol HDLC High density lipoprotein cholesterol TSH Thyroidstimulating hormone FT4 Free thyroxineUrinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ¥ μgL 0cChen Nutrition Metabolism Page of ResultsGeneral characteristics of the study populationThe general characteristics are presented in Table The mean age of the study population was nearlyone half were women About of the studypopulation were overweight or obese BMI ¥ kgm2and were current smokers The percentage of aneducational level beyond high school was and theaverage duration of diabetes was yearsCompared to those with adequate iodine nutritionsubjects with ID were slightly older and were more likelyto be women These subjects also had higher TSH lowerBMI and a lower percentage of current smokers Inaddition subjects with more than adequate and excessiodine nutrition were slightly younger and had higherBMI but comparable TSHUrinary iodine concentration and type of salt intake inthe populationThe distribution of UICs in the study population is presented in Fig The median 25th75th percentile UICof subjects with diabetes was μgL indowntown Shanghai which falls within the range of μgL that WHOUNICEFICCIDD categorize asadequate Urinary iodine measurements indicative of IDUIC μgL were present in of the studypopulation Meanwhile and of the populationshowed more than adequate UIC μgL andexcess iodine intake UIC ¥ μgL respectivelyThe distribution of type of salt intake is presented inFig As high as of the study population consumed noniodized salt consumed iodized saltand consumed both Logistic regression analysisshowed that compared to those who consumed iodizedsalt subjects consumed noniodized salt were morelikely to be women OR 95CI and havea higher educational attainment OR 95CI but a comparable age OR 95CI Association of urinary iodine concentration with elevationof UACR reduction of eGFR and DKDThe association of UIC with elevation of UACR reduction of eGFR and DKD is shown in Table Comparedwith those with adequate iodine nutrition subjects withID had an increased risk of elevation of UACR OR 95CI reduction of eGFR OR 95CI and DKD OR 95CI Adjustment for age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 did not attenuate the association of ID with UACRand DKD However multivariable adjustment weakenedthe association between ID and reduction of eGFR further such that it was no longer significant Meanwhilesubjects with more than adequate and excess iodine nutrition did not have an increased risk of elevation ofUACR reduction of eGFR and DKD after multivariableadjustmentAssociation of urinary iodine concentration with DRTable presents the association of UIC with nonproliferative and proliferative DR Compared with thosewith adequate iodine nutritionthe ORs of nonproliferative and proliferative DR in subjects with morethan adequate iodine nutrition were 95CI Fig Distribution of UICs in the study population 0cChen Nutrition Metabolism Page of Fig Distribution of type of salt consumed in the study populationthan adequate and 95CI respectively Multivariable adjustment weakened the association betweenmoreiodine nutrition and nonproliferative DR further such that the association was nolonger significant There was no significant associationobserved between DR and ID and excessiodinenutritionDiscussionIn this study among over communitydwellingChinese adults with diabetes we found that of thesubjects consumed noniodized salt and had IDIodine deficiency was significantly associated with ahigher prevalence of elevated UACR and DKDindependently of age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 To the best of our knowledge this is the first studyto investigate the current status of iodized salt consumption and iodine nutrition status in a relatively largepopulation with diabetes and further investigate the association between UIC and diabetic microvascularcomplicationsChina was once severely affected by IDD and hence amandatory Universal Salt Iodization USI program wasTable Association of urinary iodine with elevation of UACR and reduction of eGFRUrinary iodineHigh UACRAdequateLowMore than adequateExcessivePrevalenceOdds RatioModel Model Reduced eGFRPrevalence Odds RatioModel Model DKDPrevalence Odds Ratio Ref Ref Ref Ref Model Model Ref Ref Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with elevation of UACR reduction of eGFRand DKD P Model was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4High UACR was defined as UACR ¥ mgg reduced eGFR as eGFR mlmin173 m2 and DKD as UACR mgg or eGFR mLmin173 m2Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ¥ μgL 0cChen Nutrition Metabolism Page of Table Association of urinary iodine with DRUrinary iodineNonproliferative DRAdequatePrevalence Odds RatioModel Model Proliferative DRPrevalence Odds RatioModel Model Ref Ref Ref RefLowMore than adequateExcessive P Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with DRModel was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ¥ μgLintroduced in and was successful in eliminatingIDD However since the prevalence of thyroid diseaseshas markedly increased in recent years some concernsabout the USI have circulated especially among coastalresidents in urban areas [] In the present analysis themedian UIC of residents with diabetes in downtownShanghai has fallen to marginal levels of iodine sufficiency μgL and more surprisingly more thanhalf of the subjects consumed noniodized saltand were iodine deficient Compared with a studyconducted by the Shanghai Municipal Center for DiseaseControl and Prevention CDC in of participants used iodized salt and were iodine deficientat that time [] During Zhongyan Shanand her colleagues performed a crosssectional study ineastern and central China and reported that the medianUIC was μgL in schoolaged children and μgLin the total cohort population [] Our previous studyconducted on the general population found that consumed noniodized salt in the urban area of Shanghai in []The present study indicates that an increasing numberof urban residents in downtown Shanghai prefer to usenoniodized salt in recent years and suffer from IDWomen and those with a higher educational level weremore tended to consume noniodized salt WhyChanges in the reported spectrum and growing incidence of thyroid disorders have been linked to the increased iodine intake resulting from USI in the localmedia and international medical literature [] Thosewith high educational attainment were more likely to beconfused by these information and worry about theirthyroid health and call for liberalizing provincial controlof sales of noniodized salt Formerly needed a prescriptionthe sale of noniodized salt has now beenunofficially allowed by some coastal city authorities []In addition T2DM is associated with an increased riskof multiple thyroid disorders such as thyroid nodule[] thyroid cancer [] and autoimmune thyroid diseases [] Since the prevalence of thyroid abnormalitieswere found to be much higher in females than males[] it is reasonable to deduce that these women wouldhave a higher tendency towards consuming noniodizedsalt after diagnosis of thyroid abnormalitiesseafood alone to provide sufficientConsidering that Shanghai is a coastal city local residents believe that they should never suffer from IDDwith high iodineenriched aquatic products consumption However it may not be true to rely on consumption ofiodineActually the environmental levels of iodine in Shanghaiare deficient μgL [] Furthermore based on theresearch initiated by Shanghai Municipal CDC iodizedsalt contributed of the total dietary iodine inShanghai[] Aquatic products which residentsthought to be rich in iodine accounted for only ofthe total dietary iodine []Thus iodized salt is still themain source foriodine supplementation in coastalpopulationsDKD wascommonlydefinedby ADA asUACR¥30mgg or eGFR60 mlmin per m2 []We found that ID was associated with elevated UACRand DKD The mechanism underlying the association ofID with DKD is not yet fully understood Deficiency ofiodine could reduce thyroid hormone production andelevate TSH It has been reported that lower free triiodothyronine and elevated TSH have significant associationwith risk for albuminuria in T2DM [ ] Moreoverinadequate iodine intake is significantly correlated withan increase in oxidative stress [] Recent evidence hasshown thattumorinflammatory cytokinessuch as 0cChen Nutrition Metabolism Page of necrosis factoralpha and interleukin1 play a pivotalrole in the pathogenesis of DKD [] Therefore we suppose the possible mechanism may be via inflammatoryresponseIn view of the evergrowing prevalence of T2DM andDKD all over the world successive intervention in thislarge population can have important impact on publichealth ID unlike most micronutrient deficiencies is notrestricted to people in developing countries with poordiets Since salt iodization is simple effective and inexpensive the best strategy to control ID is addition ofiodine into salt in nearly all countries [] Monitoringiodine situation of people with diabetes is of critical significance and education programs to diabetes especiallywomen with high academic background may also include information of adequate iodine intake in our clinical practice Our study shed light on the possiblebeneficial effect of iodine supplementation in reducingalbuminuria in T2DM which warrants further investigation in welldesigned randomized controlled trialOur study benefited from its welldefined communitybased participants with a relatively large sample sizeSecond regarding the novelty our study is the first toprovide iodine status and linked iodine insufficiency toan increased risk of albuminuria in people with diabetesThird we used ICPMS to detect UIC in the presentanalysis which was considered as the goldstandardmethod [] There were also some limitations weshould acknowledge First no causal relationship couldbe determined due to the crosssectional design of thestudy and thus our findings need to be validated by longitudinal prospective studies Second although UIC isrecommended by the WHOICCIDDUNICEF for evaluation of iodine status at the population level and widelyused in largescale epidemiological studies [ ] thesingle spot urine measurement may not accurately assesslongterm iodine status at the individual level Actuallyinter and intraindividual variability exists in UIC []However the application of a large sample size from to subjects per subgroup may counteract thebias related to the use of only one casual urine sample[] Future followup studies collecting 24h urine specimens twice are needed to replicate the present resultsConclusionA large proportion of diabetic patients in downtownShanghai consumed noniodized salt and had ID IDmay increase the risk of DKD independent of thyroidfunction in diabetic patients Maintaining USI at an appropriate level is indispensable for diabetic patients Cohort and intervention studies as well as basic researchexploring the effect and mechanism of iodine supplementation on renal function are warrantedAbbreviationsT2DM Type Diabetes Mellitus DKD Diabetic kidney disease DR Diabeticretinopathy ID Iodine deficiency UIC Urinary iodine concentrationTSH Thyroidstimulating hormone BMI Body mass index OR Odds ratioCI Confidence interval UACR Urinary albumintocreatinine ratioeGFR Estimated glomerular filtration rateAcknowledgementsThe authors thank Xiaojin Wang and Bingshun Wang from the Departmentof Biostatistics and Shanghai Jiaotong University School of Medicine for dataprocessingAuthors contributionsYL and NW designed the study CC YC HZ FX BH WZ YW HWand NW conducted the research CC and YC analyzed the data and wrotethe manuscript CC and YC contributed equally The final manuscript wasread and approved by all authorsFundingThis study was supported by National Natural Science Foundation of China Science and Technology Commission of ShanghaiMunicipality the Fourth Round of ThreeYear Public HealthAction Plan of Shanghai by the Shanghai Municipal Commission of Healthand Family Planning 20164Y0079 Municipal Human Resources DevelopmentProgram for Outstanding Young Talents in Medical and Health Sciences inShanghai 2017YQ053 Fundamental research program funding of NinthPeoples Hospital affiliated to Shanghai Jiao Tong university School of MedicineJYZZ099 Shanghai Sailing Program 20YF1423500Availability of data and materialsThe datasets during andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Ethics Committee of Shanghai NinthPeoples Hospital Shanghai Jiao Tong University School of Medicine Writteninformed consent was received from all participantsConsent for publicationNACompeting interestsNo potential conflicts of interest relevant to this were reportedReceived May Accepted August ReferencesKahm K Laxy M Schneider U Rogowski WH Lhachimi SK Holle R Healthcare costs associated with incident complications in patients with type diabetes in Germany Diabetes Care Chen C Chen Q Nie B Zhang H Zhai H Zhao L Trends in bonemineral density osteoporosis and oste ia among US adults withprediabetes Diabetes Care Cho NH Shaw JE Karuranga S Huang Y da Rocha Fernandes JD OhlroggeAW IDF diabetes atlas 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High glucose HG induced podocytes injury plays an important role in diabetes nephropathy DN development Long noncoding RNA cancer susceptibility candidate CASC2 was found to be decreased in serum of DN patients We aimed to explore the function and possible mechanism of CASC2 in HG induced podocytes injuryMethods Under normal glucose NG HG and mannitol stimulated podocyte conditions the levels of CASC2 microRNA95p miR95p and peroxisome proliferatoractivated receptor gamma PPARÎ were examined by quantitative realtime polymerase chain reaction qRTPCR Podocyte injury was evaluated by measuring cell viability and apoptosis of CIHP1 cells were checked by cell counting kit8 CCK8 assay and flow cytometry respectively Western blot was used to detect all protein levels Dualluciferase reporter RNA immunoprecipitation RIP and RNA pulldown assays were performed to confirm the relationship between CASC2 and miR95pResults HG stimulation inhibited the expression levels of CASC2 and PPARÎ but promoted the expression of miR95p HG could restrain cell viability autophagy and facilitate apoptosis in CIHP1 cells while CASC2 overexpression could reverse HGinduced podocytes injury Furthermore CASC2 could be used as a ceRNA to adsorb miR95p and miR95p mimic overturned the effects of CASC2 on cell viability autophagy and apoptosis in HGstimulated podocytes Additionally PPARÎ was a target gene of miR95p and CASC2 could weaken the HGinduced podocytes injury by upregulating PPARÎConclusion CASC2 increased cell viability autophagy and inhibited cell apoptosis by regulating miR95pPPARÎ axis thus reducing the HGinduced podocytes injuryKeywords High glucose Podocyte CASC2 miR95p PPARÎCorrespondence gltxs009163com Department of Nephrology Taian Campus of the 960th Hospital of the Chinese Peoples Liberation Army No217 Huanshan Road Taishan District Taian Shandong ChinaFull list of author information is available at the end of the BackgroundDiabetes is a common endocrine disease among which the prevalence of diabetes nephropathy DN is [] It is estimated that the number of DN patients is expected to increase to million by [] DN is characterized by the presence of albuminuria and a decreased glomerular filtration rate [] Podocyte cells podocytes are epithelial cells in 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 0cLi a0et a0al Diabetol Metab Syndr Page of visceral layer of renal follicles which play a key role in the pathogenesis of DN and are an important component of glomerular filtration barrier [ ] Several studies have revealed the correlation between podocytes injury death and apoptosis and albuminuria [] and reducing podocyte injury can improve DN [] However the mechanism for alleviating podocytes injury remains unclearLong noncoding RNAs lncRNAs are nonproteincoding RNA molecules longer than nucleotides which are widely regarded as the important regulators in cellular function and disease processes [] Increased evidences suggested that lncRNA could modulate DN progression For instance lncRNAs GM5524 and GM15645 could regulate the HGstimulated podocyte autophagy in DN [] LncRNA PVT1 knockdown repressed podocytes injury and apoptosis via increasing FOXA1 [] However there are still many lncRNAs in DN function and molecular mechanisms have not been studiedLncRNA cancer susceptibility candidate CASC2 located on chromosome 10q26 plays a regulatory role as an anticancer factor in various cancers such as hepatocellular carcinoma [] and pancreatic carcinoma [] Recently Wang et a0al revealed that CASC2 was specifically reduced in serum and renal tissues of type diabetes patients with chronic renal failure and followup identified that the serum of patients with low CASC2 expression had higher incidence of chronic renal failure [] MicroRNA95p miR95p is both a tumor depressor and a tumor promoter [ ] A report demonstrated that miR95p was related to complications of nephropathy in Type and Type diabetes patients [] The mechanism by which lncRNA can serve as the competing endogenous RNA ceRNA for miRNA to modulate the abundance of mRNA has been widely reported [] Peroxisome proliferatoractivated receptor gamma PPARÎ is implicated in several metabolic syndromes including DN Downregulated PPARÎ could activate catenin signaling to destroy podocyte architectural integrity and increase cell apoptosis in DN [] Furthermore lncRNA TUG1 could relieve extracellular matrix accumulation by sponging miR377 and regulating PPARÎ in DN [] Based on the above findings we speculated whether CASC2 can modulate PPARÎ expression by serving as a ceRNA of miR95p in DNIn this work we aimed to explore the effects of CASC2 on cell viability apoptosis and autophagy in high glucose HG induced podocytes and probe the relationship among CASC2 miR95p and PPARÎ providing a new perspective on the molecular mechanism of podocytes injury in DNMaterials and a0methodsCell culture and a0high glucose inductionHuman podocytes CIHP1 Ximbio London USA were cultured in a Dulbeccos modified Eagles medium DMEM Invitrogen Carlsbad CA USA containing fetal bovine serum FBS Gibco Carlsbad CA USA at a temperature of a0°C with CO2 When cells density reached about CIHP1 cells were exposed to normal glucose NG a0mM high glucose HG a0mM or mannitol a0mM and the exposure time was determined by individual experiments requiredCell transfectionCASC2 overexpressed plasmid CASC2 and its control Vector small interfering RNAs against CASC2 and PPARÎ siCASC2 siPPARÎ and matched siNC were provided by GenePharma Shanghai China miR95p mimic miR95p inhibitor antimiR95p and their corresponding references miRNC antiNC were synthesized by Beyotime Beijing China Transfection of podocytes was performed by using Lipofectamine InvitrogenQuantitative realtime polymerase chain reaction qRTPCRThe RNA in CIHP1 cells was extracted by TRIzol Invitrogen and the complementary DNA cDNA was synthesized via reverse transcription using HiScript Q RT Super Mix Vazyme Piscataway NJ USA The reverse transcription was performed at a0°C for a0min and at a0°C for a0s qRTPCR analysis was conducted on RealTime PCR System Applied Biosystems Foster City CA USA using the SYBR premix Ex TaqIIkit TaKaRa Wuhan China Glyceraldehyde3phosphate dehydrogenase GAPDH and U6 were used as endogenous controls for CASC2PPARÎ and miR95p respectively The primers used in this paper were synthesized by GenePharma and the sequences were used as below CASC2 forward ²GCA CAT TGG ACG GTG TTT CC3² reverse R ²CCC AGT CCT TCA CAG GTC AC3² miR95p F ²GTG CAG GGT CCG AGGT3² R ²GCG CTC TTT GGT TAT CTA GC3² PPARÎ F ²AGA GCC TTC CAA CTC CCT CA3² R ²AAC AGC TTC TCC TTC TCG GC3² U6 F ²TTG GTG CTC GCT TCG GCA ² R ²GTG CAG GGT CCG AGGT3² GAPDH F ²GGA GTC CAC TGG TGT CTT CA3² R ²GGG AAC TGA GCA ATT GGT GG3²F Cell viability and a0apoptosis detectionCIHP1 cells were tiled into the 96well plates and exposed to different treatments HG NG HG Vector HG CASC2 and so on At given points in time a0h a0h and a0h a0µL cell counting kit8 CCK8 0cLi a0et a0al Diabetol Metab Syndr Page of Beyotime was added to the cells and cultured for another a0h at a0°C Finally the absorbance at a0nm was measured by BiotekEpoch2 Beijing ChinaThe apoptosis of podocytes CIHP1 was estimated at a0h after exposure to different treatments by using an Annexin V fluorescein isothiocyanate FITC and propidium iodide PI apoptosis detection kit Keygen Beijing China Briefly podocytes were collected and were then suspended in a0µL FITC and a0µL PI in the absence of light for a0 min The apoptosis of CIHP1 cells was checked by a flow cytometer BD Biosciences Franklin Lake NJ USAWestern blot assayTotal protein from CIHP1 cells was extracted by RIPA Beyotime and denatured at a0°C for a0min before separation and then transferred to polyvinylidene difluoride PVDF Beyotime membranes Membranes were sealed with milk for a0 h before incubation with primary antibodies against Bcell lymphoma2 BCL2 Abcam Cambridge MA USA Cleavedcaspase3 Abcam Light chain 3II LC3II Abcam LC3I Abcam Beclin Beyotime PPARÎ Abcam or GAPDH Beyotime overnight at a0 °C HRPconjugated secondary antibody Abcam was employed to incubate the membranes for another a0h And the proteins were visualized by using BeyoECL Moon BeyotimeDualluciferase reporter assayCASC2 wild type CASC2wt with miR95p binding sites and its mutant type CASC2mut without binding sites were cotransfected into CIHP1 cells with miR95p or miRNC respectively Transfection was continued for a0h and luciferase activity was evaluated through a Dualluciferase reporter kit Promega Madison WI USA In the same manner PPARÎ ²untranslated region ²UTRwt with miR95p binding sites and PPARÎ ²UTRmut were cotransfected into cells with miR95p or miRNC respectively and the luciferase activity was detectedRNA immunoprecipitation RIP assay and a0RNA pulldown assayRIP detection was conducted using a Magna RIP RNABinding Protein Immunoprecipitation Kit Millipore Billerica MA USA CIHP1 cells were treated with miR95p or miRNC a0h later cells were lysed in RIP Lysis Buffer containing protease inhibitors Then Argonaute2 Ago2 or ImmunoglobulinG IgG antibody Abcam were added to the cell lysates overnight at a0°C and the immunoprecipitated RNAs were obtained CASC2 and miR95p levels were estimated using qRTPCR analysisCIHP1 cells were transfected with Biotin labeled BiomiR95p and BiomiRNC respectively At a0 h postobtained by using a Pierce¢ Magnetic RNA PullDown transfection cells were collected and the bound RNA was Kit Thermo Fisher Scientific Waltham MA USA according to the instructions Finally CASC2 enrichment was assessed by qRTPCRStatistical analysisData were acquired from at least three independent repetitions and displayed as mean ± standard deviation SD Difference analysis was conducted by Students ttest with two groups and oneway analysis of variance ANOVA with multiple groups using GraphPad Prism The P value less than was regarded as statistically distinctResultsCASC2 alleviated the a0HGinduced podocytes injuryFirstly we examined the expression of CASC2 in human podocytes treated with NG HG or mannitol by qRTPCR The results showed that HG significantly decreased CASC2 expression in CIHP1 cells compared with NG and mannitol treatment Fig a0 1a In addition a timedependent reduction in CASC2 expression was displayed in HGtreated CIHP1 cells and a0h Fig a01b In view of the expression of CASC2 was substantially reduced at a0h of HG stimulation we then overexpressed CASC2 in HGstimulated CIHP1 cells for a0 h and overexpression efficiency was identified by qRTPCR As shown in Fig a01c CASC2 expression was obviously promoted in HGstimulated CIHP1 cells after transfection of CASC2 for a0 h CCK8 and flow cytometry results indicated that overexpression of CASC2 induced cell viability Fig a01d and retarded apoptosis Fig a01e in HGtreated CIHP1 cells To confirm the results of apoptosis we detected the expression of apoptosis marker proteins BCL2 and Cleavedcaspase3 Western blot assay demonstrated that upregulation of CASC2 enhanced BCL2 expression and silenced Cleavedcaspase3 expression Fig a0 1f which was in agreement with the results of Annexin VFITCPI Furthermore HG could reduce the ratio of LC3IILC3I and Beclin expression in CIHP1 cells and CASC2 overexpression reversed the effects of HG on the expression of autophagy related proteins Fig a01g The above findings indicated that CASC2 could alleviate the HGinduced podocytes injury by affecting cell viability apoptosis and autophagyCASC2 directly interacted with a0miR5pLncRNA generally functions as a sponge for miRNA in human diseases [] We speculated whether CASC2 could also act as miRNA sponge to regulate 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 alleviated the HGinduced podocytes injury a The expression of CASC2in CIHP1 cells treated with normal glucose NG high glucose HG or mannitol was detected by qRTPCR b After CIHP1 cells were treated with HG mM for h h and h respectively CASC2 expression was measured by qRTPCR c CIHP1 cells were divided into four groups which were control NG mM HG mM HG vector and HG CASC2 CASC2 expression was detected by qRTPCR d Cell viability was assessed by CCK8 assay e Cell apoptosis was examined by flow cytometry f g Western blot assay was used to determine the expression levels of apoptosisrelated proteins BCL2 and Cleavedcaspase3 and autophagy related proteins LC3II LC3I and Beclin P 0cLi a0et a0al Diabetol Metab Syndr Page of HGinduced podocytes injury As shown in Fig a0 2a we found that miR95p was upregulated in HGtreated CIHP1 cells compared to cells treated with NG or mannitol and miR95p expression was drastically augmented in HGtreated CIHP1 cells in a timedependent manner Fig a0 2b Interestingly there were complementary sites between miR95p and CASC2 by bioinformatics website starBase v20 Fig a0 2c Dualluciferase reporter assay showed that the luciferase activity of CASC2wt was obviously decreased in CIHP1 cells transfected with miR95p than that cells transfected with miRNC whereas it was no significant difference in luciferase activity of CASC2mut Fig a02d RIP assay indicated that the enrichments of CASC2 and miR95p were higher in CIHP1 cells incubated with Ago2 Fig a02e RNA pulldown assay further revealed that the enrichment of CASC2 in BiomiR95p group was aggrandized relative to that BioNC group Fig a02f These results strongly supported that CASC2 could specifically bind to miR95p Meanwhile qRTPCR data showed that CASC2 knockdown in CIHP1 cells elevated miR95p expression and CASC2 overexpression degraded miR95p expression Fig a02g h These results suggested that CASC2 could act as a ceRNA to negatively regulated miR95p expression in podocytesCASC2 regulated the a0HGinduced podocytes injury via a0targeting miR5pAs presented in Fig a0 3a miR95p mimic miR95p could reverse the inhibitory effect of CASC2 overexpression on miR95p expression in HGinduced CIHP1 cells As expected the impact of CASC2 on promoting cell activity Fig a03b and inhibiting cell apoptosis Fig a03c in HGstimulated CIHP1 cells was offset by miR95p Simultaneously the inhibition of CASC2 on the protein expression of Cleavedcaspase3 and the promotion of CASC2 on LC3IILC3I ratio as well as the levels of BCL2 and Beclin could be weakened by transfection of miR95p in HGinduced CIHP1 cells Fig a03d e The obtained data proved that CASC2 attenuated the HGinduced podocytes injury by downregulating miR95pFig CASC2 directly interacted with miR95p a The expression of miR95pin CIHP1 cells treated with normal glucose NG high glucose HG or mannitol was measured by qRTPCR b After CIHP1 cells were treated with HG mM for h h and h respectively miR95p expression was examined by qRTPCR c StarBase v20 was used to predict the target miRNAs of CASC2 df Dual luciferase reporter RIP and RNA pulldown assays were utilized to assess the combination of CASC2 and miR95p g CASC2 expression in CIHP1 cells transfected with siNC or siCASC2 was determined by qRTPCR h The expression of miR95pin CIHP1 cells transfected with siNC siCASC2 Vector or CASC2 was measured using qRTPCR analysis P 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 regulated the HGinduced podocytes injury via targeting miR95p The HGtreated CIHP1 cells were divided into four groups Vector CASC2 CASC2 miRNC and HG miR95p a The expression of miR95p was examined by qRTPCR b c Cell viability and apoptosis were evaluated by CCK8 assay and flow cytometry respectively d e The expression levels of apoptosisrelated proteins BCL2 and Cleavedcaspase3 and autophagy related proteins LC3II LC3I and Beclin were detected by western blot assay P CASC2 acted as a0a a0ceRNA by a0sponging miR5p to a0facilitate PPARÎ expressionAs appeared in Fig a04ad HG inhibited the mRNA and protein levels of PPARÎ in CIHP1 cells compared to NG and mannitol stimulation At a0 h after the induction of HG the mRNA and protein levels of PPARÎ were dwindled in CIHP1 cells The effect of HG treatment on PPARÎ expression was the opposite of that of miR95p thus we speculated whether there was a connection between miR95p and PPARÎ As presented in Fig a04e there were binding sites for miR95p in the ²UTR of PPARÎ Dualluciferase reporter assay showed that miR95p markedly decreased the luciferase activity of PPARÎ ²UTRwt in CIHP1 cells than that PPARÎ ²UTRmut Fig a0 4f suggesting PPARÎ was the target mRNA of miR95p Then we examined the effect of miR95p on PPARÎ expression the interference efficiency of antimiR95p on miR95p expression was first examined by qRTPCR Fig a0 4g Western blot data showed that the overexpressed miR95p could restrain the protein expression of PPARÎ while the decreased miR95p could raise PPARÎ protein expression Fig a04h Additionally we found that CASC2 depletion reduced the protein expression of PPARÎ and cotransfection of antimiR95p could reverse this effect Fig a04i The above findings revealed that CASC2 positively regulated PPARÎ expression by acting as a ceRNA for miR95p in podocytesCASC2 alleviated the a0HGinduced podocytes injury by a0increasing PPARÎConsidering CASC2 could act as a sponge of miR95p to regulate the expression of PPARÎ we further investigated whether PPARÎ was involved in regulation of HGinduced podocytes injury mediated by CASC2 Western blot results indicated that cotransfection of siPPARÎ neutralized the promoting effect of CASC2 on PPARÎ protein expression Fig a0 5a The data of CCK8 and Annexin VFITCPI assays indicated that the effects of CASC2 on cell viability Fig a0 5b and apoptosis Fig a0 5c could be abolished by silencing 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 acted as a ceRNA by sponging miR95p to facilitate PPARÎ expression a The mRNA expression of PPARÎ in NG HG or mannitoltreated CIHP1 cells was analyzed by qRTPCR b qRTPCR assay was used to measure the mRNA expression of PPARÎ in CIHP1 cells treated by HG mM at different times c The protein expression of PPARÎ in NG HG or mannitoltreated CIHP1 cells was analyzed by western blot assay d Western blot assay was used to measure the protein expression of PPARÎ in CIHP1 cells treated by HG mM at different times e StarBase v20 predicted that there were binding sites between miR95p and PPARÎ f Dual luciferase reporter assay was conducted to detect the interaction between miR95p and PPARÎ in CIHP1 cells g The expression of miR95p in CIHP1 cells transfected with antiNC or antimiR95p was measured by qRTPCR h The protein expression of PPARÎ in CIHP1 cells transfected with miRNC miR95p antiNC or antimiR95p was assessed using western blot assay i PPARÎ protein expression in CIHP1 cells transfected with siNC siCASC2 siCASC2 antiNC or siCASC2 antimiR95p was estimated by western blot P PPARÎ in HGinduced CIHP1 cells Similarly the effects of CASC2 on levels of Cleavedcaspase3 BCL Beclin and LC3IILC3I ratio were rescued by siPPARÎ implying PPARÎ knockdown could increase Cleavedcaspase3 protein and decrease the expression levels of BCL2 and Beclin as well as the ratio of LC3IILC3I Fig a05d e To sum up CASC2 alleviated the HGinduced podocytes injury by upregulating PPARÎOverall it could be concluded that HG inhibited cell viability autophagy but promoted cell apoptosis by downregulating CASC2 and PPARÎ expression as well as upregulating miR95p in CIHP1 cells Fig a0DiscussionPodocytes are terminally differentiated visceral epithelial cells which are important components of the glomerular filtration barrier Podocyte viability and apoptosis as well as autophagy can affect glomerular function [] A large number of studies have shown that high glucose induction can cause podocytes injury []Several lncRNAs such as lncRNA MALAT1 [] and lncRNA PRINS [] have been found to be involved in the development of DN they regulated mRNA expression at the posttranscriptional level In this study we found that CASC2 expression was prominently downregulated in high glucosestimulated podocytes in a 0cLi a0et a0al Diabetol Metab Syndr Page of Fig CASC2 alleviated the HGinduced podocytes injury by increasing PPARÎ The HGtreated CIHP1 cells were transfected with Vector CASC2 CASC2 siNC and siPPARÎ respectively a PPARÎ protein expression was examined by western blot b c Cell viability and apoptosis were determined by CCK8 assay and Flow cytometry respectively d e The expression levels of BCL2 Cleavedcaspase3 LC3II LC3I and Beclin were checked by western blot assay P timedependent manner and doseresponse manner Autophagy is a doubleedged sword and its excessive activation or repression can cause podocytes injury [] Autophagy activity is impaired in DN patients so promoting autophagy to some extent can reduce podocytes injury [] and Beclin LC3I and LC3II have been shown to be autophagy specific proteins [] In accordance with previous data high glucose could inhibit cell viability and autophagy and promote cell apoptosis while overexpression of CASC2 could attenuate the effect of high glucose on podocytes injury suggesting the protective effect of CASC2 on podocytes injury Similarly Yang et a0al observed that CASC2 was enormously decreased in DN patients while there was no significant difference in CASC2 expression in DN patients as compared to those with DM without complication DN [] Besides Wang et a0 al reported that the development of type diabetes might have no significant effects on CASC2 expression in renal tissue whereas CASC2 expression in renal tissues was found to be evidently lower in patients with type diabetes complicated with chronic renal failure [] These data suggested that 0cLi a0et a0al Diabetol Metab Syndr Page of Fig Schema presented the mechanism that HG repressed cell viability autophagy and promoted cell apoptosis by regulating the CASC2miR95pPPARÎ axis in CIHP1 cellsCASC2 inhibition was very likely to be involved in the pathogenesis of DN Moreover lncRNA often functions as ceRNA and we speculated that CASC2 might also be involved in the regulation of podocytes injury by sponging miRNACompared with nondiabetic subjects the level of miR95p was higher in serum of patients with gestational diabetes mellitus [] and serum miR9 might be an underlying marker for poor prognosis of DN [] In our data the abundance of miR95p was increased in high glucoseinduced podocytes and miR95p was validated to be the target miRNA for CASC2 and CASC2 could inversely modulate miR95p expression in podocytes Recovery experiments showed that CASC2 mitigated podocytes injury by decreasing miR95p via acting as a miR95p sponge similar to the work of Zhang et a0al who indicated that lncRNA SOX2OT could reduce the high glucosestimulated podocytes damage by autophagy induction through binding to miR9 [] Therefore it is reasonable to infer that CASC2 regulated podocyte activity apoptosis and autophagy through sponging miR95pPPARÎ agonists have been widely reported to improve glycemic status in diabetes patients [] and PPARÎ has favorable renal protective effects [] As expected high glucose treatment obviously retarded PPARÎ expression Importantly miR95p directly targeted PPARÎ ²UTR and negatively modulated its expression In addition CASC2 could regulate PPARÎ expression by sponging miR95p based on these results we hypothesized whether CASC2 implicated in podocytes injury by regulating PPARÎ The results showed that PPARÎ knockdown neutralized the effect of CASC2 on podocytes injury Besides PPARÎ has been shown to restore podocyte integrity to improve proteinuria []ConclusionIn summary we believed that CASC2 mainly upregulated the expression of PPARÎ by acting as the ceRNA of miR95p thus alleviating HGinduced podocytes injury through increasing cell viability autophagy and reducing cell apoptosis This study provided a new molecular regulatory mechanism for podocytes injury induced by HG in DNAbbreviationsHG High glucose DN Diabetes nephropathy CASC2 Cancer susceptibility candidate NG Normal glucose RIP RNA immunoprecipitation PPARÎ Peroxisome proliferatoractivated receptor gammaAcknowledgementsThe authors sincerely appreciate all members participated in this study Authors contributionsFL designed the experiments performed the experiments and analyzed and collected the data wrote the manuscript BD analyzed interpreted the data performed the experiments and wrote the manuscript XN performed the experiments and analyzed the data All authors read and approved the final manuscript FundingThis work was supported by Key Research Development Program of Shandong Province China [Grant No2019GSF108172] Natural Science Foundation of Shandong Province China [Grant No2016ZRA08005] and Science and Technology Development Plans of TCM of Shandong Province China [Grant No ] Availability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author on reasonable request 0cLi a0et a0al Diabetol Metab Syndr Page of Ethics approval and consent to participateNot applicablePatient consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestAuthor details Department of Nephrology Heze Mudan Peoples Hospital Heze Shandong China Department of Nephrology Liaocheng Peoples Hospital Liaocheng Shandong China Department of Nephrology Taian Campus of the 960th Hospital of the Chinese Peoples Liberation Army No217 Huanshan Road Taishan District Taian Shandong China Received February Accepted July References Dronavalli S Duka I Bakris GL The pathogenesis of diabetic nephropathy Nat Clin Pract Endocrinol Metab Ogurtsova K da Rocha Fernandes JD Huang Y Linnenkamp U Guariguata L Cho NH et al IDF Diabetes Atlas Global estimates for the prevalence of diabetes for and Diabetes Res Clin Pract Afkarian M Zelnick LR Hall YN Heagerty PJ Tuttle K Weiss NS et al Clinical manifestations of kidney disease among US adults with diabetes JAMA Ziyadeh FN Wolf G Pathogenesis of the podocytopathy and proteinuria in diabetic glomerulopathy Curr Diabetes Rev Berthier CC Zhang H Schin M Henger A Nelson RG Yee B et al Enhanced expression of Janus 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Z Wan J Hou X Geng J Li X Bai X MicroRNA27a promotes podocyte injury via PPARgammamediated betacatenin activation in diabetic nephropathy Cell Death Dis 201783e2658 Duan LJ Ding M Hou LJ Cui YT Li CJ Yu DM Long noncoding RNA TUG1 alleviates extracellular matrix accumulation via mediating microRNA377 targeting of PPARgamma in diabetic nephropathy Biochem Biophys Res Commun Liang W Sun F Identification of pivotal lncRNAs in papillary thyroid cancer using lncRNAmRNAmiRNA ceRNA network analysis PeerJ 20197e7441 Li D Lu Z Xu Z et al Spironolactone promotes autophagy via inhibiting PI3KAKTmTOR signalling pathway and reduce adhesive capacity damage in podocytes under mechanical stress Biosci Rep 2016364e00355 Susztak K Raff AC Schiffer M Bottinger EP Glucoseinduced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy Diabetes Chen Z Ma Y Yang Q et al AKAP1 mediates high glucoseinduced mitochondrial fission through the phosphorylation of Drp1 in podocytes J Cell Physiol https doi101002jcp29646 Jiang L Cui H Ding J Smad3 signalling affects high glucoseinduced podocyte injury via regulation of the cytoskeletal protein transgelin Nephrology https doi101111nep13701 Hu M Wang R Li X Fan M Lin J Zhen J et al LncRNA MALAT1 is dysregulated in diabetic nephropathy and involved in high glucoseinduced podocyte injury via its interplay with betacatenin J Cell Mol Med Jiao H Xie D Qiao Y LncRNA PRINS is involved in the development of nephropathy in patients with diabetes via interaction with Smad7 Exp Ther Med Kim H Dusabimana T Kim SR Je J Jeong K Kang MC et al Supplementation of abelmoschus manihot ameliorates diabetic nephropathy and hepatic steatosis by activating autophagy in mice Nutrients Wu F Li S Zhang N Huang W Li X Wang M et al Hispidulin alleviates highglucoseinduced podocyte injury by regulating protective autophagy Biomed Pharmacother Sc | Thyroid_Cancer |
"clinicopathological characteristics with risk factors of breast cancer patients in NigeriaMethods Newly diagnosed female patients with breast cancer were assessed over months Patients were reviewed using a predesigned proforma which focused on sociodemographic information clinical information risk factors and tumor biologyResults A total of women were identified their mean age was years More than half are premenopausal at presentation with Eastern Cooperative Oncology Group ECOG score of and right side as the most common primary site of disease Less than half of them are estrogen receptor ER positive are progesterone receptor PR positive and are hormone receptor positive and triple negative respectively Most patients presented at the latter stage of the disease stage III and stage IV Only are well differentiated and almost all had invasive ductal histological type Obesity and physical inactivity are the most common risk factors for the disease A significant relationship was found between immunohistochemistry status and family history of breast cancer tumor site previManuscript submitted June accepted July Published online August aOncology and Radiotherapy Department Lagos University Teaching Hospital Lagos NigeriabMolecular and Anatomical Pathology Department College of Medicine University of Lagos Lagos NigeriacRadiotherapy Radiobiology Radiodiagnosis and Radiography Department College of Medicine University of Lagos Lagos NigeriadWest Cancer Centre and Research Institute Memphis TN USAeArrive Alive Diagnostics and Imaging Services Ltd Lagos NigeriafCorresponding Author Adeoluwa Akeem Adeniji Oncology and Radiotherapy Department Lagos University Teaching Hospital Lagos Nigeria Email godscrownbestyahoocom 1014740wjon1303ous breast surgery previous lump and alcohol intakeConclusion Findings from this study showed that Nigerian breast cancer patients differ from their counterparts in the high human development index HHDI countries in terms of the patients and disease characteristics In view of this prevention and treatment options should consider this uniqueness to ensure better outcomeKeywords Breast cancer Subtypes Tumor biology Risk factors Correlation NigeriaIntroductionCancer is a major public health concern globally [] According to GLOBACAN cancer is the single most important factor impacting life expectancy worldwide [] In women worldwide breast cancer is the most common malignancy [] Every year about million new cases are diagnosed worldwide and this represents of the female population [] In alone there were million new cases of breast cancer worldwide and over deaths and this represents new cases of cancer and of all cancerrelated deaths []One of the indicators that reflect the development of each country the status and their living conditions is the human development index HDI The HDI is defined as the average achievement of three factors including life expectancy at birth gross national income per capita and mean and expected years of schooling Low HDI level includes countries that are the least developed and the very high HDI level includes the most developed countries [] Although low human development index countries LHDI like Nigeria have a lower incidence of breast cancer when compared to high human development index HHDI countries like the United States of America USA mortality rates are higher [] The incidence rate in the LHDI countries is rising likely because of westernization and its lifestyle choices []The high mortality rate is seen because of late stage presentation misdiagnosis and poor health seeking behavior s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjonThis 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 0cBreast Cancer Among Nigerian WomenWorld J Oncol among other factors of the African population in general The screening rates is still low ranging from to for reasons ranging from cost quality assurance and fear of radiation [] Studies have also shown that the genetic and histopathologic subtypes in African women are likely to be more aggressive than those seen in their Caucasian counterparts []Worldwide the treatment of breast cancer is now personalized dependent on the patient the stage and grade of disease histological type immunohistochemistry drug preference surgery and radiation impact and techniques for best outcomes When the pathology immunohistochemistry and tumor biology types are not factored into treatment modalities for these patients coupled with late stage at presentation poverty and lack of funding for treatment there are worse outcomes for patients and this accounts for the high incidence of morbidity and mortality that is seenAccording to the Central Intelligence Agency fact book there is prevalence rate of poverty in Nigeria [] There is paucity of studies detailing biology or genetics of breast cancer in SubSaharan Africa likely because of the difficulty involved in obtaining and processing tissue samples usually because of financial constraint [] and due to the lack of laboratory facilities to carry out these investigations []Currently the management of Nigerian women with breast cancer is dependent on protocols imported from developed countries like the USA even though the patient population and disease profile may differ Understanding the profile of Nigerian women with breast cancer helps to create prevention and treatment in a more personalized approach in management of the disease in NigeriaThis study therefore focuses on exploring those characteristics in Nigerian patients the differences seen when compared to their counterparts in HHDI countries and hopes that these findings could impact prevention and management of breast cancer patients in NigeriaMaterials and MethodsStudy designThis is a noninterventional prospective study among participants recruited from the Radiotherapy Unit of Lagos University Teaching Hospital IdiAraba Nigeria Participants were selected newly histologically diagnosed with tumor staging according to American Joint Committee on Cancer 8th edition breast cancer patients who attended the outpatient clinics for treatment for the first time from July to July Participants were all females aged years or more Patients who were acutely ill Eastern Cooperative Oncology Group ECOG score were excluded from the study A structured intervieweradministered proforma was used to obtain required data from all study participants during the study period The proforma collected data on sociodemographic and disease characteristics Neutr ia and febrile neutr ia was graded using the Common Terminology Criteria for Adverse Events CTCAE version The CTCAE is a set of criteria for the standardized classification of adverse effects of drugs used in cancer therapyMeasuresStudy proforma Sociodemographic and socioeconomic informationParticipants were administered questionnaires aimed at gathering information about their age marital status level of education occupation partners occupation and economic statusPatients occupation was categorized under three domains unemployed including student housewife minimally skilled artisan civil servant trader and skilledprofessional doctor lawyer accountantPatients marital status was defined into two categories married or unmarried divorced separated single and widowed Clinical information and risk factorsParticipants were asked questions about their past medical history including parity first symptom menopausal status and duration of illness Risk factors like alcohol use smoking family history use of contraceptive breastfeeding and previous history of benign breast lesions were also elicited Some clinical data were obtained by reviewing the patients hospital folder with a specific focus on cancer diagnosis staging surgery and ECOG performance of participantsBody mass index BMI of each patient was calculated using the height and weight recorded in their medical case files at first presentation to the hospital A BMI of kgm2 or more was defined as obesity and kgm2 or more was considered overweightPresence of comorbidities including hypertension diabetes human immunodeficiency virus and peptic ulcer disease was recorded Positive family history of breast cancer is defined as breast cancer both in first and second degree of patients family Physical inactivity is measured by inability to move around carry out day to day activities or at least min of moderate intensity physical activity per week as recommended by the World Health anization [] Early menarche is defined as first menstrual period in a female adolescent before the age of years [] while late first pregnancy is defined as above years [] Previous lump is the presence of a benign lump that was removed before the onset of the breast malignancy Pathology and immunohistochemistryParticipants hospital folders were reviewed for data on pathologic staging of disease pathologic information including histologic type tumor grade and immunohistochemistry classifis The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol Frequency ± Mean ± SD Range Living with a partner Not living with a partnerTable Characteristics of Breast Cancer PatientsCharacteristicsAge years Marital status Occupation Education level Unemployed Minimally skilled Skilled and professional None Primary Secondary Tertiary Table Immunohistochemistry Distribution Among Breast Cancer Patients Negative Positive Negative PositiveReceptor statusEstrogen receptor status Progesterone receptor status HER2 receptor status Hormonal receptor status Triple negativity Equivocal Negative Positive Negative PositiveFrequency SD standard deviationHER2 human epidermal growth factor receptor cation of disease Human epidermal growth factor receptor HER2 is defined by immunohistochemistry onlyData analysisData analysis was done using Statistical Package for Social Sciences software for Windows version SPSS Chicago IL Univariate analyses were presented in the forms of tables as descriptive frequency distribution of the sociodemographic and immunohistochemistry of the patients Correlation and association analyses were conducted using Chisquared and analysis of variance ANOVA with a precision index of ¤ Ethical considerationsEthical approval was sought from the ethics committee of the Lagos University Teaching Hospital and the study was conducted according to the principles of the Declaration of Helsinki Informed consent was sought from every participant before undertaking to participate in the studyResultsA total of patients were seen as outpatients with histologically diagnosed breast cancer The mean age of the patients studied was years with a range of years Table Majority live with a partner were unemployed and attained tertiary level of educationTable summarizes the immunohistochemical status of the patients Estrogen receptor ER and progesterone receptor PR positivity were cases and respectively About one in every five had HER2 positivity Almost half have triple negative subtypeThe majority of participants sampled suffered from right breast cancer Table The mean age at diagnosis and body mass index BMI at first presentation to the clinic were years and kgm2 A total of were premenopausal A total of had preexisting comorbidities while have had breast surgery before and more than half presented with ECOG performance score ¥ The most common primary site of tumor was the rightThe most frequent histological type was invasive ductal with cases Table Of these cancers were grade were grade and were grade Stages I II III and IV were and respectively with having confirmed cases of an metastasis and two cases did not have documented investigation of an metastasis in their medical case filesThe most common risk factors identified with the participants were overweightobesity and physical inactivity About of patients studied had a family history of breast or any other type of cancer Table A significant relationship was found between the HER status and history of breast surgery P tumor site P Table family history of breast cancer P and previous lump P Table There was also a significant relationship between HR status and alcohol intake P and family history of breast cancer P Table The only significant relationship seen in triple negative subtype was with family history of breast cancer P Table Immunohistochemistry status correlations with the age of the patients age at diagnosis menopausal status and the histologic type were not statistically significants The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol eulavP ± n ± eulavP lacoviuqE n evitageN n evitisoPn ± ± ± ± ± ± eulavP evitageNn ± ± ± ± ± ± yregrus tsaerb fo yrotsiHlasuaponemerPlasuaponemtsoP sutats lasuaponeMerocs GOCEmgk IMBseitidibromoCseY oN laretaliBthgiRtfeL etis romuT scitsiretcarahCsisongaid ta egA DS±nae M evitagen elpirTsutats REHsutats lanomroH evitisoP n ycneuqerFepytbuS romuT yb scitsiretcarahC lacniilC fo noitubirtsDi lebaTpu ygoocnO evitlarepooCnre tsaE GOCE xedni ssam ydob IMB noitiaved dradnats DS rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol eulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerF evitagen elpirTsutats REHsutats lanomroHepytbuS romuT yb scitsiretcarahC cgoohtaPli amonicrac latcud evisavnIamonicrac ralubol evisavnIasrehtO rotpecer rotcaf thw lamredpei namuh REH iamoncrac yrallipap dna suoncumi yralludem srehOat fo noitubirtsDi lebaTscitsiretcarahCezis romuT sutats ladoN sisatsatem romuTegats esaesiDedarg romuTIIIIII epyt ygolotsiHIIIIIIVI s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol evitagen elpirTsutats REHsutats lanomroHeulavPn eulavP lacoviuqE n evitageN n evitisoPn eulavP evitageNn evitisoPn ycneuqerFepytbuS romuT yb srotcaF ksRi fo noitubirtsDi lebaTpmul tsaerb tnangilamngineb suoiverPdeeftsaerb ton diDehcranemylraE ycnangerp tsrfi etaLytisebothgiewrevOytirapilluNesu evitpecartnoc larOerusopxe noitaidaRytivitcani lacisyhPyrotsih ylimaFekatni lohoclAgnikomSscitsiretcarahCDiscussionAfricanAmericans in USA have more metastatic breast cancer when compared to other races and the same said for highgrade disease larger tumor size and hormone receptor negativity in the blacks [] These are significantly increased among the blacks living in Africa [ ]This study clearly itemizes the sociodemographic clinical histological and immunohistochemistry characteristics of the Nigerian breast cancer patients in a hospitalbased study In this study the mean age was years with majority of women aged between and years similar to the findings in previous studies in Nigeria but in contrast to western countries where most of the breast cancer patients are postmenopausal [ ] Some studies have postulated a decrease in levels of circulating estrogen levels as responsible for the decreasing age of breast cancer patients worldwide [ ] This finding emphasizes on the need for preventive health education and screening programs not only targeted at the elderly because of the assumption that they are the prime age group at risk while this might be true for western countries and the pattern of disease presentation in Nigerian patients clearly highlights the need to begin screening for the disease before yearsThe previous studies conducted in Africa and Nigeria are largely hospitalbased studies and with small sample sizes making it difficult to predict associations between patients and risk factors The finding of obesity and physical inactivity as the largest risk factors for breast cancer is new in the premenopausal group although this was always true for many western countries mostly for postmenopausal women [] This finding is new in LHDI countries like Nigeria and the predominance of the triple negative subset may account for this finding compared to the hormone receptor positive subset predominance in the western countries In a similar study carried out in Nigerian women in early menarche and not breast feeding were the risk factors associated with increased risk of development of breast cancer In this study early menarche was only seen in of breast cancer patients []Family history is not a common risk factor in our patients as compared to their counterparts in HHDI countries [ ] The same is true for early menarche and nulliparity Not surprisingly the profile of risk in Nigerian cancer patients has evolved to mirror their Caucasian counterparts in the areas of obesity and physical inactivity [ ] This finding helps to focus healthcare professionals during screening exercises not to rule out likely patients because of the absence of traditional risk factors like family history early menarche or nulliparityIn this study like many other studies conducted in SubSaharan Africa patients are seen in locally advanced and advanced stages of their diseases [ ] This finding is not true for women in developed countries as patients tend to present at earlier stages [] Majority of the respondents were of moderate economic status which suggests that funds may not be the reason for late stage presentation as seen in previous studies [ ] Finding the reason why these patients presented late despite the fairly stable economic status is beyond the scope of this study and is for further review Perhaps the reason P rotpecer rotcaf thw liamredpe namuh REHs The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cAdeniji et alWorld J Oncol may be associated to the painless nature of their first symptom which may have affected their health seeking behaviorIn recent times targeted therapies based on grade histology and immunohistochemistry have resulted in better outcomes for patients [ ] Globally the invasive ductal carcinoma is the commonest histologic subtype of breast cancer [] This is true for breast cancer patients in this study representing of the breast cancer patients seenTriple negative was the commonest subtype seen in these patients and differed from the less aggressive subtypes seen in Caucasian women [ ] This subtype is associated with high rates of tumor invasion and metastases and is associated with a poorer prognosis [] This may explain the high mortality rates seen in the Nigerian breast cancer patients despite the comparatively lower incidence ratesConclusionNigerian breast cancer patient are likely to be premenopausal obese or overweight with no family history of higher tumor grade triple negative subtype late stage and hormone receptor negative These findings explain the high mortality rates seen in the Nigerian breast cancer patients and can be modified or useful in targeted treatment to ensure a better outcome Supplementary Material wwwwjonla Samuel Olalekan Keshinro Financial support Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Bashir Mariam Adebola Samuel Olalekan Keshinro Administrative support Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Bashir Mariam Adebola Michael Martin Provision of study materials or patients Adeoluwa Adeniji Akeem Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Collection and assembly of data Adeoluwa Adeniji Akeem Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebola Samuel Olalekan Keshinro Data analysis and interpretation Adeoluwa Adeniji Akeem Timilehin Gabriel Fagbenro Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Manuscript writing all authors Final approval of manuscript all authors Accountable for all aspects of the work all authorsData AvailabilityThe data supporting the findings of this study have been deposited in OneDrive and can be accessed via the link at cuttlyadenijibreastcancerSupplementary MaterialReferencesSuppl Data of All Study Participants During the Study PeriodAcknowledgmentsWe acknowledge the entire staff of the department especially the medical record officers nurses and the resident doctorsFinancial DisclosureNone to declareConflict of InterestThe authors declare that they have no conflict of interest regarding this workInformed ConsentInformed consent was obtainedAuthor ContributionsConception and design Adeoluwa Adeniji Akeem Olayemi Olubunmi Dawodu Muhammad Yaqub Habeebu Ademola Oluwatosin Oyekan Michael Martin Bashir Mariam Adebo Ginsburg O Bray F Coleman MP Vanderpuye V Eniu A Kotha SR Sarker M et al The global burden of women's cancers a grand challenge in global health Lancet 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 Ghoncheh M Momenimovahed Z Salehiniya H Epidemiology incidence and mortality of breast cancer in Asia Asian Pac J Cancer Prev 201617S34752 Shrivastava S Singh N Nigam AK et al Comparative study of hematological parameters along with effect of chemotherapy and radiotherapy in different stages of breast cancer Int J Res Med Sci Soheylizad M Khazaei S Jenabi E Delpisheh A Veisani Y The relationship between human development index and its components with thyroid cancer incidence and mortality using the decomposition approach Int J Endocrinol Metab 2018164e65078Igene H Global health inequalities and breast cancer an impending public health problem for developing countries Breast J Brinton LA Figueroa JD Awuah B Yarney J Wiafe S Wood SN Ansong D et al Breast cancer in SubSaharan Africa opportunities for prevention Breast Cancer Res Treat Akhigbe AO Omuemu VO Knowledge attitudes and practice of breast cancer screening among female health workers in a Nigerian urban city BMC Cancer Lawal O Murphy FJ Hogg P Irurhe N Nightingale J s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0cBreast Cancer Among Nigerian WomenWorld J Oncol Mammography screening in Nigeria A critical comparison to other countries Radiography Akinola R Wright K Osunfidiya O Orogbemi O Akinola O Mammography and mammographic screening are female patients at a teaching hospital in Lagos Nigeria aware of these procedures Diagn Interv Radiol MO O Ayodele SO Umar AS Breast cancer and mammography Current knowledge attitudes and practices of female health workers in a tertiary health institution in Northern Nigeria Screening Agboola AJ Musa AA Wanangwa N AbdelFatah T Nolan CC Ayoade BA Oyebadejo TY et al Molecular characteristics and prognostic features of breast cancer in Nigerian compared with UK women Breast Cancer Res Treat Factbook CI The world factbook Available at wwwciagovlibrarypublicationstheworldfactbook Accessed on January 2nd Fregene A Newman LA Breast cancer in subSaharan Africa how does it relate to breast cancer in AfricanAmerican women Cancer AkaroloAnthony SN Ogundiran TO Adebamowo CA Emerging breast cancer epidemic evidence from Africa Breast Cancer Res 201012Suppl 4S8 Fuzeki E Banzer W Physical activity recommendations for health and beyond in currently inactive populations Int J Environ Res Public Health Ibitoye M Choi C Tai H Lee G Sommer M Early menarche A systematic review of its effect on sexual and reproductive health in low and middleincome countries PLoS One 2017126e0178884 Lampinen R VehvilainenJulkunen K Kankkunen P A review of pregnancy in women over years of age Nurs J DeSantis CE Ma J Gaudet MM Newman LA Miller KD Goding Sauer A Jemal A et al Breast cancer statistics CA Cancer J Clin Ntekim A Nufu FT Campbell OB Breast cancer in young women in Ibadan Nigeria Afr Health Sci Henderson BE Ross R Bernstein L Estrogens as a cause of human cancer the Richard and Hinda Rosenthal Foundation award lecture Cancer Res Bray F McCarron P Parkin DM The changing global patterns of female breast cancer incidence and mortality Breast Cancer Res Wolin KY Carson K Colditz GA Obesity and cancer Oncologist Huo D Adebamowo CA Ogundiran TO Akang EE Campbell O Adenipekun A Cummings S et al Parity and breastfeeding are protective against breast cancer in Nigerian women Br J Cancer Adesunkanmi AR Lawal OO Adelusola KA Durosimi MA The severity outcome and challenges of breast cancer in Nigeria Breast Adebamowo CA Adekunle OO Casecontrolled study of the epidemiological risk factors for breast cancer in Nigeria Br J Surg Porter P Westernizing women's risks Breast cancer in lowerincome countries N Engl J Med McPherson K Steel CM Dixon JM ABC of breast diseases Breast cancerepidemiology risk factors and genetics BMJ Awofeso O Roberts AA Salako O Balogun L Okediji P Prevalence and pattern of latestage presentation in women with breast and cervical cancers in Lagos University Teaching Hospital Nigeria Niger Med J JedyAgba E McCormack V Adebamowo C DosSantosSilva I Stage at diagnosis of breast cancer in subSaharan Africa a systematic review and metaanalysis Lancet Glob Health 2016412e923e935 Vanderpuye V Grover S Hammad N PoojaPrabhakar Simonds H Olopade F Stefan DC An update on the management of breast cancer in Africa Infect Agent Cancer Ibrahim NA Oludara MA Sociodemographic factors and reasons associated with delay in breast cancer presentation a study in Nigerian women Breast Lannin DR Mathews HF Mitchell J Swanson MS Swanson FH Edwards MS Influence of socioeconomic and cultural factors on racial differences in latestage presentation of breast cancer JAMA Sohn YM Han K Seo M Immunohistochemical subtypes of breast cancer correlation with clinicopathological and radiological factors Iran J Radiol 2016134e31386 Beral V Bull D Doll R Peto R Reeves G Collaborative Group on Hormonal Factors in Breast C Breast cancer and abortion collaborative reanalysis of data from epidemiological studies including women with breast cancer from countries Lancet Li CI Anderson BO Daling JR Moe RE Trends in incidence rates of invasive lobular and ductal breast carcinoma JAMA Wu X Baig A Kasymjanova G Kafi K Holcroft C Mekouar H Carbonneau A et al Pattern of Local recurrence and distant metastasis in breast cancer by molecular subtype Cureus 2016812e924s The authors Journal compilation World J Oncol and Elmer Press Inc¢ wwwwjon 0c" | Thyroid_Cancer |
Genomic characterization of malignant progressionin neoplastic pancreatic cystsMicha«l No«et alIntraductal papillary mucinous neoplasms IPMNs and mucinous cystic neoplasms MCNsare noninvasive neoplasms that are often observed in association with invasive pancreaticcancers but their origins and evolutionary relationships are poorly understood In this studywe analyze samples from IPMNs MCNs and small associated invasive carcinomas from patients using whole exome or targeted sequencing Using evolutionary analyses weestablish that both IPMNs and MCNs are direct precursors to pancreatic cancer Mutations inSMAD4 and TGFBR2 are frequently restricted to invasive carcinoma while RNF43 alterationsare largely in noninvasive lesions Genomic analyses suggest an average window of overthree years between the development of highgrade dysplasia and pancreatic cancer Takentogether these data establish noninvasive IPMNs and MCNs as origins of invasive pancreatic canceridentifying potential drivers of invasion highlighting the complex clonaldynamics prior to malignant transformation and providing opportunities for early detectionand interventionA list of authors and their afï¬liations appears at the end of the paperNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178Pancreatic cancer is a deadly disease with a dismal prognosisthat is predicted to soon be the second leading cause ofcancer death in the United States1 However like otherepithelial malignancies pancreatic cancer arises from noninvasiveprecancerous lesions that are curable if detected and treated earlyenough Although the majority of pancreatic cancers are believedto originate in microscopic precancerous lesions pancreaticintraepithelial neoplasia or PanIN a significant minority arise inassociation with larger cystic neoplasms that can be detectedusing currently available imaging technologies2 These neoplasmswhich includeintraductal papillary mucinous neoplasmsIPMNs and mucinous cystic neoplasms MCNs are frequentlydiagnosed incidentally on abdominalidentifying acohort of atrisk patients with an important opportunity forprevention of invasive pancreatic cancer2 However preventionmust be balanced with potential overtreatment of lowrisk lesionsas pancreatic resection carries significant morbidity and evenoccasional mortality3 There is a critical need to understand themolecular alterations that are associated with the development ofinvasive cancer as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thusrequiring clinical interventionimagingAlthough genomic analyses have been performed on hundredsof invasive pancreatic cancers relatively few noninvasive neoplasms have been analyzed comprehensively Whole exome andtargeted sequencing of small cohorts of IPMNs and MCNs haverevealed driver genes characteristic of each type of cystic neoplasm4 while targeted analyses in larger cohorts have conï¬rmed the prevalence of speciï¬c driver gene mutations thatcorrelate with grade of dysplasia or histological subtype7 Thesestudies have conï¬rmed that hotspot mutations in the oncogenesKRAS and GNAS occur in lowgrade lesions while mutations inother driver genesincluding CDKN2A TP53 RNF43 andSMAD4 occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma8 Targeted nextgeneration sequencing has been used to analyze pancreatic drivergenes in different regions of IPMNs revealing a surprising degreeof intratumoral genetic heterogeneity even with respect to wellcharacterized driver gene mutations9 However the aboveanalyses were based on studies of either single regions from eachneoplasm or a limited number of genes from multiple regionsand did not provide an analysis of the evolutionary relationshipbetween different regions of pancreatic cysts and associatedcancers These limitations highlight the need for comprehensivegenomic analysis of these cysts and associated invasive cancers tounderstand the molecular alterations that underlie the transitionto invasive carcinomaIn this study we perform whole exome sequencing of IPMNsand MCNs and their associated invasive carcinomas Importantlywe focus our study on small invasive carcinomas cm inorder to more precisely analyze the genetic alterations that occurat malignanttransformation in pancreatic tumorigenesis Inaddition in a subset of our samples we perform deep targetednext generation sequencing on a larger set of additional tissuesamples in order to assess mutated loci through entire neoplasmsincluding areas of lowgrade dysplasia highgrade dysplasia andinvasive carcinoma These analyses reveal important features ofpancreatic tumorigenesisincluding evolutionary relationshipsbetween different regions within cystic neoplasms as well asmolecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancerResultsOverall approach In order to dissect the molecular relationshipsbetween noninvasive dysplastic lesions and invasive pancreaticcancers we performed whole exome sequencing of neoplastictissue samples from patients with small invasive carcinomas cm associated with neoplastic pancreatic cysts including patients with IPMNs and patients with MCNs Supplementary Data Whole exome sequencing was performed onone sample from the noninvasive component with highgradedysplasia and one sample from the invasive cancer in each caseand for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencingMatched normalsamples were analyzed by whole exomesequencing in each case to exclude germline variants and toidentify somatic mutations Whole exome sequencing was performed with an average total coverage of distinct coverageof generating TB of sequencing data SupplementaryData In addition to whole exome analyses we performed targetednext generation sequencing of microdissected tissue samplesfrom seven of the above cases six IPMNs and one MCN Forthese targeted analyses we performed laser capture microdissection to isolate neoplastic cells from every available tissue block ofthe noninvasive cyst and cancer specimens Separate sampleswere microdissected based on grade of dysplasia cell morphollocation This resulted in ogy architecture and spatialadditional samples per case The targeted panel analyses includedall mutated loci identiï¬ed in the whole exome sequencing of theseseven cases as well as the entire coding regions of wellcharacterized pancreatic driver genes Supplementary Data The targeted sequencing had an average coverage of distinct coverage of Supplementary Data We developed an integrated mutation calling pipeline torigorously assess mutations in all sample types in our analyses inorder to conï¬dently identify even subclonal alterations in sampleswith low neoplastic purity see Methods Fig Supplementary Data In addition we utilized both on target and off targetreads to examine focal copy number changes as well as loss ofheterozygosity throughout the genome Fig SupplementaryData and From our whole exome sequencing analyses weidentiï¬ed an average of somatic mutations in samples fromnoninvasive components range and an average of somatic mutations in invasive carcinoma samples rangeFig 1a Supplementary Data An average of somatic mutations were shared between the noninvasive andinvasive components while somatic mutations were unique tosamples from noninvasive components and somatic mutationswere unique to samples from invasive cancer Fig 1a We alsoidentiï¬ed an average of ï¬ve shared copy number alterationsbetween noninvasive and invasive components as well as anaverage of one copy number alteration unique to samples frominvasive cancer A similar mean proportion of somatic mutationsand copy number alterations were unique to invasive samples for somatic mutations for copy number alterationsAnalysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCNtumorigenesis In every analyzed case there were multiple sharedmutations between the noninvasive and invasive componentsThese included both driver and passenger mutations indicatingthat they shared a common phylogenetic ancestor Fig 1a b Inaddition accumulation of unique mutations in both noninvasiveand invasive components demonstrated independent evolutionafter the divergence of the subclone that gave rise to the invasiveFig Supplementary Figs S1S18 Analysis ofcanceradditional adjacent lowgrade or highgrade samples from thesame lesions revealed a subset of shared mutations suggestingthat these dysplastic lesions preceded the development of theinvasive carcinoma Fig Supplementary Figs S1 S2 S3 S5and S16 Evolutionary analyses showed a branched phylogeny inNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178asnoitatuMCancerization onlyDuctal cancer onlyMucinous cancer onlySharedIPMN onlyMCN onlyPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMbKRASTP53CDKN2ASMAD4RNF43GLI3GNASMUC16PTPRTTGFBR2ATMCNTN5PIK3CAALKAPCRYR2STK11CTNNB1ERBB4EGFRSF3B1NOTCH1KEAP1ERBB2MYCRETTSC2PrecursorSharedCancerFig Somatic mutations identiï¬ed in matched noninvasive and invasive cancer samples a In each patient sample multiple mutations were sharedbetween the noninvasive and invasive cancer samples gray In addition some mutations were limited to the noninvasive bluegreen while others werelimited to the cancer redpink Darker colors indicate alterations that were likely restricted to one component but where sequencing coverage in thesecond component was limited The proportions of shared and distinct mutations varied between different lesions b Somatic mutations in the mostfrequently mutated genes are categorized as shared between noninvasive and cancer gray limited to noninvasive blue or limited to cancer redMutations in some genes such as KRAS were always shared while others were enriched in samples from noninvasive RNF43 or cancer SMAD4MutationsMTP1MTP2MTP3MTP4MTP5MTP6MTP7MTP8MTP9MTP11MTP13MTP14MTP18MTP19MTP23MTP24MTP26MTP30chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12chr13chr14chr15chr16chr17chr18chr19chr20chr21chr22Copy number log2SamplesLG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationFig Copy number alterations identiï¬ed in matched noninvasive and invasive cancer samples Chromosomal gains red and losses blue are shownfor each chromosome in each patient with noninvasive samples on the left and cancer samples on the rightNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP19HEbefore LCMafter LCMMTP8KRASG12DKRASG12RKRASQ61H1225380275_TGKRASQ61H1225380275_TAKRASG12VTMEM56RWDD3c5662TC [SP]KCNA5A451AC17orf98T142MDUSP27T652TADAMTS12P429LCOL12A1R933HSCAF8G961VTMEM246A372AMCM10E613KPNLIPRP1T465TC13orf35L33LSMAD6L179LGRIN2AS397SAP1G1T798TTP53E171FCHO1c22471GC [SP]LRFN1S632SLTBP4P243LLOHchr17001ABRF645SALPK1R873RLOHchrX275DELchr92182377CDKN2ABSND861NSUPT6HI104TPLEKHF1D258DLOHchr221716LTBRR425GACOT11R306LOR6N1Y120YENPP5Y341YITGB3C562BTKP116TSLC1A7G465DFGF3R104QZBED4P1100LSIM1R192CPHC1S627CMS4A1T41TLOHchr712725SH2D5R199WRNPEPL1G413VGAL3ST2P85PJADE2N352SHHLA1A97VTMEM141Q61QBPTFR296HKLHL22R603RGALEQ261LANKS4BQ368RKRI1S326SOR8I2C240CLRTM2G319VLOHchr97123LAMB3R887LSPTAN1I1484FTP53R175HTissue sourceLG IPMNHG IPMNMUCINOUS CANCERDUCTAL CANCERCANCERIZATIONSequencingTargeted SeqWhole Exome SeqDriver MutationHotspotInactivating_OtherDELLOH µm µmMPT19 LG IPMN µm µm µmMPT19 HG IPMN µm_________ µm µm µmMPT19 ductal cancer µm µm µmMPT19 cancerization T2 T1 µm µmMPT8 LG IPMN µm µm µmMPT8 HG IPMN µm__ µm µm µmMPT8 mucinous cancer T1 T2 TTC39AN83ILRRC8DI741MOBSCNR2837QRGS7A195AOR14A2A156ACCDC13S534SEGFLAMG553VITGA1V27LBDP1N622NKIAA0319V824FRIMS1R211QPRDM13A303VIFNGR1M1PHACTR2A348PGPR85F208FGNED83HHTR7T240MGRM5T946KKRASG12DUNC13CN497SBNIP2D58DCCDC33D354NCLDN9P187PNOTUMR308CPIEZO2R2407QC19orf24G52AMUC16P9201QCCNE1R85WGNASR201HPHF21BA116TTNS1R894QNDUFAF6L320ISLC15A1E26KCLUHR433PRR23AA151TBRD3R313HUSP34R3136HRHOAK27ESIM1A654TNRG1A540VVRK3S129NCCDC166A84TST3GAL4V165MTSPAN17P63PMAD1L1S327SC17orf47E142GCHERPL47ITRPC5E418DZFP37R18WGNASD464NMED12E1497KBAG2T93TRNF43R371CACNG2V255MMAGEB18A63VBRINP3A748APDE1BE401EQRICH2R160RRNF43P370fsINTS1G527VMYOM3G152SPRRT3P407STGFBR2R553HPDZD2I810IFAM120BR646CEPHB4V682MNOBOXR302CCEP164R897QMAB21L1T171MZCCHC14T80MCD68L295LGAS2L2S728LRUNDC3AE49EPHBI122VDNMT1N109NZNF865A721ACASS4S376SKCNJ6R322SLC5A4L390PLRRC8CR355HNPR1R439HPPP1CBV263LBIRC6E4424VIL1Q468QMOGAT3c2359GAUSP20A675TMEX3BS256YMUC13T30AZBTB10R456QSLC26A3V54IC9orf84E8DPPP1R36R303CPLEKHA6Q171SSH3P639PDSCAML1G1252DSLCO1C1R639KENDOVA2VCOL9A1A680TMC2RF228LBPIFB2A427SLOHchr812546DELCDKN2ARNF43P660fsRNF43C471fsRNF43E39fsRNF43M1_G4delRNF43S321RNF43P231LFig Somatic mutations identiï¬ed in MTP19 and MTP8 in targeted and whole exome sequencing We show mutations identiï¬ed in each sampleincluding lowgrade IPMN light blue highgrade IPMN dark blue ductal cancer red mucinous cancer pink and cancerization purple The type ofsequencing analysis targeted or whole exome performed for each sample is indicated in a track on the bottom Representative images of neoplastic tissuestained by hematoxylin and eosin as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP2MTP8MTP3MTP19MTP1MTP5MTP24LG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationMutationsFig Evolutionary reconstruction of samples analyzed by whole exome and targeted sequencing In all cases noninvasive samples bluegreenprecede invasive samples redpink in the evolutionary history In MTP5 different invasive cancer samples are placed in different regions of thephylogeny highlighting multiple independent invasion events in this lesion In MTP19 a sample of cancerization purple has descended from invasivecancer sampleseach case with multiple clonally related but distinct dysplasticsamples from each neoplasm Fig Importantly removal ofdriver gene mutations from these analyses did not significantlyalter the resulting phylogenies indicating that the evolutionaryrelationships are supported by mutations in addition to driveralterations which might be shared by chance Thus the inferredevolutionary relationships between IPMNMCNs and cancersamples are robust as the probability of sharing a nonhotspotmutation due to chance alone is vanishingly small in million while the mean number of shared point mutations was the vast majority of which did not occur in hotspotsAnother potentialexplanation forshared mutationsthis sample impurityinnoninvasive and invasive samples is the presence of a smallnumber of cancer cells contamination in IPMNMCN samplesOur detailed pathological characterization and macro or microdissection minimized the risk ofInaddition variant allele frequencies VAFs of shared mutationsin IPMNMCN and cancer samples can also help to evaluate thelikelihood of such contamination In mutations shared betweenthe IPMNMCN and cancer the mean VAF in the noninvasivesamples was with only of shared mutations having aVAF below in the noninvasive samples These high VAFsindicate that the shared mutations were not the result of a smallnumber of cancer cells contaminating the noninvasive samplesOverall these data provide evolutionary evidence that IPMNs andMCNs were precursors to invasive pancreatic cancer with lowgrade regions usually preceding highgrade regions and ultimatelyresulting in invasive carcinomaDriver genes of IPMNMCN tumorigenesis Through wholeexome and targeted sequencing analyses of IPMNsMCNs andassociated invasive carcinomas we conï¬rmed the high prevalenceof mutations in previously identiï¬ed pancreatic driver genesincluding mutations of KRAS of cases GNAS CDKN2A TP53 SMAD4 and TGFBR2 Fig 1b Somatic mutations were also identiï¬ed in RNF43 which has been previously highlighted for its role as a driver inmucinproducing pancreatic cysts4 Somatic mutations wereobserved at low prevalence in key positions in the PI3K PIK3CATSC2 and WNT APC CTNNA2 CTNNB1 signaling pathwaysas well as in STK11 Fig 1b Supplementary Data Alteration ofthese genes and pathways has been previously reported in afraction of IPMNs471314 The two MCNs analyzed were similarto the IPMNs in the cohort with hotspot mutations in KRAShomozygous deletion of CDKN2A and inactivating mutations inRNF43 among others but as expected these MCNs did not haveGNAS alterations Supplementary Figs S3 S76In addition to driver genes previously reported in IPMNs ourdata provide an opportunity to discover novel drivers of IPMNtumorigenesis We identiï¬ed somatic mutations in the DNAdamage response gene ATM in of lesions including onenonsense mutation Fig 1b Supplementary Data In additionwe identiï¬ed alterations in Hedgehog pathway member GLI3 in of cases Fig 1b Supplementary Data We alsoidentiï¬ed somatic mutations in a previously described hotspot inSF3B1 which encodes a protein critical for RNA splicing Fig 1bSupplementary Data Ampliï¬cations of the wellcharacterizeddriver genes ERBB2 and MYC were each observed in a single caseand have not been previously reported in IPMNs SupplementaryData Other altered genes with a previously unknown role inIPMN tumorigenesis include MUC16 four cases PTPRT fourcases and CNTN5 three cases Fig 1b Intriguingly in onecase an STK11 mutation was found in combination with bialleliclosstheATM lossand cancerspeciï¬c biallelic KEAP1NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178combination ofreported in lung cancers Supplementary Fig S315these three mutations has previously beenAlthough KRAS mutations occur in the majority of IPMNs andare thought to initiate tumorigenesis in these lesions two IPMNslacked mutations in this gene One case contained a hotspotmutation in codon of GNAS another potential initiator ofIPMN tumorigenesis as well as alterations in TP53 and RNF43Supplementary Fig S15 The other case lacked mutations in anyof the frequently altered pancreatic driver genes but containedhotspot mutations in both CTNNB1 S45P and SF3B1 H662QSupplementary Fig S10 These cases highlight alternativepathways of initiation and progression in IPMNs lacking KRASmutationsOrder of genetic alterations in IPMNMCN tumorigenesis Ourmultiregion sequencing approach of IPMNsMCNs and associated invasive carcinomas provided insights into the order ofspeciï¬c genetic alterations in pancreatic tumorigenesis In ofthe cases at least one somatic mutation in the initiating drivergenes KRAS and GNAS was shared between the noninvasivecomponent and associated invasive cancer with the remainingcase lacking mutations in these genes Somatic mutations in TP53and CDKN2A were also shared in the noninvasive componentand associated invasive cancer in the majority of cases In contrast SMAD4 had alterations conï¬ned to the invasive carcinomain three cases and was shared between noninvasive and invasivesamples in four cases Fig 1b The majority of SMAD4 alterations in all sample types were biallelic including in noninvasive samples and in invasive samples SupplementaryData Alteration of TGFBR2 which functions in the samesignaling pathway as SMAD4 was also restricted to the cancer inone case Fig 1b The other genes with mutations restricted tothe invasive cancer CDKN2A CNTN5 PIK3CA KEAP1 andRET only had this pattern in a single sample Fig 1bOur study also identiï¬ed driver mutations in subclones ofnoninvasive neoplasms that diverged from and were not presentin invasive cancer These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumorsuppressor geneseg PIK3CA pE545K CTNNB1 pS45FSMAD4 pE33fs and multiple inactivating RNF43 mutations inpatient MTP3 Supplementary Fig S3 Mutations in RNF43were a particularly striking ï¬nding in these cases as somenoninvasive components contained several different RNF43mutations each limited to a small number of sections and noneinvolving the invasive cancer Fig Supplementary Fig S3 Inaddition to heterogeneity in RNF43 in early lesions we alsoidentiï¬ed two cases with multiple mutationsin KRAS inprecursor lesions of which only one was present in the invasivecancer For example in MTP19 KRAS pG12V was present in themajority of IPMN samples as well as all the invasive cancersamples but there were an additional four other KRAS mutationsall occurring in hotspot positions that were present in a smallnumber of sectionsin lowgrade IPMN samples Fig Intriguingly these three lowgrade IPMN regions shared nomutations with the invasive cancertheyrepresented genetically independent clonessuggesting thatNotably while there were often many differences in thesomatic point mutations identiï¬ed in the matched noninvasiveand invasive samples the copy number proï¬les were quite similarbetween IPMNMCNs and invasive cancers Fig Supplementary Data and the proportion of copy number alterationsunique to cancer samples was similar to that observed forsomatic mutations While homozygous deletion of some genesoccurred in the invasive cancer but notthe noninvasivecomponent such as CDKN2A in MTP8 Fig analyses ofchromosomal gains and losses through assessment of allelicimbalance revealed that an average of of the genome wassimilar in copy number in matched noninvasive and invasivesamples Fig Supplementary Data Insights into pancreatic neoplasia revealed by sequencing Thesamples analyzed by targeted sequencing were characterizedmorphologically and meticulously isolated using laser capturemicrodissection Even with this process we identiï¬ed samples intwo cases that were characterized morphologically as IPMNs butthrough genomic and evolutionary analyses were determined tobe identical to or descendants of the associated invasive cancersFor example in MTP19 some of the samples originally identiï¬edmorphologically as noninvasive IPMN and T1 sharedall the mutations present in the invasive cancer sample T2 andcontained additional mutations suggesting that these samplesdescended from the cancer Figs Evolutionary analysessuggested that some of the morphologically identiï¬ed IPMNsamples in this case as well as MTP1 actually representedintraductal spread of invasive carcinoma also referred to ascancerization of the ducts In these cases after invading thestroma the carcinoma invaded back into and colonized the cystsuch that it was morphologically indistinguishable from IPMNwith highgrade dysplasiaIn one case MTP5 we also identiï¬ed an interesting pattern ofmultifocal invasion of the carcinoma In this case we analyzedï¬ve different samples from invasive cancerthree samples wereisolated from a mucinous carcinoma and two samples wereisolated from a ductal carcinoma Based on evolutionary analysesof the patterns of shared and distinct mutations in the cancersand IPMNs we conclude thatthere were multiple separateinvasion events in this lesion as represented by the mutationsshared between the invasive cancers and noninvasive componentsas well as those that were unique to the speciï¬c invasive cancersFig Supplementary Fig S5As our study represents the largest cohort of comprehensivelysequenced IPMNsMCNs we also analyzed mutational signaturesin our dataset Intriguingly our data contrast somewhat with themutational signatures previously reported in pancreatic ductaladenocarcinoma PDAC1617 Like PDAC the most prominentmutational signature was associated with age Signature 1Awhich was identiï¬ed in almost every case SupplementaryFig S19 However we also identiï¬ed signatures associated withAPOBEC enzymes four cases smoking three cases andmismatch repair deï¬ciency cases Although smoking isconsidered a risk factor for pancreatic cancer until now themutational signature associated with smoking has not beenreported in pancreatic neoplasia18Evolutionary timeline of highgrade IPMN to PDAC To estimate the time between the development of highgrade IPMN andPDAC we evaluated Bayesian hierarchical models for the numberof acquired mutations under a range of possible mutation ratesThese models estimate the time interval between a founder cell ofa PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division timesare constantseeMethods We performed this analysis on the paired WES datafrom of our cases Supplementary Fig S20 We excludedMTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such we lacked WESdata from an IPMN sample in this case In the analyzed casesthe average median time to progression from IPMN to PDAC was years but the models showed a bimodal distribution Thismedian time was nearly years for patients but nearly yearsthis period of developmentthroughoutNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178for patients with more than acquired mutations highlightingpotential variability in progression time between patients Forexample in patient MTP1 most models suggested an average of years between the development of the IPMN and the PDAC CI years In contrast for patient MTP2 with additional mutations acquired in the PDACthe transitionappears to have been slower with an average estimate of years CI years from the Bayesian models Overall theseanalyses suggest that for most patients there is a significantwindow of time between development of highgrade dysplasiaand pancreatic cancer providing an opportunity for surveillanceand interventionDiscussionThis study representsthe largest dataset of whole exomesequencing of IPMNs and MCNs to date Importantly our dataestablished that both IPMNs and MCNs are direct precursors ofinvasive pancreatic cancer Fig This conclusion has beenpreviously suggested by the morphological relationship betweenthe noninvasive neoplasms and invasive cancer on traditionalhistologic sections as well as shared driver gene mutations intargeted sequencing studies679 However the presence ofmany shared driver and passenger mutations clearly demonstrated the common origin of IPMNsMCNs and invasive pancreatic cancers in our study and evolutionary analyses revealedthat dysplastic lesions precede invasive cancers Evolutionaryanalyses suggested that highgrade noninvasive lesions occur over years before invasive carcinoma providing a window ofopportunity for early detection and interventionIn this study we identiï¬ed somatic mutations in driver genesthat had not been previously implicated in IPMNsMCNs Forexample we identiï¬ed alterations in the DNA damage responsegene ATM in of the analyzed cases Germline mutations inATM have been recently reported in patients that developedIPMNs highlighting the potential importance of this gene inIPMN risk19 In addition mucinous colloid carcinomas aresignificantly more common than typical ductal carcinomas inpatients with germline ATM mutations further highlighting thelink between mutations in this gene and IPMNs20 Although theATM gene is large potentially increasing the likelihood of passenger or artifactual mutations even larger genes such as TTNhad a lower mutation prevalence suggesting that at least some ofthe alterations identiï¬ed in ATM are likely to be bona ï¬desomatic mutations Somatic mutations in GLI3 which encodes acomponent of the Hedgehog signaling pathway were identiï¬ed in of cases Somatic mutations in GLI3 were recently reportedin a distinct morphological variant of pancreatic carcinomaundifferentiated carcinoma with osteoclastlike giant cells aswell as at a low prevalence in sporadic PDAC suggesting that theimportance of GLI3 mutations and its signaling pathway inpancreatic tumorigenesis may extend beyond IPMNsMCNs21The hotspot mutations in SF3B1 which encodes a protein criticalfor RNA splicing are also potential drivers in the IPMN pathwayHowever somatic mutations in this gene have been reported in avariety of other neoplasms including hematologic malignanciesand uveal melanoma24We highlight somatic alteration of the SMAD4 pathway as aputative driver of progression to invasive cancer as mutations inSMAD4 or TGFBR2 occurred only in invasive cancer samples in of the cases analyzed SMAD4 was the only gene with cancerspeciï¬c mutations in more than one case highlighting thepotentially unique role this gene plays in pancreatic carcinogenesis This role has been previously suggested by next generationsequencing of highgrade PanINs showing an absence of SMAD4mutations in precancerous lesions as well as cancerspeciï¬cSMAD4 mutationsreported in a paired PanINcarcinomaanalyses2728 Loss of SMAD4 expression limited to invasivecarcinomas has been reported in MCN and IPMNassociatedinvasive cancers and targeted sequencing of a small number ofIPMNs and matched cancers identiï¬ed a single case with aSMAD4 mutation occurring only in the cancer72829 In our datathere were also four cases where mutations in SMAD4 wereshared between noninvasive and invasive cancer samples and twowhere SMAD4 mutations were limited to the noninvasive component Although our wholeexome approach could not detect alltypes of SMAD4 alterations such as rearrangements or epigeneticchanges the majority of SMAD4 mutations observed in bothnoninvasive and invasive components affected both alleles Takentogether this suggests that the role of SMAD4 mutations may notbe universal and may depend on other factors including cellintrinsic such as somatic mutations in other driver genes andcell extrinsic such as stromal and immune microenvironmentmechanismsAlthough some of our cases had SMAD4 mutations limited tothe invasive cancer most of the IPMNMCNassociated cancerslacked driver gene alterations that were associated with invasivedisease suggesting that malignant progression is not universallydriven by point mutations Previous studies have speciï¬callydemonstrated the importance of copy number alterations andchromosomal rearrangements in pancreatic tumorigenesis30 Wedid not identify large differences in the copy number proï¬lesbetween noninvasive components and associated invasive cancers suggesting that global genomic instability may be importantas an early feature of tumorigenesis but is not likely to drivemalignant transformation in many casesOur study also revealed prevalent genetic heterogeneity indriver gene mutations in early lesions demonstrating morecomplex processes than previously suggested by traditional lineartumorigenesis models Similar to our recently reported polyclonalorigin of IPMNs12 we identiï¬ed multiple independent clonesinitiated by distinct KRAS mutations in two cases in the currentstudy In addition our study identiï¬ed multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones apattern previously observed by our group and not shared by othergenes in our whole exome sequencing analyses1231 In most casesRNF43 mutations were enriched in noninvasive components andabsent from the associated invasive cancers More generally weobserved multiple instances of clear driver mutations includinghotspot mutations in oncogenes as well as inactivating mutationsin tumor suppressor genes that were limited to the noninvasivecomponents and not present in the associated invasive cancerThus these mutations occur and clonally expand in the IPMN orMCN but are not present in the subclone that subsequentlyinvades Such independent evolution of premalignant lesions hasbeen observed in other an sites and does not diminish theconclusion of our evolutionary analyses that IPMNsMCNs aretheseprecursors ofobservations suggest unique selective processes at different timepoints in tumorigenesis such that mutations selected in theprecancerous lesion are not selected for or are even selectedagainst in the invasive cancerinvasive pancreatic cancer32 RatherIn addition to these observations about clonal evolution innoninvasive lesions our data also provide genetic evidence formultiple underappreciated processes in pancreatic neoplasiaFirst we provide genetic evidence for intraductal spread ofinvasive carci | Thyroid_Cancer |
"conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patients native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of 109L and platelets count of 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors cant rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it cant work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]TuÄcu D KarakaÅ Z G¶k§e M AÄaoÄlu L Un¼var A SarıbeyoÄlu E Ak§ay A DevecioÄlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c" | Thyroid_Cancer |
Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1 Sujuan Liu2 Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal musclesmetabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancerammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modiï¬cation on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodiï¬cations of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classiï¬ed into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classiï¬edas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle ï¬tness and cardiacmuscle disease which needs further clariï¬cation of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle ï¬ber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates speciï¬c sites toinhibit the expression of musclespeciï¬c genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespeciï¬c genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespeciï¬c genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespeciï¬cgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy ï¬ux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle ï¬bersin which class I HDACs deï¬ciency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespeciï¬c single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD PTEN FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD MEF2D NFκBp65MEF2D PCAF Ampd3RCAN1 MEF2ACpt1bMEF2 Glut4 Myosin HeavyChain PGC1α and Hsc70Pax7MEF2 Glut4 Baf60cAtg7 Beclin1 LC3Dach2Myog Gdf5MEF2Pax7 MFN1 Fam65bdysferlin and MAFbxHDAC10SIRT1PGC1α STAT3IIAIIBIIIMnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylaseNFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I ï¬ber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesityRodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanï¬ Xiao Cui Samant of muscle massHDAC11IVActivation inhibition unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespeciï¬cknockout CR caloric restriction HFD high fat diet IR insulin resistanceheart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in speciï¬c tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myoï¬bers Moresi Therefore these ï¬ndingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter speciï¬cally knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the dissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the ï¬rst ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis ï¬ux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesisAc deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisï¬nding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 ï¬ows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites ï¬rst and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I ï¬berthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I ï¬ber is signiï¬cantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the ï¬nding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I ï¬ber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I ï¬ber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy ï¬ux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespeciï¬c genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspeciï¬c inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are conï¬rmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is signiï¬cantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitï¬ies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespeciï¬c SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is signiï¬cantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneï¬cial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespeciï¬c knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspeciï¬c knockouthep and skeletal musclespeciï¬c knockoutskmmice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in speciï¬c physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet deï¬nedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could signiï¬cantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that speciï¬callyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration signiï¬cantly improved enduranceperformance and signiï¬cantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers puriï¬ed SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample veriï¬cationCONCLUSIONThe very ï¬rst mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modiï¬cations establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the speciï¬c downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and diï¬cult issues in this ï¬eld Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with speciï¬cHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and diï¬culty in speciï¬c inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more speciï¬cHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more speciï¬cityand trials for the treatment of metabolic diseases may have greatpotential as well | Thyroid_Cancer |
Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligandreceptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimers diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdalauencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individuals social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In ï¬eld tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These ï¬ndings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modiï¬ed theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on speciï¬camygdala molecular proï¬les and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentiï¬cation of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe ï¬ndings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat ¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of median tissueculture infectious dose TCID50 diluted in sterile Dulbeccosmodiï¬ed Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pï¬zer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome proï¬les from older pigs could beconfounded with changes in diet and environment associatedwith weaning while proï¬les from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out ï¬ash frozen on dry ice and stored at ¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturersinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ ï¬les were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ ï¬les are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIAsex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identiï¬ed by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe BenjaminiHochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical signiï¬cance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delï¬no Serao Delï¬no and RodriguezZas signiï¬cance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identiï¬cation of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were ï¬ltered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoeï¬cient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module proï¬les were identiï¬edusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module proï¬le was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identiï¬ed by full nodes with sizereï¬ecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was ¦C standard error ¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coeï¬cient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIAsex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The proï¬le ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression proï¬le between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction proï¬le characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the ï¬ndingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identiï¬edin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identiï¬er and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Inï¬uenza AES GO0051050¼Positive regulation oftransportGO0051049¼Regulation of transportGO0050801¼Ion homeostasisGO0048878¼Chemical homeostasisGO0030001¼Metal ion transportES GO0048871¼Multicellular anismalhomeostasisES GO0061564¼Axon developmentGO0007420¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identiï¬er and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBP 10E10 10E10 10E10 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179¼Hormone activityGO0006970¼Response to osmoticstressGO0019221¼Cytokine mediatedsignaling pathwayGO1901385¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway 12E01GO0085029¼Extracellular matrix 18E01assembly 91E02 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identiï¬edby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and EpsteinBarr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented theï¬ndings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i | Thyroid_Cancer |
Body fat distribution predicts cardiovascular events better than bodymass index BMI Waist circumference WC and neckcircumference NC are inexpensive anthropometric measurements We aimed to present the conditional distribution of WC andNC values according to BMI stratiï¬ed by age and sex from the Brazilian Longitudinal Study of Adult Health ELSABrasilbaseline data We analyzed ELSABrasil participants with complete data We used spline quantile regression modelsstratiï¬ed by sex and age to estimate the NC and WC quantiles according to BMI To test a putative association between ageand median NC or WC values we built sexspeciï¬c median regression models using both BMI and age as explanatoryvariables We present estimated 25th 50th 75th and 90th percentiles for NC and WC values according to BMI age and sexPredicted interquartile intervals for NC values varied from to cm and for WC values from to cm Median NCwas not associated with age in men P011 nor in women P079 However median WC increased with advancing age inboth sexes Po0001 for both There was significant dispersion in WC and NC values for a given BMI and age strata for bothmen and women WC but not NC values were associated with increasing age The smaller uence of advancing age on therelationship between BMI and NC compared to WC values may be useful in longitudinal studiesKey words Nomograms Adiposity Bodymass index Waist NeckIntroductionThe prevalence of obesity is increasing worldwideand a growing body of evidence shows that body fatdistribution might add important information for predictingcardiovascular events above and beyond bodymass indexBMI itself A seminal work published in the 1950s already reportedthat differences in the localization of adiposity between menand women are linked to their different cardiovascularproï¬le The study of fat tissue distribution and cardiovascular risk has gained more attention recently and ithas been hypothesized that speciï¬c fat depots couldincrease vascular damage through mediators thatcan uence glucose homeostasis and lipid metabolismammation and coagulation Certain locations of fataccumulation have been linked to diverse cardiometabolic proï¬les suggesting that regionalfat distribution could play an important role in the development ofCorrespondence CP Baena cristinabaenapucprbrReceived March Accepted June Braz J Med Biol Res 1015901414431X20209815the consensus point outcardiovascular diseases in both nonobese and obesepeople Recently the Consensus Statement of theInternational Atherosclerosis Society argued for theinclusion of waist circumference WC as a vital sign giventhat the prevalence of abdominal obesity is increasing anddysfunctional adipose tissue could be estimated moreaccurately by WC than BMI as shown in recent studiesAdditionallythe gap inknowledge with a recommendation for description of WCvalues for a given BMI category across different agesby sex as the limitations of BMI have been increasingly demonstrated in different populations especially indemonstrating changes in adiposity during aging Moreover it is difï¬cult to measure body fat mass directlywhile WC and neck circumference NC are inexpensiveand easily obtainable anthropometric measurementsfor 0cNeck and waist circumference percentilesAnother study looked at one Brazilian population ofthe Baependi Heart Study in a crosssectional analysisand showed that WC in men discriminated the hypertensives better than visceral and body adiposity indexes Body fat distribution patterns vary and the positivecorrelations between BMI and both NC and WC do notfollow perfectlinear associations and despite beingassociated with higher cardiovascular risk in differentpopulations factors as sex age physical activitysmoking habits number of pregnancies and geneticpredisposition have also been linked to body fat distribution Hingorjo studied young universitystudents in Pakistan and found that approximately of NC variance in males and of NC variance infemales was not explained by BMI values In additionthese discrepancies may vary in different populationsand in different time periods Stern showed thatthe predicted WC according to BMI in Chinese men andwomen increased from to In the same countryand time interval Du reported that the prevalence of central obesity in adults with normal o25 kgm2BMI increased from to during yearsThis underlines the importance of studying the WCand NC values according to BMI in large epidemiologicstudies conducted within different populations To datethere is no such study conducted in large samples Therefore our aim was to present the conditional distribution ofWC and NC values according to BMI stratiï¬ed by ageand sex among midadult and elderly men and womenparticipants at the baseline assessment of the BrazilianLongitudinal Study of Adult Health ELSABrasil studya large multicenter cohort study in BrazilMaterial and MethodsStudy designELSABrasil is a multicenter prospective cohort study that enrolled civil servants aged to years from Brazilian cities Belo Horizonte PortoAlegre Rio de Janeiro Salvador S£o Paulo and Vitria In this crosssectional analysis we used baselinedata Approvals were obtained from theinstitutional review boards of all the centers and all thesubjects signed an informed consent formStudy sampleFrom ELSABrasil participants at baseline weexcluded that did not have complete BMI WCor NC data Our sample comprised menand women with complete dataStudy variablesHeight and weight were measured using a standardized scale and a ï¬xed stadiometer and BMI was calculated by dividing body weight by the squared height inBraz J Med Biol Res 1015901414431X20209815meters kgm2 WC was measured using an inelastic tapeof cm MabisGulick USA at the midpoint betweenthe lowest rib margin and the iliac crest NC wasmeasured with an inelastic tape mm right under thethyroid cartilage and perpendicular to the long axis of theneck with the participant in a sitting position All measurements were performed by trained nurses The intraclass correlation coefï¬cient for repeated measurementswas 95CI Age is presented as a continuous variable and alsostratiï¬ed as years years years and years Race was selfreported as White BrownBlack Asian and Native Educational level was stratiï¬edas up to incomplete high school highschool and collegeor above Smoking status was selfreported as neverformer and current smoker Monthly family income atbaseline was converted from Brazilian reals BRL to USdollars USD at a rate USD1 BRL2 and stratiï¬edas oUSD1245 USD1245 and XUSD3320Excessive alcohol drinking was deï¬ned as gweekfor men and gweek for women Blood pressurewas obtained in the sitting position after a minimum restperiod of min Three consecutive readings were obtainedfor each participant after oneminute interval between eachone The mean of the two last measurements was deï¬nedas the casual blood pressureLaboratory measurements were obtained after anovernight fast Fasting glucose was determined enzymatically by the hexokinase method Total cholesterol highdensity cholesterol HDLcholesterol lowdensity cholesterol LDLcholesterol and triglycerides were determinedby the enzymatic colorimetric method Hypertension was deï¬ned as the use of medications to treathypertension systolic blood pressure X140 mmHg ordiastolic blood pressure X90 mmHg at baseline Diabeteswas deï¬ned by a medical history of diabetes use ofmedications to treat diabetes a fasting glucose X126 mgdL glycated hemoglobin HbA1C levels X65 or a 2horal glucose tolerance test X200 mgdL Dyslipidemiawas deï¬ned as use of lipidlowering treatment or a LDLcholesterol level X130 mgdLStatistical analysisCategorical variables are reported as absolute countsand proportions Continuous variables are reported asmeans±SD or median interquartile range We usedspline quantile regression models stratiï¬ed by sex andage to estimate the conditional distribution of NC and WCaccording to BMI These models were used to estimatethe 25th 50th 75th and 90th percentiles for NC and WCvalues in the BMI range between and kgm2 To testa putative association between age and median NC orWC values in men and women we built sexspeciï¬cmedian regression models using both BMI and age asexplanatory variables Analyses were performed using theR software Signiï¬cance level was set at 0cNeck and waist circumference percentilesResultsTable details the characteristics of the sample according to sex The mean age was years Most of theparticipants selfreported being of White race having a college education and never havingsmoked Table shows the estimated 25th 50th75th and 90th percentiles for NC values in cm accordingto BMI age strata and sex Predicted interquartile intervals75th25th percentile for NC values varied from to cm to of predicted median values Similarly Table shows the predicted conditional distribution for WC valuesTable Characteristics of the study samplein cm also according to BMI age strata and sexPredicted interquartile intervals for WC values varied from to cm to of predicted median valuesGraphical presentations ofthe results are available inFigures and We built sexspeciï¬c median regression models usingboth BMI and age as explanatory variables to testifmedian NC or WC were associated with age in men andwomen We found median NC was not associated withage in men P011 nor women P079 Howevermedian WC increased with advancing age in both sexesPo0001 for both Figure shows predicted median WCAge years mean±SD years N years N years N years N RaceWhite N Brown N Black N Other N Educational levelIncomplete high school N High school N College or above N Monthly family incomeoUSD1245 N USD12453319 N XUSD3320 N Hypertension N Diabetes N Dyslipidemia N SmokingNever N Past N Current N Excessive drinking N Systolic blood pressure mmHg mean±SDDiastolic blood pressure mmHg mean±SDBodymass index kgm2 mean±SDNeck circumference cm mean±SDWaist circumference cm mean±SDFasting plasma glucose mgdL mean±SDTotal cholesterol mgdL mean±SDLDLcholesterol mgdL mean±SDHDLcholesterol mgdL mean±SDTriglycerides mgdL median [P25P75]MenN6879± WomenN8206± AllN15085± ±±±±±±±±± [] ±±±±±±±±± [] ±±±±±±±±± []LDL lowdensity cholesterol HDL highdensity cholesterolBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesTable Neck circumference predicted quantiles for sex age and body mass index BMIAge years years years yearsBMIMenWomenP25P50P75P90P25P50P75P90and NC values for men and women with BMIs of and kgm2DiscussionWe presented the conditional distribution of WC andNC values according to age sex and BMI values in alarge sample of Brazilian adults There was a significantvariance in WC and NC values for a given BMI and agestrata for both men and women In addition we found thatWC but not NC values were associated with increasingage Some mechanisms have been proposed to explaindifferent fat tissue distribution within individuals with thesame BMI such as dysfunctional adipose tissue sedentary lifestyle or both As mentioned above local bodyfat mass and its clinical markers as NC and WC areassociated with multiple phenotypes of higher cardiovascular risk The association between these phenotypes andNC or WC cannot be explained exclusively by higherBMIs Evidence from the Framingham Study shows thatbody fat distribution and fat depots could be betterpredictors of cardiovascular diseases CVD than BMI Braz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesTable Waist circumference predicted quantiles for sex age and body mass index BMIAge years years years yearsBMIMenWomenP25P50P75P90P25P50P75P90Population data from the European Prospective Investigation into Cancer and Nutrition of the Norfolk cohort showedthat WC and waisttohip ratio were more consistentpredictors of coronary heart disease than BMI Theresults of the Framingham Heart Study showed that NCwas associated with CVD risk factors after adjustment forBMI In addition for speciï¬c scenarios the associationbetween these anthropometric measurements and cardiovascular risk may be heterogeneous or even additive In acomparison of the clinical usefulness of NC and WC inindividuals with severe obesity mean BMI meanage years NC values had stronger associations withtype diabetes insulin resistance metabolic syndromeand hypertension compared to WC values In theELSABrasil NC was significantly associated withcardiometabolic risk factors as insulin resistance hypertriglyceridemia and higher blood pressure after adjustment for WC and BMI The study of body fat distribution patterns in subjectswith similar BMI may be importantfor both identifyingindividuals at a higher cardiovascular risk compared topeers with the same BMI and understanding the factorslead to unfavorable fat distribution proï¬les Therethatis evidence that WC values are increasing more thanBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesFigure Predicted 25th 50th 75th and90th percentiles for waist circumferenceWC values according to age body massindex and sexBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesFigure Predicted 25th 50th 75th and90th percentiles for neck circumferenceNC values according to age body massindex and sexBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesFigure Median waist WC and neck circumference NC values according to age strata for men and women with body mass index of and kgm2 Data are reported as meansexpected for the increase in BMI values in recent decadesin different populations Stern analyzed datafrom Chinese men and women aged to yearsin and Chinese men and women with thesame age range in They found that for every agestrata and in both sexes predicted WC for individuals witha BMI of or kgm2 were higher in than in Janssen compared data from subjectsaged to years in to individuals also aged to years who were evaluated in inCanada They found that for individuals with a BMI of kgm2 the predicted WC values in were to cm higher than in In addition each kgm2 increasein BMI value was associated with higher WC increases in compared to Walls comparedNHANES data from participants and participants and found that WC valuesin American adults younger than years of age but notin older individuals increased cm more than expectedfor the rise in BMI values during this period Another studywith aggregated data from three crosssectional surveystaken in and n8313 and respectively looked at WC change inAustralians and found an independent increase of WCshowing that the proportion of obese people detected byWC increased for women and for men On the other hand Elobeid analyzed adifferent timeframe in the United States anddid not ï¬nd a slope for the relationship between WCand BMI over time significantly different from zero Therelationship between NC and BMI is less studied and toour knowledge there are no large epidemiological studies describing the conditional distribution of NC valuesaccording to BMI and age strata Our results highlight theimportance of such descriptions as we found that therelationship between BMI and NC values was uencedless by age strata than the relationship between BMI andWC values Future longitudinal analysis of ELSABrasildata will provide important information about the clinicalrelevance of this ï¬ndingA study by Stern shows predicted WCvalues for Chinese adults with a BMI of kgm2 and forthose with a BMI of kgm2 We compared their data which matches the inclusion period for ELSABrasilto our predicted median WC values for men and womenwith the same BMI values We found slightly higher predicted WC values for men and lower predicted WC values for women in ELSABrasil compared to the Chinesepopulation In all cases estimates did not differ by morethan cm Some differences between these two studiesmay be partially accountable for this ï¬nding First Stern used linear regression which is a least squareBraz J Med Biol Res 1015901414431X20209815 0cNeck and waist circumference percentilesmodel for mean values and in our study we used quantileregression which is a linear mathematical optimizationtechnique for estimating quantile valuesincluding themedian Although we aimed to compare similar agestrata it is possible that heterogeneity in age stratiï¬cationcutoffs may also have yielded different estimates as bothstudies point to higher WC values according to BMI withincreasing ageOur study had some limitations As it is a descriptivestudy with crosssectional design causal inferences werenot focused Although inexpensive both WC and NC maybe prone to measurement errors and NC values may alsobe uenced by neck muscular volume Thereforemeasurements in other samples should be studied beforeusing these values as a screening tool As strengths ourstudy described the distribution oftwo anthropometricmeasurements in a very large multicenter epidemiologic study in Brazil The conditional distribution of thesevalues according to BMI may be used as markers ofbody fat distribution in future prospective ELSABrasilanalyses To our knowledge analyses of large samplesfocusing on NC values distributions conditioned to BMIand age were not previously published Although it mustbe conï¬rmed by prospective data the smaller uence ofadvancing age on the association between BMI and NCcompared to WC values may be useful to help understand distribution of body fat in longitudinal studies Webelieve our study contributes to ï¬ll the gap of evidencementioned in the recent statement ofthe InternationalAtherosclerosis Society in terms of providing a description oftwo adiposity measures by different BMI ageand sex Moreover our study adds to the previous bodyof evidence on the change of waist and neckcircumferences according to BMI age and sex as aneasy and reproducible tool to identify adverse fat depotsphenotypesIn this study we estimated sex and agespeciï¬cquantile values for NC and WC according to BMI Therewas significant dispersion in WC and NC values for agiven BMI and age strata for both men and women WCbut not NC values were associated with increasing ageAcknowledgmentsThis work was supported by the Brazilian Ministry ofHealth and CNPq grant numbers 0106001000RS 021200BA 0106030000ES 0106027800MG 00SP 0106007100RJ The authors would like to thank the participants of the ELSABrasil who made this studypossibleReferences Canoy D Boekholdt SM Wareham N Luben R Welch ABingham S Body fat distribution and risk of coronaryheart disease in men and women in the European prospective investigation into cancer and nutrition in Norfolk cohorta populationbased prospective study Circulation 101161CIRCULATIONAHA106673756 Vague J The degree of masculine differentiation ofobesities a factor determining predisposition to diabetesatherosclerosis gout and uric calculous disease Obes Res 101002j155085281996tb00536x Lim S Meigs JB Ectopic fat and cardiometabolic and vasInt J Cardiol riskcularjijcard201308077 Lim S Meigs JB Links between ectopic fat and vasculardisease in humans Arterioscler Thromb Vasc Biol 101161ATVBAHA114303035 Anothaisintawee T Sansanayudh N Thamakaison SLertrattananon D Thakkinstian A Neck circumference asan anthropometric indicator of central obesity in patients withprediabetes a crosssectional study Biomed Res Int Preis SR Massaro JM Hoffmann U DAgostino Sr RBLevy D Robins SJ Neck circumference as a novelmeasure of cardiometabolic riskthe Framingham heartstudy J Clin Endocrinol Metab 101210jc20091779 Ross R Neeland IJ Yamashita S Shai I Seidell J Magni P Waist circumference as a vital sign in clinical practicea Consensus Statement from the IAS and ICCR WorkingBraz J Med Biol Res 1015901414431X20209815Group on Visceral Obesity Nat Rev Endocrinol 101038s4157401903107 Amankwah N Brunetti R Kotha V Mercier C Li L Ding J Abdominal obesity index as an alternative centralobesity measurement during a physical examination OpenNutr J Gearon E Tanamas SK Stevenson C Loh VHY Peeters AChanges in waist circumference independent of weightImplications for population level monitoring of obesity PrevMed Baltim 101016jypmed2017 Lohman TG Roche AF Martorell R Anthropometric standIL Humanardization reference manual ChampaignKinetics De Oliveira CM Ulbrich AZ Neves FS Dias FAL HorimotoARVR Krieger JE Association between anthropometric indicators of adiposity and hypertension in a Brazilianpopulation Baependi heart study PLoS One e0185225 101371journalpone0185225 Liang J Wang Y Li H Liu X Qiu Q Qi L Neckcircumference and early stage atherosclerosis the cardiometabolic risk in Chinese CRC study Cardiovasc Diabetol 101186s129330140107x Zhou J Ge H Zhu M Wang L Chen L Tan Y Neckcircumference as an independent predictive contributor tocardiometabolic syndrome Cardiovasc Diabetol Baena CP Lotufo PA Santos I de S Goulart AC BittencourtMS Duncan BB Neck circumference is associatedwith carotid intimalmedia thickness but not with coronary 0cNeck and waist circumference percentiles Schmidt MI Duncan BB Mill JG Lotufo PA Chor D BarretoSM Cohort proï¬le longitudinal study of adult healthELSABrasil Int J Epidemiol 101093ijedyu027 Mill JG Pinto K Griep RH Goulart A Foppa M Lotufo PA Medical assessments and measurements in ELSABrasil [in Portuguese] Rev Saude Publica 101590S003489102013047003851 Schmidt MI Griep RH Passos VM Luft VC Goulart ACMenezes GM de S Strategies and development ofquality assurance and control in the ELSABrasil [in Portuguese] Rev Saude Publica S003489102013047003889 Despr©s JP Lemieux I Abdominal obesity and metabolic syndrome Nature nature05488 Britton KA Massaro JM Murabito JM Kreger BE HoffmannU Fox CS Body fat distribution incident cardiovasculardisease cancer and allcause mortality J Am Coll Cardiol 101016jjacc201306027 Assyov Y Gateva A Tsakova A Kamenov Z A comparisonof the clinical usefulness of neck circumference and waistcircumference in individuals with severe obesity Endocr Res Janssen I Shields M Craig CL Tremblay MS Changes inthe obesity phenotype within Canadian children and adults to Obesity oby2011122 Walls HL Stevenson CE Mannan HR Abdullah A ReidCM McNeil JJ Comparing Trends in BMI and WaistCircumference Obesity 101038oby Elobeid MA Desmond RA Thomas O Keith SW AllisonDB Waist circumference values are increasing beyondincreases Obesity those expected from BMI 101038oby2007282artery calcium Results from The ELSABrasil Nutr MetabCardiovasc Dis 101016jnumecd Baena CP Lotufo PA Fonseca MGM Santos IS GoulartAC Bensenor IMJ Neck circumference is independentlyassociated with cardiometabolic risk factors crosssectionalanalysis from ELSABrasil Metab Syndr Relat Disord 101089met20150083 Bouchard C Tremblay A Genetic uences on theresponse of body fat and fat distribution to positive andnegative energy balances in human identical twins J Nutr 943S947S 101093jn1275943S Chantler S Dickie K Micklesï¬eld LK Goedecke JHGoedecke JH Micklesï¬eld LK Determinants of change inbody weight and body fat distribution over years in asample of freeliving black South African women Cardiovasc J Afr 105830CVJA2016038 Suder A Socioeconomic and lifestyle determinants of bodyfat distribution in young working males from Cracow PolandAm J Hum Biol 101002ajhb20687 Hingorjo MR Qureshi MA Mehdi A Neck circumference asa useful marker of obesity a comparison with body massindex and waist circumference J Pak Med Assoc Stern D Smith LP Zhang B GordonLarsen P Popkin BMChanges in waist circumference relative to body mass indexin Chinese adults Int J Obes 101038ijo201474 Du T Sun X Yin P Huo R Ni C Yu X Increasing trends incentral obesity among Chinese adults with normal bodymass index BMC Public Health Aquino EML Barreto SM Bensenor IM Carvalho MS ChorD Duncan BB Brazilian Longitudinal Study of AdultHealth ELSABrasil objectives and design Am J Epidemiol 101093ajekwr294Braz J Med Biol Res 1015901414431X20209815 0c' | Thyroid_Cancer |
properly citedIntroduction Endogenously produced antiganglioside antibodies could aï¬ect the evolution of cutaneous melanoma Epidemiologicaland experimental evidence suggest chronic ammation to be one of the hallmarks in skin cancers The aim of the study was tocharacterize the relation between antiganglioside antibodies and ammation in cutaneous melanoma focusing on gangliosidesGM1 GM2 GM3 GD1a GD1b GT1b GQ1b Material and Method We performed an observational study that included subjects subdivided into three groups patients with metastatic melanoma cases patients with primary melanoma casesand healthy subjects subjects The assessment of antiganglioside antibodies IgG and IgM classes against GM1 GM2GM3 GD1a GD1b GT1b GQ1b was performed using immunoblot technique EUROLine kit Results The presence of IgGand IgM antiganglioside antibodies in primary melanoma was as follows antiGM1 and GM2 and GM3 and GD1a and GD1b and GT1b and GQ1b and In metastaticmelanoma the level of antiganglioside antibodies was significantly lower compared with primary melanoma p while inthe control group they were absent Antiganglioside antibodies antiGM1 and GD1a were positively correlated while antiGM3GD1b and GT1b were negatively associated with the ammatory markers interleukin IL8 and C reactive protein CRPConclusions Tumour ganglioside antigens generate an immune response in patients with primary melanomas The hosts ability toelaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a dangersignal from the tumour microenvironment Antiganglioside antibodies associated with ammation markers could be used asdiagnostic monitoring and treatment tools in patients with cutaneous melanoma IntroductionGangliosides are a group of bioactive glycolipids located onthe outer face of cell membranes These glycolipids play amajor role in cell proliferation diï¬erentiation migrationapoptosis signal transduction cell adhesion modulatinggrowth factor or hormone receptor antigen recognition protein traï¬cking viral transformation and oncogenesis []Atypical expression of some ganglioside antigens associatedwith certain tumours neuroblastomas melanomas gliomaslymphomas small cell lung cancer and prostate cancer andfurthermore could play an importantrole in cancer 0cJournal of Immunology Researchimmunotherapy [] Gangliosides that are released inextracellular spaces could have dual action antitumor andprotumour eï¬ect [] Data regarding the endogenousimmune response directed toward tumour gangliosides andthe signiï¬cance of this response are limited A series of studiesperformed in in vivo experimental models and in vitro inmurine and human cancer cells have shown that monoclonalantiganglioside antibodies have antitumor potential Theseantibodies exert numerous antitumor eï¬ects through variousmechanisms An important mechanism is the translocationof gangliosides from the plasmatic membrane into theintracellular spaces so binding of antibodies to the surfaceof the tumor cells and complement activation that leads to celllysis mediated by complementdependent cytotoxicity andantibodymediated cellular cytotoxicity [ ] Antiganglioside antibodies modulate ceramide synthesis [ ]reception and transduction of the cytotoxic signal [] theyare involved in suppression or induction of cell death throughdiï¬erent pathways apoptosis necrosis oncogeneslike structural and functional changes of mitochondria accumulationof reactive oxygen species acetylation of gangliosides accumulation of sphingosine sphingamine ceramides [ ]Proteomic studies showed that antiganglioside antibodiescould induce changes like the disruption of signalling systemsP38MAPK PARP JNK123 METc ERK12 P13KAKTand FAK modulation of the level and function of transcription factors P53 SP1 MYCN and HSF1 regulating the balance between apoptosisinducing and apoptosissuppressingfactors cysteineaspartylproteases Bax Bcl2 [ ]These antibodies stimulate the cytotoxicity of chemotherapeutic drugs and small molecule inhibitors [ ] As a result antiganglioside antibodies could be used as diagnostic monitoringand treatment tools in cancer patients [ ]Ganglioside levels are increased in malignant melanocytes and represent an important topic of research [ ]Several researchers have emphasized the role of glycolipidsas markers of melanoma A study analysing the expressionof gangliosides in melanocyte lines and melanoma cell linesfound out an increased expression of GD3 synthase genesin melanoma cells but not in melanocytes The same resultswere obtained for GM2GD2 synthase [] It seems thatgangliosides induce cell proliferation and invasion throughp130Cas and paxillin in melanoma cells []Inï¬ammatory mechanisms play an important role inmelanoma Multiple studies have shown that plasma levelsof C reactive protein CRP increase during tumor proliferation and several relations have been evaluated CRPsurvivalrelationship CRPresponse therapy CRPammationNowadays CRP is considered a true marker for assessingammation in melanoma as well as a marker for responseto treatment Prospective studies have provided consistentresults in the predictive value of CRP in neoplastic diseaseproving high sensitivity and speciï¬city [] In addition inmelanoma elevated levels of CRP may reï¬ect the amountand activity of circulating proammatory cytokines eginterleukin IL8 IL8 plays a crucial role in regulating cellfunction for host defence and for developing natural immunity [ ] Moreover IL8 is released by various cell typesincluding polymorphonuclear neutrophils PMNs monoSerum dilution Incubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceIncubation with conjugated enzyme degrees min with balanceWashing with universal tampon times min each with balanceIncubation with fixed on strip antigens degrees min with balanceWashing with universal tampon times min each with balanceEUROLine scan evaluationFigure Antiganglioside detectioncytes T lymphocytes and endothelial cells upon exposureto ammatory stimuli Melanoma cells have been reportedto express IL8 and this uences their oncogenic properties[ ] IL8 follows the evolution of melanoma progression and regression under treatment reï¬ecting the stage ofthe disease []Based on these accumulating data we have investigatedantiganglioside antibodies in correlation with other ammatory markers IL8 CRP and the clinical evolution of the melanoma patients Clarifying these relations could significantlyimprove the prediction of clinical outcomes Furthermore itcan lead to the development of appropriate therapeutic strategies in patients with cutaneous melanoma Material and Method Patients We performed an observational prospectivestudy during years in Clinical Hospital for Infectious andTropical Diseases Victor BabesDermatology Department Bucharest The study was approved by the Ethics Committee of the Hospital All participants agreed to be includedin research studies without prejudice of the diagnosis orpersonal image and signed the informed consent accordingto the Declaration of HelsinkiThe study included adult patients with cutaneous melanoma with no other pathologies and no treatment for theprimary disease Exclusion criteria were age under yearspregnancy alcohol use melanoma under treatmentWe performed an observational study that included subjects subdivided in three groups patients with metastaticmelanoma cases patients with primary melanoma cases and healthy subjects with matching sex and age subjects Patients were selected and examined accordingto ESMO Clinical Practice Guidelines for melanoma 0cJournal of Immunology ResearchTable Test strips coated with parallel lines of puriï¬ed antigensAntigenGM1GM2GM3GD1aGD1bGT1bGanglioside typeSourceMonosialoganglioside GM1Monosialoganglioside GM2Monosialoganglioside GM3 Dog erythrocytesBovine brainBovine brainDisialoganglioside GD1aDisialoganglioside GD1bTrisialoganglioside GT1bBovine brainBovine brainBovine brainGQ1bTetrasialoganglioside GQ1bBovine brainStructureGal3GalNAc4[Neu5Ac3]Gal4GlcCerGalNAc4[Neu5Ac3]Gal4GlcCerNeu5Ac3Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac3]Gal4GlcCerGal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCerNeu5Ac8Neu5Ac3Gal3GalNAc4[Neu5Ac8Neu5Ac3]Gal4GlcCer Glcglucose Gal galactose GalNAc Nacetylgalactosaminediagnosis based on clinical histopathological immunohistochemical and imagistic data All the events related to theprogression of the disease were recorded relapse metastasisneurotoxicity hyper reactivation of the immune system upontreatment The group characteristics were similar for age andsex the primary melanoma group included women and men with a mean age of ± years the metastaticmelanoma group included women and men with a meanage of ± years and the control group included women and men with mean age of ± years Materials and Reagents In this work the assessment ofantiganglioside antibodies was made by the immunoblottechnique using EUROLine kits Figure This methodallows the evaluation of antibodies IgG and IgM classesagainst GM1 GM2 GM3 GD1a GD1b GT1b and GQ1bfrom serumplasma The kit contains strips marked withpuriï¬ed antigens Table Theevaluation ofantigangliosideantibodies wasperformed using the EUROLine Scan software After readingthe signal intensity on the strips marked with ganglioside antigens the results were evaluated and the results are presented asoptical sensibility The assessment of IL8 was performed bythe ELISA method using Enzo Life Science reagents withTECAN analyser and the results are presented as pgdl CRPwas assessed by immunoturbidimetry using Human reagentsand HumaStar300 analyser the results are presented as mgdl Statistical Analysis All the results were analysed usingIBM SPSS Statistics We evaluated the normality of datadistribution using the KolmogorovSmirnov test The variationbetween groups was determined using the parametric teststtest when two groups were compared or ANOVA test whenmore groups were compared and nonparametric tests likeMannWhitney or Wilcoxon The correlation between groupswas evaluated using linear regression and Pearson coeï¬cientp was considered with statistical signiï¬cance ResultsAntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b autoantibodies determined in primary metastaticmelanoma had diï¬erent serological proï¬les compared tothe control group The presence of IgG and IgM antiganglioside antibodies in primary melanoma was as followsantiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and antiGQ1b and Inmetastatic melanoma IgG and IgM antiganglioside antibodies had the following proï¬le antiGM1 and antiGM2 and antiGM3 and antiGD1a and antiGD1b and antiGT1b and and antiGQ1b and In the control group antiganglioside autoantibodies were absentThe assessment for IgG antiGM1 antiGM2 antiGM3antiGD1a antiGD1b antiGT1a and antiGT1b showedextremely low signal intensity in all groups Figure Whencomparing the mean of signal intensity for IgG no statisticaldiï¬erences were observed between groups We obtained a statistically significant diï¬erence in IgM antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1a and antiGT1b when comparing primary melanoma respectively metastatic melanoma to the control group and once more whencomparing primary versus metastatic melanoma Table To evaluate if the presence of IgM antibodies was associatedwith melanoma development we determined their relation toammatory factors IL8 and CRP recommended by AJCCfor melanoma staging Table IL8 levels were statistically significantly increased in primary melanoma ± pgmland in metastatic melanoma ± pgml when compared with the control group ± pgml CRP levelswere found in primary ± ngml and metastaticmelanoma ± ngml significantly higher when compared with the control group ± ngml IL8 andCRP had no statistically significant variation when comparedto primary versus metastatic melanoma groups Positive correlations with statistical signiï¬cance were determined betweenantiGM1 and CRP respectively IL8 between antiGD1aand CRP respectively IL8 Figure Negative significant correlations were observed between antiGM3 antiGT1b andCRP respectively IL8 Figure High levels of CRP and IL were associated with an increase in antiGM1 antiGD1aand a decrease in antiGM3 antiGM2 antibodies of IgM typeTable DiscussionsMelanoma the most aggressive skin tumour is a multifactorial cancer being the result of the interplay between geneticimmunological and environmental factors [] Gangliosides due to their expression on tumor cells have beeninvolved in tumor biology and immunogenicity and hence 0cJournal of Immunology ResearchAntiGM1 IgM and IgG signalintensity in all groups AntiGM2 IgM and IgG signalintensity in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM1 IgM AntiGM1 IgGaAntiGM3 IgM and IgGintensity levels in all groups AntiGM2 IgM AntiGM2 IgGbAntiGD1a IgM and IgGintensity signal in all groups ControlPrimary melanomaMetastatic melanomaControlPrimary melanomaMetastatic melanomaAntiGM3 IgM AntiGM3 IgGcControlPrimary melanomaMetastatic melanomaAntiGD1b IgM and IgG signalintensity in all groupsControlPrimary melanomaMetastatic melanomaAntiGD1b IgM AntiGD1b IgGAntiGD1a IgM AntiGD1a IgGdAntiGT1b IgM and IgGintensity levels in all groups AntiGT1b IgM AntiGT1b IgGefAntiGQ1b IgM and IgGintensity levels in all groupsControlPrimary melanomaMetastatic melanomaAntiGQ1b IgM AntiGQ1b IgGgFigure Antiganglioside signal intensity in all groups p have been considered as targets for cancer immunotherapy[] The probability thatsome tumourassociatedganglioside determinants induce a human immune responsegenerated much interest in medical research [ ]If endogenously synthesized antiganglioside antibodies reactonly with human cancer cells these antibodies could play animportant role in the hosts protective immunity to thetumor There is little information about quantitative variations of serum antigangliosides their origin and progressionof melanoma Tumour ganglioside antigens generate a significantly increased immune response in patients with primarymelanoma versus metastatic melanoma In our study thehosts ability to generate an early antiganglioside responseis supported by a significantly increased titter of IgMantibodies in patients with primary versus metastatic melanoma and the control groupfor antiGM1 antiGM2antiGM3 antiGD1a antiGD1b antiGT1b and antiGQ1b Figure The range of antiganglioside antibodiescould serve as an indicator of diï¬erentiation between patientswith primary melanoma and metastatic melanoma Based on 0cJournal of Immunology ResearchTable IgM antiganglioside signal intensity in all groupsAntibodies Classp signiï¬cancePM vs MM PM vs control MM vs controlIgMAntiGM1IgMAntiGM2IgMAntiGM3AntiGD1aIgMAntiGD1b IgMAntiGT1bIgMAntiGQ1b IgMMM metastatic melanoma PM primary melanoma psigniï¬cancestatisticalTable IL8 and CRP in all studied groupsStudy groupPrimarymelanomaMetastaticmelanomaControlgroupCRPmgdl ± ± ± psigniï¬canceIL8pgml ± ± ± psigniï¬canceour ï¬ndings we estimate that the levels of the antigangliosideantibodies could provide information regarding the clinicalstaging of melanomaIn addition the capacity of patients to develop an antiganglioside response in the early stage of development of melanoma could be understood as a mean of defence of the bodythrough eliminating a danger signal from the tumour microenvironment represented by the stimulation of glycosphingolipid synthesis [ ] Synthesis of antiganglioside antibodiescould confer a survival advantage in patients with primarymelanoma [] In metastatic melanoma patients we observeda reduction of antiganglioside antibody synthesis a result thatcould suggest the immunosuppressive eï¬ect exerted by theoverproduction of gangliosides associated with tumour metastasis andor due to the overall decreased immune responseIt has been shown in a previous study that in patients withuntreated primary melanoma there is a significant statisticalcorrelation between antiGM1 type IgM level and clinicalstage of the disease Breslow index Clark level tumour localization histologic type presenceabsence of ulceration [ ]In our study patients with cutaneous melanoma had detectable levels of antiGM1 in primary stages The presence of apositive significant relationship between IgM antiGM1 leveland IL8 and CRP in our study justiï¬ed our statement regarding the involvement of these antibodies in tumour proliferation by stimulating ammation [ ] The presentstudy is the ï¬rst one that evaluated the relation betweenantiganglioside antibodies and IL8 and CRP based on theirrole in melanoma diagnosis progression and outcome [] in metabolic disorders [ ]Previous studies in patients with prostate cancer [] orsarcoma [] have shown that antiGM1 antibodies had nodiagnostic or prognostic value in these pathologies In patientswith diï¬erentiated thyroid cancer antiGM1 type IgG andIgM were associated with carcinogenesis but the lack of correlation between antibody level and clinical status indicated thatantiGM1 had no diagnostic value in diï¬erentiated thyroidcancer []Another study performed by our group showed thatpatients with primary melanoma with a high level of IgMantiGM3 had a favourable prognosis compared withpatients displaying a low antibody titer [] In a study onpatients with primary untreated melanoma stages I andII lymph nodes clear of metastasis it was shown that theantiGM2 antibody titer for IgMtype was not diï¬erentiatedin correlation to the tumor thickness For antiGM3 it wasobtained a direct relationship between the serum titer andthe thickness of the tumor [] AntiGM2 and antiGM3antibodies have no diagnostic signiï¬cance in thyroid cancerdue to the low prevalence of these antibodies [] In ourpresent study antiGM3 negative correlations with IL8and CRP are suitable with the hypothesis that patients withprimary melanoma with a high level of IgM antiGM3 havea favourable prognosisGD1a was thought to generate an immune response inpatients with earlystage melanoma [] In patients withT1T2 stage prostate cancer there were identiï¬ed increasedIgM antiGD1a values compared to the T3T4 stage whichsustains the development of an early endogenous immuneresponse able to eliminate the danger signal from the tumormicroenvironment These data support the role of antiGD1ain the early diagnosis of localized prostate disease [] TheantiGD1a IgMtype titer was deï¬ned as a negative predictivefactor of survival in patients with soft tissue sarcoma [] andin patients with primary melanoma [] In patients serumdiagnosed with ovarian cancer an increased titer of IgMantiGD1 was found the authors pointing out that theseantibodies could represent immunological markers associated with ovarian cancer progression [] In cutaneousmelanoma IgM antiGD1a could be considered a markerassociated with melanoma progression based on the negativecorrelation with CRP and IL8 as shown in our studyThe antiGD1b immune response in patients with gastricneoplasm can be used as a prognostic marker [] On thecontrary the lack of correlation between the presence ofantiGD1b and the clinical status of patients with thyroidcancer has indicated that antigangliosides do not have diagnostic signiï¬cance in this neoplasm []The antiGT1b titer can be an overall positive factor associated to global survival in sarcoma [] AntiGD1b GT1band GQ1b antibodies that are negatively correlated with IL8and CRP suggest that they could indirectly suppress tumorgrowth and angiogenesis AntiGD1b GT1b and GQ1bIgM type uence the progression of melanoma [ ]soft tissue sarcomas [ ] Ehrlich subcutaneous solidtumors [ ] Ehrlich carcinomas accompanied by ascites[ ] and gastric cancer []Our study limitations could be considered the semiquantitative assessment method of antiganglioside antibodies as aquantitative determination technique could oï¬er more sensitive data ing the door for further studies One more 0cJournal of Immunology ResearchMGitnAMGitnAaDGitnA CRP CRP CRP MGitnAMGitnAaDGitnAIL8IL8IL8Figure AntiGM1 antiGM3 and antiGD1a in relation to CRP and IL8 in the primary melanoma groupimportant limitation of the study is that we evaluated thecorrelation between antiganglioside antibodies and ammation markers in melanoma IL8 and CRP only after orbetween the surgical treatment of melanoma newer therapiesbeing also in place This could be the ï¬rst study which otherresearchers and clinicians can use and analyze in order toevaluate the uence of diï¬erent melanoma treatments onantiganglioside antibodies The pathogenic mechanismsinvolved in melanoma are complex [] therefore theevaluation during the followup period of melanoma patientsat diï¬erent points is needed for a better antigangliosideproï¬le characterization 0cJournal of Immunology ResearchTable AntiGM1 GM2 GM3 GD1a GD1b GT1b andGQ1b relation with ammatory markers in primary melanomagroupAntiGM1AntiGM2AntiGM3AntiGD1aAntiGD1bAntiGT1bAntiGQ1bCRPr p ¤ r p r p r p ¤ r p ¤ r p ¤ r p IL8r p ¤ r p r p r p ¤ r p ¤ r p ¤ r p ConclusionsAntiganglioside antibodies antiGM1 and GD1a werepositively correlated while antiGM3 GD1b and GT1bwere negatively associated with the ammatory markersIL8 and CRP The hosts ability to elaborate an early antiganglioside response could be considered as a defence mechanism directed toward eliminating a danger signal from thetumour microenvironment Moreover our results suggest thattumour ganglioside antigens generate a significantly increasedimmune response in patients with primary versus metastaticmelanoma Antiganglioside antibodies associated with ammation markers could be used as diagnostic monitoring andtreatment tools in patients with cutaneous melanomaData AvailabilityThe data used to support the ï¬ndings of this study areincluded within the articleConflicts of InterestThe authors declare no conï¬icts of interestAuthors ContributionsAll authors have equally contributed to the writing and editing of the manuscriptAcknowledgmentsThis research and article processing charges were funded by agrant of the Romanian Ministry of Research and InnovationCCCDIUEFISCDI project number 61PCCDI2018 PNIIIP112PCCDI2017034References[] YH Xu S Barnes Y Sun and G A Grabowski Multisystem disorders of glycosphingolipid and ganglioside metabolism Journal of Lipid Research vol no pp [] I Horwacik and H Rokita Targeting of tumorassociatedgangliosides with antibodies aï¬ects signaling pathways andleads to cell death including apoptosis Apoptosis vol no pp [] J L Daniotti R D Lardone and A A Vilcaes Dysregulatedexpression of glycolipids in Tumor cells from negative modulator of Antitumor immunity to promising targets for developing therapeutic agents Frontiers in Oncology vol p [] I Nicolae A Caragheorgheopol S Schipor Gnagliosides and sex hormones in human melanoma Acta Endocrinologica vol no pp [] Y Ohmi M Kambe Y Ohkawa 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"Optimizing Telemedicine Encounters for Oral and Maxillofacial Surgeons During the COVID19 Pandemic Hwi Sean Moon DDS MD1 Tim T Wang BA23 Karthik Rajasekaran MD4 Ryan Brewster BA5 Rabie M Shanti DMD MD46 Neeraj Panchal DDS MD MA7 1Resident Department of Oral Maxillofacial Surgery University of Pennsylvania Philadelphia PA 2DMD Candidate School of Dental Medicine University of Pennsylvania Philadelphia PA 3MPH Candidate Perelman School of Medicine University of Pennsylvania Philadelphia PA 4Assistant Professor of Otorhinolaryngology University of Pennsylvania Philadelphia PA 5MD Candidate Stanford University School of Medicine Stanford University Stanford CA 6Assistant Professor of Oral and Maxillofacial Surgery University of Pennsylvania Philadelphia PA 7Assistant Professor and Section Chief of Oral and Maxillofacial Surgery Philadelphia Veterans Affairs Medical Center Penn Presbyterian Medical Center University of Pennsylvania School of Dental Medicine Philadelphia PA Corresponding Author Neeraj Panchal DDS MD MA Tel Mailing Address N 39th St Philadelphia PA Email Address npanchalupennedu Disclosures None to report Abstract Word Count Manuscript Word Count Number of References Number of Figurestables Number of Supplements 0c The COVID19 pandemic has changed conventional medical practice patterns across all health disciplines including oral and maxillofacial surgery practices The use of telemedicine has rapidly expanded to uphold safety strategies of physical distancing and disease transmission reduction while maintaining uninterrupted care of patients To date there are no specific guidelines to optimize telemedicine encounters in the oral and maxillofacial surgery practice The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond Statement of Clinical Relevance The goal of this paper is to provide best practices for both oral and maxillofacial surgeons and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond INTRODUCTION The COVID19 pandemic has disrupted society in a multitude of ways Healthcare is no exception the SARSCoV virus rapid transmission and high hospitalization rate have strained the availability of medical resources including personal protective equipment PPE respiratory ventilators and hospital beds[] The virus also poses a major threat to healthcare personnel whose risk of exposure are compounded by the aforementioned PPE shortages[] In response the American Association of Oral and Maxillofacial Surgeons AAOMS recommended delaying elective surgeries in accordance with the Centers for Disease Control and Preventions calls to 0c postpone elective medical and dental procedures[] Similarly four out of five dental offices have closed for all except emergency procedures[] In the face of these challenges the medical and dental communities have remained steadfast in caring for patients with nonelective health needs and innovating alternate ways to deliver care One of the most important and popular alterations in the delivery of care is the increased utilization of telemedicine which allows surgeons and patients to connect virtually[ ] This has enabled patients to access muchneeded medical care while preserving PPE and minimizing exposure to pathogens Though studies have found telemedicine to decrease costs and save time without compromising patient satisfaction it was not widely used in healthcare before the COVID19 pandemic[ ] Similarly teledentistry was deemed to be in its infancy by the founder of the American Teledentistry Association in [] Nevertheless telemedicine has shown promise and has been incorporated into the workflow of various oral and maxillofacial surgery institutions and practices across the country Virtual visits are particularly useful in triaging patients For example patients with dentoalveolar infections can meet virtually with surgeons and receive prescriptions for appropriate analgesics and antibiotics without going to the emergency department Also patients with oral lesions can take images and show their surgeon before their inperson visit to expedite the diagnosis and treatment planning workflow This enables patients to access timely attention of providers while lightening the load on the healthcare system by reducing the number of inperson visits 0c Associated with the recent rise in telemedicines popularity is a learning curve for both surgeons and patients The incorporation of technology and the shift to virtual visits can be jarring for the patientsurgeon relationship and must be navigated thoughtfully While there have been helpful telehealth guides for surgeons and patients in other surgical specialties we do not know of any such guidelines for oral and maxillofacial surgery[ ] As such we detail best practices for both oral and maxillofacial surgeons OMS and their patients to effectively utilize telemedicine for the duration of COVID19 and beyond GENERAL CONSIDERATIONS FOR TELEMEDICINE In accordance with the AAOMS White Paper on Telehealth and Remote Treatment virtual management of any oral and maxillofacial surgical condition should only be provided by appropriately licensed oral and maxillofacial surgeons as regulated by the state law[] The delivery of patient care through telemedicine must continue to follow evidencebased guidelines to ensure quality and safety for all patients All providers must comply with the latest telehealth requirements outlined by the United State Health and Human Services to protect patient privacy and comply with the Health Insurance Portability and Accountability Act HIPAA[] Furthermore providers are ethically obligated to inform all patients about the potential benefits limitations and risks of telemedicine[] Patients requiring emergency or urgent services must be directed to the nearest hospital 0c SETTING UP FOR TELEMEDICINE While there are several modalities to conduct a telemedicine encounter we strongly recommend a live synchronous twoway interaction between the patient and the OMS incorporating both audio and visual telecommunications tools This can be achieved with a desktop computer laptop or smartphone The United States Census Bureau reports that approximately percent of households have computers or smartphones and percent have broadband internet subscriptions[] Of these options though we recommend using a desktop or a large screen laptop with a highresolution camera over smartphones even if the latter meets the minimum technical requirements Ideally telemedicine visits can offer a clinic experience that closely simulates inperson encounters Trained administrative staff members should call patients beforehand to discuss the virtual setup and basic expectations for the visit Prasad et al created Figure which exemplifies a patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the encounter [] In the following we will detail key aspects and considerations for both OMS and patients to maximize their telehealth visits 0c Insurance Coverage and Billing In an effort to reduce the burden posted on healthcare entities and facilitate mitigation efforts the Centers for Medicare Medicaid Services CMS broadened access for Telemedicine coverage with private payers following suit CMS expansion included voiceonly visits which is critical for patients without access to a smartphone or computer video capabilities Furthermore CMS allowed for parity of payment for Telemedicine visits and inperson visits so providers can bill Medicaid and Medicare at the same rate as they would for an inperson visit[] This new policy is especially relevant for older patients covered by Medicare for they are generally at higher risk for COVID19 complications[] Therefore before telehealth encounters providers should confirm if the patients insurance plan has Telemedicine coverage and also whether the insurance plan waives all copays for nonCOVID19 related visits to avoid a scenario where a patient receives a bill unknowingly The American Association of Oral and Maxillofacial Surgeons provide additional detailed information about telehealth billing relevant for OMSs including updated links to AMA and ADA billing codes on its website at httpswwwaaomsorgpracticeresourcestelehealthfaqs 0c Professionalism and provider attire The visit should replicate the same level of professionalism as that of inperson appointments Providers should dress professionally as they would in their office or hospitalbased practice Before the patient comes in providers should review the patients relevant medical records and chief complaints to save time and maximize the efficiency of the visit Also in the interest of both time and professionalism OMS and patients should both be mindful to start the visit on time During the visit OMS should communicate with patients to maintain transparency For instance if an OMS needs to document something during the visit they should respectfully inform the patient of the task to prevent any misunderstanding At the end of the visit it is important for OMS to summarize what they accomplished during the visit and provide a clear plan for appropriate next steps Physical background When possible OMS and patients should conduct virtual encounters in welllit spaces Lighting specifically can have a profound effect on video quality As such overhead lights can be helpful while lights behind the person should be avoided Care should also be taken to prevent other sources of potential disruptions such as background noise or visual distractions It may be helpful for OMS to evaluate their surroundings from the perspective of their patients 0c Technological background It is important for OMS to test their video and audio quality before visits to anticipate any potential technical difficulties that may interrupt the encounter A strong WiFi internet connection is preferred over cellular data to ensure a stable signal With the exception of electronic health records OMS should close any unnecessary programs or internet browser tabs to preserve internet bandwidth In addition OMS should be mindful that their patients may have varying internet speeds As such OMS should give approximately seconds of lag time after patients stop speaking to allow all of their words to come through completely Patient and camera position If possible patients should sit upright on a chair in front of a computer placed on top of a desk They should sit close enough to the camera so that their entire head and neck area are within the video frame The camera should be at approximately eyelevel for both OMS and patients to maintain eye contact and remain engaged during the virtual encounter For patients using a smartphone the device can be propped up at a to 90degree angle from the table surface to allow patients to free both of their hands for physical exam tasks Patient clothing Patients must be notified of the appropriate clothing well in advance prior to the appointment Ideally clothing should allow patients entire head and neck regions to be visualized while maintaining patient comfort and professionalism Any hat or scarves should be removed if at all possible maintaining appropriate cultural and religious norms Patient items Prior to the appointment patients should prepare the following items which can help aid OMS in visualization and retraction during the virtual physical exam Most of these items are commonplace inexpensive and available at the patients home 0c Flashlight A flashlight or a penlight can enhance visualization of certain obscure head and neck structures particularly those in the oral cavity The builtin flashlight of a smartphone can also be used Ruler A ruler or a measuring tape can be used to measure the patients maximal mouth opening and mandibular range of motion Napkins A napkin can be used to touch any intraoral landmarks and also to clean up after any inadvertent salivation from the virtual physical exam Spoon A spoon can be used to retract the cheeks or depress the tongue to evaluate structures of the oropharynx such as the soft palate and tonsillar pillars Cheek retractors A fun way for patients to achieve cheek retraction can be to use the plastic props from the board game Speak Out which was created in [] This game provides horseshoeshaped plastic retractors shown in Figure that can be placed along the patients upper and lower lips to lateralize the cheeks thus allowing handsfree visualization of the dentition and oral cavity soft tissue Patient assistant If available patients should ask a family member or friend to accompany them to the telehealth visit The assistant can help patients perform the physical tasks required during the virtual physical exam Assistants can also help position the web camera to improve the providers view In fact these assistants are essentially mandatory for pediatric patients or patients with disabilities Feedback It is a good practice for providers to seek feedback from patients after a visit This will help OMS hone their telehealth skills and ensure that their patients are receiving an appropriate quality of care 0c THE VIRTUAL HISTORY AND PHYSICAL Thorough patient assessment proper medical documentation and appropriate diagnostic testing are critical components of OMS practice that enable proper diagnosis and treatment planning OMS should obtain patients medical histories in a similar manner as they would in their offices New patients must be asked for comprehensive histories that include chief complaint history of present illness past medical history past surgical history dental history medications allergies pertinent family history social history and complete review of systems For patients of record their medical histories should be updated to reflect their current chief complaint All patients should also be screened with an up to date COVID19 questionnaire If an infection is suspected OMS should refer patients to their primary care physicians or local emergency department depending on the severity of symptoms for appropriate workup Vitals should be obtained if the patient has access to a thermometer blood pressure cuff pulse oximeter or weighing scale Even without any of these devices patients can still measure their pulse by applying two fingers on the patients carotid counting the number of beats per minute Also patients can calculate their respiratory rate by observing the number of chest rises in one minute Finally oxygen saturation can be measured with certain mobile health applications though OMS should not solely rely on their results for major medical decisions Finally patients with fever body temperature F warrants further work up in an emergency setting for a differential diagnosis that includes COVID19 infection The virtual physical exam will be limited to a head and neck exam and a cranial nerve exam While inspection and palpation are the basis of a focused physical examination in oral and maxillofacial surgery OMS must learn to work together with patients to achieve the same goals 0c virtually Patients must perform maneuvers on themselves with the OMSs guidance To this end a printed stepbystep schematic as illustrated in Figure can be helpful for patients to receive before the visit During the visit the OMS can reinforce the diagram with clear verbal instructions that avoids medical jargon The exam itself must be conducted systematically with a topdown outsidein approach as is typical in an oral and maxillofacial surgery practice The exam can be further divided into head and neck subsites The OMS should ask for specific symptoms related to each subsite and carefully inspect for any abnormalities while guiding the patient or the patients assistant through the exam The following section offers additional details and considerations for each subsite Head The OMS should ask about any history of head trauma The head is assessed to ensure that it is normocephalic and atraumatic Face The OMS should ask the patient about any facial pain swelling weakness numbness or history of trauma to the region Then OMS can start the facial exam by asking the patient to lean close to the camera First the face is examined for any skin lesions along the forehead eyelids external ears nose malar region vermilion of the lips and the chin Patients left and right sides of the face should be compared for any gross asymmetry or deformities OMS can then guide patients through palpating their own face for any bony discontinuity or soft tissue swelling Patients can also tap their own face with two fingers to reveal any tenderness in the sinuses Regarding the eyes OMS can assess if the pupils are equal The extraocular muscles along with the oculomotor supratrochlear and the abducens nerves can be tested by having the 0c patient look up down left and right without moving the head The sensory portion of the trigeminal nerve can be tested by asking patients to close their eyes and slide both of their index fingers horizontally along the ipsilateral forehead ophthalmic branch cheek maxillary branch lip and chin mandibular branch The branches of the facial nerves can be tested by asking patients to raise their eyebrows close their eyes tightly puff out their cheeks smile widely and show their bottom teeth Temporomandibular joint TMJ The OMS should ask the patient about any facial jaw or ear pain trismus difficulty mastication clicking or locking of the joint Then the TMJ exam begins by asking patients to palpate their mandibular condyles and muscles of mastication to look for any tender spots Providers can ask patients to open and close their mouth while palpating the condyles to feel for any clicks or crepitus Also maximal interincisal opening can be roughly estimated by the number of fingerbreadth or precisely measured using a ruler The mandibular range of motion can be assessed or measured in protrusive and lateral excursive positions Neck The OMS should ask for any difficulty breathing dysphagia sore throat odynophagia hoarseness or new neck swelling The neck exam begins with inspection looking for any asymmetry or tracheal deviation Patients can be asked to turn the head from side to side look upwards and shrug the shoulders to assess the spinal accessory nerves OMS should ask the patients assistant if possible to stand right behind the patient and palpate the patients neck Using their fingertips on both hands the assistant can palpate the neck in a unidirectional manner from superior to inferior and then from lateral to medial Ask them to note any palpable bumps or tender spots It is particularly important to palpate the lateral neck for enlarged lymph nodes 0c Lastly OMS can identify the thyroid by asking patients to swallow while palpating the appropriate area on the neck to rule out thyromegaly Oral cavity and oropharynx The OMS should ask for any oral pain oral swelling or sores tongue numbness difficulty with tongue movement or dry mouth Examination of the oral cavity exam can be challenging because intraoral structures can be difficult to retract and illuminate The aforementioned cheek retractors whether from a board game or makeshift spoons can be helpful for retraction of soft tissue and visualization In addition patients friends and family can help a great deal by adjusting the camera while also properly angling an additional light source For each intraoral structure the OMS must carefully inspect for ulcers raised lesions abnormal white leukoplakic or bright red erythroplakic lesions In general OMS can best visualize structures near or at the level of the maxilla when patients lift their heads up to degrees Likewise structures near or at level of the mandible are best observed with the patient dropping the chin approximately degrees OMS may find it useful to practice these examination techniques on their own cameras before the visit Patients should be recommended to wash their hands or to use gloves before touching any intraoral landmarks The exam begins by sliding the patients index finger along the maxillary and mandibular vestibule to look for any swelling or fluctuance With the cheeks retracted the patient can palpate their buccal and labial mucosa using the thumb and index finger with one 0c finger compressing along the face extraorally When possible palpation should be bidigital Next the patient can use their index figures to palpate the tuberosity retromolar trigone and the hard palate for tenderness or irregularities The tongue is the most common site for oral cancer and must be thoroughly examined The dorsal surface of the tongue should be examined by asking the patient to fully protrude their tongue Providers should also ask patients to move their protruded tongues to the left and right to inspect the lateral tongue and ensure the function of the hypoglossal nerves The ventral tongue and the floor of the mouth can be observed by asking patients to touch the tip of their tongue to their hard palate The tongue can then be palpated for lumps or masses Next the sublingual and submandibular glands can be palpated for symmetry and lack of elevation by the patient with their extended index fingers on the floor of the mouth The examination of the oropharynx is mostly limited in virtual encounters Nevertheless the soft palate tonsils and uvula can be partially visualized with the patients mouth wide open and using a spoon to depress the tongue Although unpleasant the glossopharyngeal and vagus nerves can be tested by gently touching the soft palate using a spoon to induce a gag reflex Dentition If the patient is dentate the provider should ask about dental pain sensitivity loosening of teeth bleeding or sore gums or malocclusion The patients dentition can be evaluated after retracting soft tissue as described previously Dental caries missing teeth periodontal disease gingival lesions or swelling can be readily identified Mobility of teeth can be assessed by using the patients thumb and index finger In edentulous patients the alveolar 0c ridge should be examined for any abnormalities as part of the aforementioned oral soft tissue exam CONCLUSION The COVID19 pandemic has catalyzed an exponential increase in telemedicine usage Telemedicine helps patients maintain access to care conserves limited medical resources and protects both OMS and patients from pathogen exposure Nevertheless there is an expected learning curve that accompanies such a paradigm shift in the delivery of care As such this paper provides a guide of best practices to aid both OMS and patients to navigate this promising electronic tool In addition we provide an accessible schematic handout that can be given to patients before a telehealth appointment to help them prepare for the visit for both setting up and performing physical exam procedures Because telemedicine may have a role in oral and maxillofacial surgical care even after this pandemic we are optimistic that these best practices can be helpful and relevant for the present situation and beyond 0c FIGURE LEGEND Figure Patient informational handout with graphic illustrations detailing the set up as well as key examination steps that patients may be asked to perform during the telemedicine encounter Reprinted from [] 0c Figure Horseshoeshaped plastic lipcheek retractors REFERENCES Li Q Guan X Wu P et al Early Transmission Dynamics in Wuhan China of Novel CoronavirusInfected Pneumonia N Engl J Med httpsdoiorg101056NEJMoa2001316 Feng S Shen C Xia N et al Rational use of face masks in the COVID19 pandemic Lancet Respir Med httpsdoiorg101016S221326002030134X Li R Rivers C Tan Q et al Estimated Demand for US Hospital Inpatient and Intensive Care Unit Beds for Patients With COVID19 Based on Comparisons With Wuhan and Guangzhou China JAMA Netw Open 3e208297e208297 httpsdoiorg101001jamanetworkopen20208297 White DB Lo B A Framework for Rationing Ventilators and Critical Care Beds During the COVID19 Pandemic JAMA httpsdoiorg101001jama20205046 Heinzerling A Stuckey MJ Scheuer T et al Transmission of COVID19 to Health Care Personnel During Exposures to a Hospitalized Patient Solano County California 0c February MMWR Morb Mortal Wkly Rep httpsdoiorg1015585mmwrmm6915e5 Ng K Poon BH Kiat Puar TH et al COVID19 and the Risk to Health Care Workers A Case Report Ann Intern Med httpsdoiorg107326L200175 Halepas S Ferneini EM A Pinch of Prevention is Worth a Pound of Cure Proactive Dentistry in the Wake of COVID19 Journal of Oral and Maxillofacial Surgery httpsdoiorg101016jjoms202003036 AAOMS Member Alert COVID19 Update Healthcare Facilities Preparing for Community Transmission In Centers for Disease Control and Prevention httpswwwcdcgovcoronavirus2019ncovhcpguidancehcfhtml Accessed May CDC Guidance for Providing Dental Care During COVID19 In Centers for Disease Control and Prevention httpswwwcdcgovoralhealthinfectioncontrolstatementCOVIDhtml Accessed May Carey M Second week of HPI polling shows dentists response to COVID19 American Dental Association Hollander JE Carr BG Virtually Perfect Telemedicine for Covid19 New England Journal of Medicine httpsdoiorg101056NEJMp2003539 Prasad A Brewster R Newman JG Rajasekaran K Optimizing your telemedicine visit during the COVID19 pandemic Practice guidelines for patients with head and neck cancer Head Neck na httpsdoiorg101002hed26197 Russo JE McCool RR Davies L VA Telemedicine An Analysis of Cost and Time Savings Telemedicine and eHealth httpsdoiorg101089tmj20150055 Cain SM Moore R Sturm L et al Clinical assessment and management of general surgery patients via synchronous telehealth Journal of Telemedicine and Telecare httpsdoiorg1011771357633X16636245 Teledental Practice and Teledental Encounters An American Association of Teledentistry Position Paper Jampani ND Nutalapati R Dontula BSK Boyapati R Applications of teledentistry A literature review and update J Int Soc Prev Community Dent httpsdoiorg1041032231076297695 Wicklund E Dentists Use Telehealth to Improve Access to Care And Fight a Phobia In mHealthIntelligence httpsmhealthintelligencecomnewsdentistsusetelehealthtoimproveaccesstocareandfightaphobia Accessed May 0c Smith WR Atala AJ Terlecki RP et al Implementation Guide for Rapid Integration of an Outpatient Telemedicine Program during the COVID19 Pandemic Journal of the American College of Surgeons httpsdoiorg101016jjamcollsurg202004030 AAOMS White Paper on Telehealth and Remote Treatment Notification of Enforcement Discretion for Telehealth Remote Communications During the COVID19 Nationwide Public Health Emergency In HHSgov httpswwwhhsgovhipaaforprofessionalsspecialtopicsemergencypreparednessnotificationenforcementdiscretiontelehealthindexhtml Accessed May Ethical Practice in Telemedicine In American Medical Association httpswwwamaassnorgdeliveringcareethicsethicalpracticetelemedicine Accessed May Ryan C Computer and Internet Use in the United States United States Census Bureau Additional BackgroundSweeping Regulatory Changes to Help US Healthcare System Address COVID19 Patient Surge CMS In CMSgov httpswwwcmsgovnewsroomfactsheetsadditionalbackgroundsweepingregulatorychangeshelpushealthcaresystemaddresscovid19patient Accessed Jun Medicaid Learning Network Telehealth Services Hasbro Brings Mouth Piece Challenge to the Masses with New SPEAK OUT Game In Business Wire httpswwwbusinesswirecomnewshome20160624005633enHasbroBringsMouthPieceChallengeMassesNew Accessed May 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 |
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes | Thyroid_Cancer |
This study aimed to investigate serum matrix metalloproteinase MMP2 and MMP9levels in patients with papillary thyroid carcinoma PTCMethods Fortyone patients with PTC undergoing ultrasoundguided radiofrequency ablationRFA and controls were included Serum MMP2 and MMP9 levels were determined byenzymelinked immunosorbent assay before and after surgery Potential affecting factors wereevaluated by logistic regression analysisResults Serum MMP2 and MMP9 levels were significantly higher in PTC patients comparedwith controls and decreased significantly after surgery According to receiver operating characteristic curve analysis diagnostic values for preoperative serum MMP2 and MMP9 levels were and There was no contrastagent perfusion in the ablation zone in of lesionsand enhancement within or at the lesion edge in The volume reduction at monthsfollowup was Age microcalcification irregular shape and lesion diameter and numberwere influencing factors for PTC Age and lesion diameter and number were independent riskfactors while calcification and morphology were protective factorsConclusion Serum MMP2 and MMP9 levels have important clinical values for the diagnosis andtreatment of PTC by RFA Preoperative serum MMP2 and MMP9 levels combined with otheraffecting factors contribute to disease prognosisDepartment of Ultrasound Beilun Peoples Hospital ofNingbo Beilun Branch of the First Affiliated Hospital ofZhejiang University Ningbo Zhejiang ChinaThese authors contributed equally to this studyCorresponding authorQian Ding Department of Ultrasound Beilun PeoplesHospital of Ningbo Beilun Branch of the First AffiliatedHospital of Zhejiang University No East LushanRoad Ningbo Zhejiang ChinaEmail qianding02sohucomCreative 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 0cJournal of International Medical ResearchKeywordsPapillary thyroid carcinoma radiofrequency ablation matrix metalloproteinase2 matrix metalloproteinase9 disease prognosis serum levelDate received June accepted March IntroductionPapillary thyroid carcinoma PTCis acommon thyroid malignancy accountingfor about of systemic malignancies1PTC is currently characterized by a highincidence and low mortality2clinicalRecentimprovementsin highfrequencyultrasonic diagnostic technologies and theapplication of ultrasoundguided puncturetechniques have led to an apparent increasein the incidence of PTC year by year3Regardingthyroidtumors the palpation detection rate forthyroid microtumors in the general population is about compared with as highas to for highfrequency ultrasound6 which has thus greatly improveddisease diagnosisdiagnosistheofSurgical resection is a routine treatmentfor thyroid tumors but the recurrence rateis usually high and the consequent reduction in thyroid function can seriously affectthe patients quality of life7 Associatedwith the increasing detection rate of thyroidtumors and the pursuit of minimally invasive treatments radiofrequency ablationRFA has been gradually applied in theclinic RFA uses local hyperthermia tocause tissue necrosis The thermal effectsdo not depend on the tissue type andmost lesions can be completely eliminatedby RFA89 RFA has thus become a novellocal treatment for tumorsThe clinical diagnosis of benign andmalignant thyroid tumors currently dependson the clinical manifestations and pathological examinations Howeverthe clinicalmanifestations are mostly derived frominvolvessubjective empirical analysis while a ï¬nalpathological diagnosisinvasiveprocedures with lesssatisfactory speciï¬city It is therefore necessary to identifyappropriatepredictivetumor markersdiagnosticandforimplicationsThe collagenases matrix metalloproteinase MMP2 and MMP9 can degradetype IV collagen in the basement membranewith importanttumorangiogenesis and tumor cell invasion andmetastasis10 MMP2 and MMP9 expression levels were found to be upregulatedin thyroid cancer tissue11 however thesestudies mostly examined pathological tissuesafterinvasive surgery and lessinvasivemeasures such as serum levels of MMP2and MMP9 have been lesswell consideredIn this study we detected serum levels ofMMP2 and MMP9 in patients with PTCbefore and after ultrasoundguided RFAWe also determined the therapeutic effectsof RFA during the followup period andinvestigated the relevant prognostic factorsMaterials and methodsStudy subjectsPatients who underwent ultrasoundguidedRFA in our hospital from May toOctober were included in this studyThe inclusion criteria were as follows patients diagnosed by preoperative ï¬neneedle aspiration cytology no historyof neck surgery and patients requiringminimally invasive treatment for aestheticreasons and because of neck oppression 0cPan et alwith anxiety The exclusion criteria were asfollows benign lesions conï¬rmed by ï¬neneedle aspiration cytology history ofneck surgery and severe coagulopathyPeripheral venous blood samples wereobtained from the included patients beforeand at and months after the operation and serum levels of MMP2 andMMP9 were determined Additional subjects with conï¬rmed benign thyroid noduleswithout RFA were included as a controlgroup Prior written informed consent wasobtained from all patients and the studywas approved by the ethics review boardof our hospitalPreoperative preparationcalciï¬cationThe number size nature echo boundarymorphologysurroundinghalo and nodular blood ï¬ow distributionof the tumors were assessed before the operation After skin disinfection local anesthesia was performed with lidocainesolution A total of mL Sonovue Bracco Milan Italy was injected via the elbowvein and the bloodsupply characteristicswere then evaluated by contrastenhancedultrasound CEUS of the ablationtargetedlesions using a Mylab90 ultrasonic devicewith 10MHz probe Esaote ShenzhenGuangdong ChinaAccording to the location of the thyroidnodules the thyroid and carotid space thyroid and tracheal space thyroid and esophageal space and posterior thyroid spacerecurrent laryngeal nerve were separatedusing a saline and lidocaine mixture basedon the intraoperative conditions to form aliquid separation zone to protectthesestructures from thermal damageAblation treatmentUnder ultrasound guidancethe tip ofthe RFA needle rated power Woutput frequency kHz was accuratelypenetrated into the nodule and RFA wasperformed using an OlympusCelon PowerRFA System Germany in mobile mode12following the fromdeeptoshallow principle The lesions were subjected to multipointed and multifaceted ablation untilthe thyroid tissue layer with the noduleswas completely covered by the strong echogenerated by heat accumulation The wholeprocess was carried out under continuousultrasound monitoring A highecho areawas produced in the ablation zone duringthe ablation treatment The position of theelectrode needle was gradually adjustedaccording to the lesion size After ensuringthat there was no residual enhancement inthe ablation zone the ablation needle wasremoved and the ablation was completedAfter the operation an ice compress wasapplied for h to avoid skin burnsSerum MMP determinationFor all subjects mL venous blood wascollected from the elbow vein under fastingconditions before and after the operationrespectively Blood samples in the controlgroup were collected after ultrasound contrast examination The blood samples wereplaced at room temperature for minutesand then subjected to centrifugation at 02 g for minutes The serum washarvested and serum levels of MMP3 andMMP9 were determined using enzymelinked immunosorbentELISAkitsBoster Bioengineering WuhanHubei ChinaassayFollowup and efficacy evaluationImmediately after the operation the ablation range was evaluated by CEUS If residualtissues were detected ablation wasrepeated immediately Ultrasound detectionwas performed at and monthsafter surgery to determine the nodule sizesand volumes The volumereduction rate 0cwas calculated according to the followingformula volume reduction rate¼ preoperative volume followup volumepreoperative volume 02 Echo and bloodï¬owchanges in the ablation zone were alsoobserved and analyzed The efï¬cacy wasdetermined based on the criteria for RFAfor treating tumors13 disappearance ofnodules indicated by complete disappearance of blood ï¬ow conï¬rmed by ultrasonography indicated complete cure noduleby 15 indicatedvolumemarked effect and nodule volume reducedby to indicated improvementreducedClinicopathological featuresInformation on ultrasoundbased clinicopathologicalincluding numbersize and calciï¬cation of the lesion wereobtainedfeaturesversion Statistical analysisData were expressed as mean 06 standarddeviation Statistical analysis was performed using IBM SPSS StatisticsforWindowsIBM CorpArmonk NY USA Comparisons betweengroups were performed using v2testsPotentially related factors were analyzedby univariate or multivariate logistic regression The prognostic predictive effects ofserum MMP2 and MMP9 levels wereevaluated by receiver operating characteristic ROC curve analysis P was considered statistically signiï¬cantResultsPatientsJournal of International Medical Research men mean age 06 years range yearsSerum MMP2 and MMP9 levels beforeand after treatmentThe characteristics of the ultrasound images inthe included subjects are shown in Table Serum levels of MMP2 and MMP9 weremeasured before and after treatment Serumlevels of MMP2 and MMP9 were signiï¬cantly higher in patients with PTC comparedwith the control subjects P Serumlevels of MMP2 and MMP9 had declinedat month after the operation comparedwith before surgery but the difference wasnot signiï¬cant However serum levels ofMMP2 and MMP9 had declined signiï¬cantly in the PTC patients at and monthsall P Table These results suggestthat changes in serum MMP2 and MMP9levels between before and after surgery mayhave signiï¬cant implications for the therapyof PTCROC curve analysis of preoperative serumMMP2 and MMP9 levelsPreoperative serum MMP2 and MMP9levels were used as potential diagnostic indicators In the patients with PTC the predictive probability from theregressionmodel was used as the diagnostic resultsand the gold standard classiï¬cation criteriawere used as the pathological results ROCcurves were obtained accordingly The areaundercurve AUC values for serum MMP and MMP9 levels were and respectively Figure These results suggestthat serum levels of MMP2 and MMP9could contribute to the disease diagnosisFortyone patients with PTC lesionswere enrolledincluding women and men mean age 06 years range to þ65 years The control group included patients with conï¬rmed benignthyroid nodules including women andEvaluation of RFA efficacyWe also evaluated the efï¬cacy of RFA CEUSof the lesions before ablation showed hypoenhancement in nodules isoenhancementin nodules and slight hyperenhancement 0cPan et alTable Characteristics of thyroid ultrasound imagesPTC patientsNormal controlLesion numberOneTwoMultipleLesion size 14 cm cmCalcificationMicrocalcificationCoarse calcificationMorphologyRegularIrregularAge years 15 yearsnP compared with the control groupnTable Serum matrix metalloproteinase2 and levels in controls and inpatients with papillary thyroid carcinoma before and after treatmentControlsPTC patientsBefore surgery month after surgery months after surgery months after surgery months after surgeryMMP2 06 06 06 06 06 06 PMMP9P 06 06 06 06 06 06 MMP matrix metalloproteinase PTC papillary thyroid carcinomain nodules Ultrasound examination afterablation showed no contrastagent perfusionin the ablation zone in lesions and enhancement of different degrees at theedge or inside the lesion in the other lesions and the ablation area wasgradually reduced with prolonged ablationFigure There was no signiï¬cant changein ablation volume in any patients at month after surgery compared with beforesurgery However the volume was reducedby at months offollowupcompared with before surgery P Table These results showed that RFAtreatmentthetumor volume in patients with PTCcould effectively reduceInfluence of relevant factors on diseaseprognosisTheclinical data of patients beforeRFA were retrospectively analyzed by 0cJournal of International Medical Researchirregularlogistic regression to identify factors thatmay affectthe disease prognosis Agemicrocalciï¬cationshape anddiameter and number of lesions were significant inï¬uencing factors for PTC P The hazard ratios HRsfor age andlesion diameter and number were indicating that these represented independentrisk factors In contrast the HRs for microcalciï¬cation and irregular shape were negativeindicating that a greater degree ofcalciï¬cation and regular shape were associated with lower risks of developing the disease and were thus protective factorsTable Figure ROC analyses of serum MMP2 andMMP9 levels MMP matrix metalloproteinaseFigure Efficacy evaluation of radiofrequency ablation a Twodimensional 2D ultrasound showingobvious bloodflow signals around the tumor and fewer signals within the tumor b Preoperative contrastenhanced ultrasound showing no obvious enhancement in the lesion with lowperfusion performance c Inthe 2D imaging localization the ablation needle was inserted into the tumor for ablation d The tumor wascompletely ablated with no bloodflow signal at year after ablation 0cPan et alDiscussionitis difï¬cultPTC is a type of thyroid tumor with a highincidence14 which has been increasingrapidly worldwide1516 Mostthyroidtumor cases are currently diagnosed by hiscytological detectiontopathological orHoweverto distinguishbetween benign and malignant papillaryhyperplastic nodules and it is therefore difï¬cult to diagnose PTC There is also a lackof effective and speciï¬c prognostic molecular markers for PTC17 The relationshipbetween MMPs and tumors is a currenthotspot of modern cancer research MMPsplay important roles in pathophysiologicalprocesses such as the dynamic extracellularmatrix balance as well as in tissue remodeling and repair10 Tumor cells may inducethe matrix to secrete MMPs via a series ofsignalingprovidingpathwaysthusTable Volume reductions after radiofrequencyablation of papillary thyroid carcinomasReductionrate Lesionvolumecm3 06 06 06 06 months after surgery 06 P P compared with before surgeryBefore surgery month after surgery months after surgery months after surgeryfavorable conditions for tumor cell invasionand metastasisalternatingthe extensive surgicalSurgical resection is a traditional methodthyroid nodules18for the treatment ofHowevertraumaunsightly neck scars and risks of laryngealnerve injuries postoperative recurrenceand multiple operations mean that increasing numbers of patients are opting for minimally invasive ablation methods RFA is athermal ablation therapy that uses highfrequencyelectromagneticwaves generated by the radiofrequencyinstrument inserted into the tumor tissueto accumulate heat by rapid friction of positive and negative ions within the cells causing local coagulation in the tumor tissuewhich isthen removed by the bodyssystem19 Reduction ratesimmuneforbenign thyroid nodules of to after month of ablation and to after months of ablation have beenreported20 In this study ultrasound performed immediately after ablation of lesions showed no perfusion of contrastagents in lesions and enhancementto varying degrees at the edge or inside thelesion in of lesions Although therewere no signiï¬cant changes in lesion volumeat month after the operation the lesionvolumes were reduced by to at months after surgery Considering thatthe ablation effects might be associatedwith the heat and the ablation needle aTable Logistic regression analysis of relevant risk factors for disease prognosisAgeMicrocalcificationIrregular morphologyLesion diameterLesion numberB HRHR hazard ratio CI confidence interval95CILower limitUpper limitP 0cJournal of International Medical Researchï¬ne needle is good for mobile conformalablationthe highfrequency alternatingelectromagnetic wave only circulates in theeffective region between the two needle tipsthus allowing accurate control of the ablation zone The ablation safety zone aroundthe PTC was relatively small in the currentstudy and the nodulereduction rate afterablation was thus relatively greaterWe analyzed the serum levels of MMP2and MMP9 in PTC patients by ELISAPreoperative serum levels of both enzymeswere signiï¬cantly higher in patients withPTC compared with patients with benigninthyroid nodules Regarding changesserum MMP2 and MMP9 levelstheAUC values based on the ROC curveswere and for MMP2 andMMP9 respectively suggesting satisfactory clinical diagnostic and prognostic valuesIn the present study serum levels MMP2and MMP9 were lower in the ï¬rst monthafter surgery compared with before surgerybut the difference was not signiï¬cant Thismight be because before ablationthetumor parenchyma induced the tumorstroma to produce larger amounts ofMMP2 and MMP9 which were releasedinto the blood These results suggest thatfailure to completely ablate the tumor ortumor recurrence may result in the secretionof high levels of MMP2 and MMP9 intothe blood However serum levels of MMP and MMP9 were signiï¬cantly decreasedat and months after surgery compared with before surgery suggesting thatserum MMP2 and MMP9 were secretedby the tumor The lesions disappearedafter PTC ablation thus reducing the secretion of MMP2 and MMP9 and therebyreducing the degradation and destructionof type IV collagen protecting the basement membrane of normaltissues andinhibiting the growth and metastasis oftumorresults alsoshowed that age microcalciï¬cation irregular shape and lesion diameter and numbercells Thecurrentwere risk factors for PTC Logistic regression analysis showed that age 14 yearswas an important risk factor for PTC inline with the ï¬ndings of Yu et al21Microcalciï¬cation is caused by the deposition of calcium salts at the tip of the nippleor the secretion of calcium salts by thetumor itself and has been considered tobe the most speciï¬c sign of PTC In thisstudy the incidence of microcalciï¬cationwas higher in PTC patients compared withthe control group and logistic regressionidentiï¬ed it as an independent risk sign forthyroid PTC Our results also identiï¬edirregular morphology two nodules and anodule diameter 14 cm as danger signs forPTClargely consistent with previousï¬ndings22This study had some limitations It was asinglecenter study with a relatively smallnumber of cases Moreover the relevantthyroid hormone analysis and other serumindicators could not be followed up for alonger periodtreating PTC FurthermoreIn summary the results of this studyshowed that RFA could shrink or eliminatethyroid lesions thus representing a minimally invasive safe and effective methodforserumlevels of MMP2 and MMP9 before RFAcould provide a valuable reference for thediagnosis of PTC These serological indexes combined with relevant risk factors mayalso help to predict the prognosis of PTCafter ablationAcknowledgementsThis work wasProvincial HealthCommission Project WJ2017F102supported by the HubeiPlanningand FamilyDeclaration of conflicting interestThe authors declare that there is no conï¬ict ofinterest 0cPan et alFundingThis research received no speciï¬c grant from anyfunding agency in the public commercial ornotforproï¬t sectorsorcid000000026050ORCID iDQian DingReferences Zhang YB Zhang B Yan DG et al[Central compartment reoperation for recurrentpersistent differentiated thyroid cancer]Zhonghua Er Bi Yan Hou Tou Jing Wai KeZa Zhi [in Chinese] Pellegriti G Frasca F Regalbuto C et alWorldwide increasing incidence of thyroidcancer update on epidemiology and risk factors J Cancer Epidemiol DOI Brito JP Gionfriddo MR Al Nofal A et alThe accuracy of thyroid nodule ultrasoundto predict thyroid cancer systematic reviewand metaanalysis J Clin Endocrinol Metab DOI 101210jc2013 Zhao P Zheng D Dong X et al Clinicaldiagnosis and treatment of thyroid microcarcinoma a report of cases Chinese JGene Surg Anil G Hegde A and Chong FH Thyroidnodules risk stratiï¬cation for malignancywith ultrasound and guided biopsy CancerImaging DOI Levine RA Current guidelines for the management of thyroid nodules Endocr Pract DOI 104158ep12071co Zhang Y Zhang MB Luo YK et al Effectof chronic lymphocytic thyroiditis on theefï¬cacy and safety of ultrasoundguidedradiofrequency ablation for papillary thyroid microcarcinoma Cancer Med Liu Y Wang W Wang Y et al Ultrasoundguided percutaneous microwave ablation inthe treatment of recurrent thyroid nodulesJ Clin Otolaryngol Head Neck Surg Weslley Rosario P Franco Mourao GRegina Calsolari M et al Role of adjuvanttherapy with radioactive iodine in patientswith elevated serum thyroglobulin afterneck reoperation due to recurrent papillarythyroid cancer a monoinstitutional comparative study Endocrine Zhang WJ Song B and Yang T MMP2MMP9 TIMP1 and TIMP2 in theperipheral blood of patients with differentiated thyroid carcinoma Cancer Manag Res Wu R Luo Y Tang J et al Ultrasoundguidedradiofrequency ablation for papillary thyroidmicrocarcinomaa retrospective analysis of patients Int J Hyperthermia Zhao CK Hu HX Lu F et al Factors associated with initial incomplete ablation forbenign thyroid nodules after radiofrequencyablation First results of CEUS evaluationClin Hemorheol Microcirc Faggiano A Ramundo V Assanti AP et alThyroid nodules treated with percutaneousradiofrequency thermal ablation a comparative study J Clin Endocrinol Metab DOI 101210jc20122251 Tomayko EJ Cachia AJ Chung HR et alResveratrol supplementation reduces aorticatherosclerosis and calciï¬cation and attenuates loss of aerobic capacity in a mousemodel of uremia J Med Food DOI 101089jmf20120219 Xhaard C Rubino C Clero E et alMenstrual and reproductive factors in therisk of differentiated thyroid carcinoma inyoung women in France a populationbased casecontrol study Am J Epidemiol DOI 101093ajekwu220 Tafani M De Santis E Coppola L et alBridging hypoxia inï¬ammation and estrogen receptors in thyroid cancer progressionBiomed Pharmacother DOI101016jbiopha201310013 Bumber B Marjanovic Kavanagh MJacovcevic A et al Role of matrix metalloproteinases and their inhibitors in the development of cervical metastases in papillary 0cJournal of International Medical Researchthyroid cancer Clin Otolaryngol ofAssociation Gharib H Papini E Paschke R et alAmericanClinicalEndocrinologists Associazione MediciEndocrinologiand European ThyroidAssociation medical guidelines for clinicalpractice for the diagnosis and managementof thyroid nodules executive summary ofrecommendations J Endocrinol Invest Baek JH LeeJH Valcavi Ret alThermal ablation for benign thyroid nodules radiofrequency and laser Korean JRadiol DOI kjr2011125525 Vuong NL Dinh LQ Radiofrequency ablation for benign thyroid nodules 1yearfollowup in patients World J Surg Lang BHH Woo YC and Chiu KWIdentifying predictive factors for efï¬cacy inhigh intensity focused ultrasound HIFUablation of benign thyroid nodules a retrospective analysis Int J Hyperthermia Buryk MA Simons JP Picarsic J et al Canmalignant thyroid nodules be distinguishedfrom benign thyroid nodules in children andadolescents by clinical characteristics Areview of pediatric patients with thyroidnodules Thyroid 0c' | Thyroid_Cancer |
"Deregulated circular RNAs circRNAs are associated with the development of cancer and therapyresistance However functional research of circRNAs mostly focus on potential miRNA or protein binding and morepotential regulation of circRNA on host gene DNA in cancers are yet to be inspectedMethod We performed total RNA sequencing on clinical breast cancer samples and identified the expressionpatterns of circRNAs and corresponding host genes in patient blood tumor and adjacent normal tissues qPCRnorthern blot and in situ hybridization were used to validate the dysregulation of circRNA circSMARCA5 A series ofprocedures including Rloop dotblotting DNARNA immunoprecipitation and mass spectrum etc were conductedto explore the regulation of circSMARCA5 on the transcription of exon of SMARCA5 Moreoverimmunofluorescence and in vivo experiments were executed to investigate the overexpression of circSMARCA5with drug sensitivitiesResults We found that circRNAs has average higher expression over its host linear genes in peripheral bloodCompared to adjacent normal tissues circSMARCA5 is decreased in breast cancer tissues contrary to host geneSMARCA5 The enforced expression of circSMARCA5 induced drug sensitivity of breast cancer cell lines in vitro andin vivo Furthermore we demonstrated that circSMARCA5 can bind to its parent gene locus forming an Rloopwhich results in transcriptional pausing at exon of SMARCA5 CircSMARCA5 expression resulted in thedownregulation of SMARCA5 and the production of a truncated nonfunctional protein and the overexpression ofcircSMARCA5 was sufficient to improve sensitivity to cytotoxic drugsConclusion Our results revealed a new regulatory mechanism for circRNA on its host gene and provided evidencethat circSMARCA5 may serve as a therapeutic target for drugresistant breast cancer patientsKeywords Breast cancer circRNA DNA damage repair Rloop Host gene Correspondence kechenhusteducn chewhueducnleiweifrhotmailcom Xiaolong Xu Jingwei Zhang Yihao Tian and Yang Gao contributed equallyto this work3Department of Urology Tongji Hospital Tongji Medical College HuazhongUniversity of Science and Technology Wuhan China1School of Basic Medical Sciences Wuhan University Wuhan HubeiChinaFull 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 0cXu Molecular Cancer Page of IntroductionCircular RNAs circRNAs are novel RNAs that havebeen ubiquitously discovered in many species by highthroughput sequencing in recent years [ ] CircRNAsare generated by the backsplicing of intronic exonic orintergenic regions circRNAs are resistant to RNase Rand the stability of their structures makes these molecules ideal candidates for disease [] Extensive studieshave revealed that dysregulated circRNAs are involvedin the development of various cancers In gastric cancercircRNAssuch as circPVT1 circLARP4 has_circ_ and circ_100269 have been shown to play arole in promoting tumor growth and their expression iscorrelated with high TNM stage and poor prognosis []In colon and hepatic carcinoma ciRS7 promoted tumordevelopment and progression by activating the EGFR andPI3KAkt pathway [ ] CircRNAs such as circKIF4Ahsa_circ_0001944 hsa_circ_0001481 and circRNA_0025202have been implicated in molecular typing brain metastasisand drug resistance in breast cancer [] Although greatprogress has been made the roles of circRNA and relevantmolecular mechanisms remain largely unknownPrevious studies have shown that circRNAs exert theirfunctions in different ways As noncoding RNAs circRNAs regulate the expression of other genes by servingas sponges for microRNA and RNAbinding proteins[ ] In addition some circRNAs have been shownto be translated into functional proteins [ ] Inaddition circRNAs have also been shown to directlyinteract with the genomic DNA of the host gene inplant which results in altered parent gene expression[] However the interaction of circRNAs and hostgene DNA were less studied in human cancersSMARCA5 is a member of the SWISNF complex withATPdependent chromatin remodeling activity []In the process of DNA damage repair SMARCA5 isinvolved in chromatin remodeling in DNA damageregions providing a structural basis for the recruitmentof DNA damage repair factors [ ] In tumorsSMARCA5 is highly expressed in hepatic carcinoma andprostate cancer and its expression levelis inverselyrelated to tumor radiosensitivity [ ]In this study we established circRNAs have averagehigher expression than their host genes in peripheralblood comparing to tissues Then we identified acircRNA derived from SMARCA5 circSMARCA5 issignificantly decreased in breast cancer cell lines andbreast cancer samples Differentto previous worksrevealing circSMARCA5 can also function as a competing endogenous RNAs by binding with miRNAmoleculesour mechanism explorationdisplayed circSMARCA5 is involved in regulating DNArepair capacity by binding exon DNA directly Andfurther functionalinvestigation of this circRNA may[]contribute to the therapeutic implications for cytotoxicdrugresistant breast cancer patientsResultsIdentification of expression of circRNAs in breast cancerWe performed high throughput sequencing on tumorT and adjacent normal tissue AN and peripheralblood B of six breast cancer patients Total RNA withrRNAdepleted library wereconstructed and thencircRNAs expressed in those samples were identifiedCompared to tumor and adjacent normal tissue we observed average higher CIRCscore expression of circRNA linear host genes in blood than both tumor andadjacent normal tissue In all circRNAs which wereexpressed across all six patients we observed averageCIRCscores from to in blood which is higherthan tumor to and adjacent normal tissue to in six patients Fig 1a This result indicated average higher expression of circRNAs than theirhost genes in peripheral blood comparing to tissueswhich might contribute to the exploration of diagnosticbiomarker for breast cancer We then selected sixcircRNAs with high CIRCscores average to in patients and performed further experimental validation in patients Realtime PCR results establishedtwo circRNAs circHIPK3 and circSMARCA5 weresignificantly differentially expressed between tumor andadjacent normal tissue Fig 1b and Figure S1A Especially circSMARCA5 was lower expressed in tumorsamples and less studied in previous work Furthermorethe ratio of circtolinear expression of circRNA linearhost genes of circSMARCA5 in blood sample of healthvolunteers were significantly higher than those of breast cancer patients P Fig 1c and Figure S1BWe nextcirctolinear ofcircSMARCA5 and clinical relevance in patients withbreast cancer and observed significant difference in thedistribution of the patients according to pathologic T P Table S1 Together these results indicating thepotential function and candidate biomarker attributes ofcircSMARCA5 in breast cancerexamined theratio ofand functionallyinvestigatefound thatTo characterizecircSMARCA5 we firstly detected the expression of circSMARCA5 in cell lines circSMARCA5 is derived fromthe backsplicing of exon and exon of SMARCA5Fig 1d As expected endogenous circSMARCA5 butto RNase R digestionnot premRNA was resistantFig 1dthe ntcircSMARCA5 was further confirmed by Northern blotassaycircSMARCA5 was mainly present in the nucleus whereasits parent mRNA was present exclusively in the cytoplasm as evidenced by qPCR Northern blotting andRNA in situ hybridization Fig 1fh and Figure S2Fig 1e Furthermore wethe existence ofIn addition 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig Identification of circRNAs in breast cancer a Heatmap of CIRCscore FBPcircFBPlinear in tumor T adjacent normal tissue AN and bloodsample B from six breast cancer patients b Expression of six circRNAs with high CIRCscore were validated by RTqPCR assay in breast tumor andadjacent normal tissue represents P CircRNAs IDs are according to circBase through their genomic coordinates c The ratio of circtolinear ofcircSMARCA5 in blood sample of breast cancer patients and health volunteers Total RNA from blood sample of breast cancer patients and healthvolunteers was extracted and detected by RTqPCR The expression level was normalized with βactin as reference P was consideredstatistically significant d Schematic illustration showing the genomic region of circSMARCA5 derived from exons and of the SMARCA5 geneConvergent gray and divergent black primers were designed to amplify the linear or backsplicing products upper Total RNA from MCF7 cellswith or without RNase R treatment was subjected to RTPCR lower and further validated by Sanger sequencing Right e Northern blot using ajunctionspecific probe or an exon probe showing the endogenous existence of circSMARCA5 and SMARCA5 mRNA from MCF7 cells with orwithout RNaseR treatment R or R The bp marker indicates the SMARCA5 fulllength transcript transcribed in vitro The bp markerindicates exon and exon of SMARCA5 transcribed in vitro f The nucleus and cytoplasm mRNA of MCF7 were extracted and SMARCA5 andcircSMARCA5 expression levels were quantitated by RTPCR GAPDH and hU6 serve as internal references of the cytoplasm and nucleus respectively indicates P g The nucleus and cytoplasm mRNA of MCF7 were extracted SMARCA5 and circSMARCA5 were examined by Northernblotting and the SMARCA5 exon probe was applied in this experiment h Subcellular localization of circSMARCA5 and SMARCA5 in MCF7 cellsThe signals were examined by indirect RNA FISH and confocal microscopy The nucleus was counterstained with DAPI The circSMARCA5 probe waslabled by biotin while the SMARCA5 probe was labled by DIG They were stained with red and green fluorescent secondary antibodies respectively IThe expression of circSMARCA5 detected by northern blot MDAMB231 BT474 MCF7 SKBR3 are breast cancer cell lines MCF10A are normal breastcell line N1N2N3N4N5 are adjacent normal tissues T1T2T3T4 are breast cancer tissues indicates P Next we examined the expression of circSMARCA5 invarious breast cancer cell lines MCF7 SKBR3 BT474MDAMB231 and immortalized but nontransformedbreast epithelial cells MCF10A as well as in adjacentnormal tissues and breast cancer tissues Northern blotresultsthe expression levels of circSMARCA5 in MCF10A and normal adjacent tissuesare higher than breast cancer cell lines and cancer tissues Fig 1i These results indicated that circSMARCA5is downregulated in breast cancer tissues and cellsrevealed thatsequencecircSMARCA5 decreases the expression of SMARCA5 incancer cellsTo clarify the mechanisms of circSMARCA5 we investigated its effects on the expression of its parent geneSMARCA5 The expression levels of circSMARCA5 andSMARCA5 were detected by the primers of junctionsequence and exonsrespectivelyKnockdown of circSMARCA5 increased both mRNAand protein levels of SMARCA5 while converselycircSMARCA5 overexpression decreased SMARCA5levels Fig 2ac and Figure S3 Consistently the proteinof SMARCA5 was high expressed in breasttumorsamples as compared with the corresponding controlsFigure S4 Moreoverthe ratio of circtolinear ofcircSMARCA5 was significantly lower in breast andrenaltumor tissue than the corresponding adjacenttissue specimens Fig 2de and Figure S1C Besides asignificant negative correlation was also found betweencircSMARCA5 and SMARCA5 expression in various celllines and primary cancer tissues Fig 2df and FigureS5 which corroborates our observation that circSMARCA5 decreased the expression of SMARCA5 incancer cellscirSMARCA5 terminates the transcription of SMARCA5 atexon We further investigated the mechanism of circSMARCA5in regulating the expression of SMARCA5 Interestinglywe found that the overexpression of circSMARCA5 indeed decreased the expression of SMARCA5 exons but had minimal effects on the expression of exons Fig 3a Next we designed a primer location in exon for the amplification of ² cDNA ends by rapid amplification of cDNA ends RACE PCR Fig 3b left As shownin Fig 3b SMARCA5 can give rise to multiple isoformsImportantly we found a decrease in a band of bpupon circSMARCA5 overexpression while an bpband displayed the opposite phenomenon Fig 3b rightSanger sequencing showed that the bp band andthe bp band are derived from fulllength and truncated mRNA exons to respectively of the SMARCA5 gene Fig 3c Consistent with the RACE resultsNorthern blot assay further demonstrated that ectopiccircSMARCA5 expression decreased SMARCA5 levelsand promoted truncated mRNA levels Fig 3d The observations gathered thus far have led us to hypothesizethat circSMARCA5 prevents transcription from exon of SMARCA5 Indeed ChIP analysis indicated that thebinding of pol II to exons of SMARCA5 was higherthan that to exons Fig 3e left and the ectopic expression of circSMARCA5 decreased the binding of Pol IIto exons of SMARCA5 Fig 3e right To furtheraddress whether circSMARCA5 could terminate the transcriptional elongation of SMARCA5 we cloned a series ofexons of SMARCA5 in a luciferase plasmid reporterFig 4a upper The transient transfection of these luciferase reporters containing the exon sequence revealed that luciferase activity was significantly decreasedwhen circSMARCA5 was overexpressed Fig 4a lower 0cXu Molecular Cancer Page of Fig circSMARCA5 decreases the expression of SMARCA5 in cells a Generation of circSMARCA5knockdown and circSMARCA5overexpressingcells MCF7 cells were infected with lentiviruses expressing shRNA against circSMARCA5 shcircSMARCA5 three different oligonucleotides orcircSMARCA5 pLCDHcircSMARCA5 RTqPCR was performed to evaluate the expression of circSMARCA5 GAPDH was used as an internal controlb RTqPCR showing the levels of circSMARCA5 and SMARCA5 in MCF7 cells stably expressing shNC shcircSMARCA5 pLCDHciR control orpLCDHcircSMARCA5 c Western blot showing the levels of SMARCA5 in MCF7 cells stably expressing shNC shcircSMARCA5 pLCDHciRcontrol pLCDHcircSMARCA5 pLCDHcircSMARCA5Î without splicinginducing sequence GAPDH was used as an internal control DF Theratio of circtolinear of circSMARCA5 in tumor tissue were significantly lower than normal tissue in breast cancer samples d and RCC samplese P A negative correlation between circSMARCA5 and SMARCA5 expression was observed in breast cancer samples d RCC samplese and various cell lines fTo further confirm the effect of circSMARCA5 on thetranscriptional elongation of SMARCA5 we insertedexons of SMARCA5 between DsRED and EGFP as indicated Fig 4b upper The EGFP level was significantly decreased by circSMARCA5 when exons were present Fig 4b lower We further investigatedthe role of circSMARCA5 in the regulation of SMARCA5 at the protein level As expected circSMARCA5overexpression downregulated the protein levels ofSMARCA5 and upregulated truncated SMARCA5ÎSMARCA5 protein levels Fig 4c and Figure S6which was confirmed by mass spectrometry Fig 4dMoreover we found that ÎSMARCA5 is more susceptible to proteolysis by the proteasome than SMARCA5 Fig 4e Together these results show the roleof circSMARCA5 in the termination of transcriptionalelongation at exon of SMARCA5circSMARCA5 can form Rloops with its parent geneTo further dissect the mechanism of SMARCA5 transcriptional termination mediated by circSMARCA5 weinvestigated whether circSMARCA5 can bind genomic 0cXu Molecular Cancer Page of Fig cirSMARCA5 terminates the transcription of SMARCA5 at exon a RTqPCR analysis of the expression of SMARCA5 in MCF7 cells using aseries of paired primers indicates P b Rapid amplification of cDNA ends RACE PCR analysis of SMARCAC5 transcripts The PCRproducts were readily identified by agarose gel electrophoresis Each set of samples was repeated three times c Sanger sequencing of twotranscripts of SMARCAC5 that are regulated by circSMARCA5 overexpression d Northern blotting using the junctionspecific probes for exons and to show the expression levels of the transcripts of SMARCAC5 mRNA from MCF7 cells stably expressing control vector or pLCDHcircSMARCA5 circOE e CircSMARCA5 prevents transcription from exon of SMARCA5 ChIPseq analysis showing that the binding of pol II toexons of SMARCA5 was higher than that to exons ChIPqPCR showed that the ectopic expression of circSMARCA5 decreased thebinding of Pol II to exons of SMARCA5SMARCA5 DNA to form an Rloop Dotblotting withRloopspecific S96 antibody supported our hypothesisthat circSMARCA5 can bind exons of SMARCA5genomic DNA Fig 5a Additionally we performedDNARNA immunoprecipitation DRIP qPCR and confirmed the interaction between circSMARCA5 andexons pretreatment with RNase H ablated thisinteraction confirming that the interaction is Rloopspecific Fig b and Figure S7 The interaction of circSMARCA5 with the DNA of SMARCA5 was directlyverified by fluorescence in situ hybridization Fig 5cConsistent with previous findings [] dotblotting ofthe genome without RNA digest revealed that the binding of circRNA to genomic DNA may be widely presentin cancer cells Fig 5d We next determined the specificsequence of exons required for Rloop formationA series of fragments from exons were hybridizedwith circSMARCA5 for the dotblotting assay As shownin Fig 5e the bp fragment of the ² end of exon plays important role in interacting with circSMARCA5Moreoverthe secondary structure of circSMARCA5was determined by the software MFOLD [] whichrevealed the sequence ²AACAAAAUUGGGAAAGAUGAAAUGCUUCAAAU3² from the ² end of exon located in the loop region of circSMARCA5 Fig 6aWe thus hypothesized that this sequence might play akey role in mediating the circSMARCA5DNA interaction To this end we synthesized the wildtype andmutant phosphorylated DNA fragments ANT andANTmut respectively corresponding to this sequenceFig 6b Dotblotting demonstrated that wildtypeoligonucleotides ANT can bind to circSMARCA5 but 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig cirSMARCA5 blocks the transcription of SMARCA5 and promotes the generation of a truncated SMARCA5 protein ÎSMARCA5 aSchematics of luciferase reporter constructs containing the SMARCA5 exon sequence as indicated upper The SMARCA5 exon sequenceplays an important negative role in mediating the effect of circSMARCA5 overexpression on luciferase activity lower b Schematics offluorescence reporter constructs containing the SMARCA5 exon sequence as indicated upper MCF7 cells were transiently transfected withthese fluorescence reporters along with or without circSMARCA5 cooverexpression After transfection for hours the reporter transcriptionactivities were measured by flow cytometry assay c circSMARCA5 overexpression downregulated the protein levels of SMARCA5 whileupregulating truncated SMARCA5 ÎSMARCA5 protein levels MCF7 cells stably overexpressing circSMARCA5 or control cells were treated withDMSO or MG132 Western blot analysis was performed using an antibody targeting the Nterminus of SMARCA5 to evaluate the expression ofSMARCA5 and ÎSMARCA5 GAPDH was used as an internal control d The ÎSMARCA5 protein was identified by mass spectrometry and detectedSMARCA5 peptides were showed in the map The redlabeled portion is the amino acid sequence of the translated defective transcript e MCF7cells expressing FlagSMARCA5 and FlagÎSMARCA5 were treated with cycloheximide CHX μgml The cell lysates were subsequentlyharvested at sequential time points or h after treatment and then the cell lysates were immunoblotted with antiFlag or antiActin antibodymutant oligonucleotides ANTmut cannot bind to circSMARCA5 Fig 6c As expected DRIPqPCR showedthat ANT inhibited circSMARCA5 binding to the DNAat exons whereas ANTmut had no effect on thisinteraction Fig 6d Furthermore the transfection ofANT prevented the decrease in SMARCA5 proteinlevels in MCF7 cells stably expressing circSMARCA5whereas ANTmut had no effect on SMARCA5 proteinlevels Fig 6e Importantly the mutation of the keysequence in circSMARCA5 impaired the interactionwith its parent gene which was confirmed by dotblotting and DRIPqPCR assays Fig 6fh and FigureS8 Unlike circSMARCA5 circSMARCA5mut had littleeffect on SMARCA5 protein levels Fig 6i Theseresults suggested that circSMARCA5 formed Rloopswith its parent gene to inhibit the expression of SMARCA5 in cancer cellscircSMARCA5 inhibits DNA damage repair functionTo explore the roles of circSMARCA5 in cancer progression we overexpressed and depleted circSMARCA5in MCF7 cells by lentiviral vectors and then examinedthe effect of circSMARCA5 on cell proliferation migration and apoptosis However the results showed thatboth overexpressed and depleted circSMARCA5 had noeffect on these three activities Figure S9 Previousstudies have indicated that SMARCA5 plays an important role in regulating the DNA repair process and main[ ]taining theConsistent with previous reports SMARCA5 overexpression improved DNA repair capacity and reducedthe expression of Chk1 and Chk2 after DNA damagerepair Figure S10A Given that circSMARCA5 canpromote the production of the truncated ÎSMARCA5protein we tested whether the truncated protein is alsofunctional The overexpression of FlagÎSMARCA5had a minimal effect on the expression of Chk1 andChk2 after DNA damage repair Figure S10B suggesting that ÎSMARCA5 isa nonfunctional proteinproduct We next assessed whether circSMARCA5 canthe genomestability ofshowedlowerthan thatsignificantlyformation assaysaffect the function of DNA damage repair capacityCCK8 and clonerevealed thatcircSMARCA5 overexpression increased sensitivity tocisplatin or bleomycin in MCF7 cells Fig 7a b NextMCF7 cells were treated with the indicated concentration of cisplatin or bleomycin for h and then theDNA damage was evaluated by single cell gel electrophoresis SCGE at and h MCF7 cells expressingcircSMARCA5repaircapacity than did control cells Fig 7c In parallelDNA damage was examined after h of treatmentwith cisplatin or bleomycin by using an antiγH2AXantibody Consistent with the SCGE results the γH2AXin MCF7 cells expressing circSMARCA5 wassignalsignificantly higherin MCF7 cells asevidenced by immunostaining Fig 7d Consistentlycisplatin significantly enhanced the levels of DNAdamage response proteins Chk1 and Chk2 in MCF7cellsexpressing circSMARCA5 Fig 7e whereasseveral key cellcycle genes were reduced specificallyupon circSMARCA5 overexpression Fig 7f To testwhether circSMARCA5 Rloop formation is necessaryfor its DNA repair function we transfected ANT orANTmutinto circSMARCA5expressing cells TheSCGE assay and γH2AX measurement showed thatANT significantly enhanced the DNA repair capacitywhile ANTmut had no effect on this activity Fig 7gand Figure S11 Furthermore ANT significantlydecreased the degree of colocalization between circSMARCA5 and its cognate DNA locus Figure S12In addition unlike circSMARCA5 the overexpressionof circSMARCA5mut had little effect on the DNArepair rate Fig 7h and Figure S13A B Next we determined whether SMARCA5 could mediate the effects ofcircSMARCA5 in preventing DNA damage repair Asshown in Fig 7i the γH2AX signal was much lower incircSMARCA5expressing cellscomplemented withSMARCA5 than that in cells expressing circSMARCA5alone As expected ÎSMARCA5 could not rescue theinhibition of DNA damage repair function induced by 0cXu Molecular Cancer Page of Fig See legend on next page 0cXu Molecular Cancer Page of See figure on previous pageFig circSMARCA5 interacts with its site of transcription a circSMARCA5 interacts with the exon sequence of the SMARCA5 locus A seriesof exon DNA fragments were hybridized with circSMARCA in vitro The DNARNA hybridization strength was quantified by dotblot with Rloopspecific S96 antibody Hybridization stringency was altered by decreasing ionic strength mM NaCl b DRIPqPCR analysis of the exon sequence of SMARCA5 to detect the association of circSMARCA5 in MCF7 cells RNase Htreated andor DRIPqPCR analysis of the exon sequence as a control c CircSMARCA5 partially localized at its site of transcription Double FISH of circSMARCA5 red and its parent DNAregion green The nucleus was stained by DAPI d Dotblot of Rloops in MCF7 cell genomic DNA preparations treated with DNase I RNase Hor RNase R The DNARNA hybrids in genome DNA were analyzed by S96 antibody e Mapping of the Rloop formation region of circSMARCA5A series of exon deletion mutants were hybridized with circSMARCA5 for the dotblotting assay The DNARNA hybridization intensity wasanalyzed by dotblot with an S96 antibody targeting the DNARNA hybrid strand Hybridization stringency was altered by decreasing ionicstrength mM NaClcircSMARCA5 Figure S13C D Moreover the overexpression of SMARCA5 could significantly rescue thegrowth defects of cells expressing circSMARCA5 asdemonstrated by a colony formation assay Fig jTogetherthese results demonstrated the roles ofcircSMARCA5 in regulating the DNA repair process inMCF7 cellstheevaluatecircSMARCA5 overexpression enhances the cisplatinresponse in breast cancerTo furthertherapeutic potential ofcircSMARCA5 in breast cancer in vivo we establishedcircSMARCA5 overexpression clones in MCF7 cells Asshown in Fig 8a the overexpression of circSMARCA5efficiently enhanced the sensitivity of MCF7 xenograftsto concurrent cisplatin treatment Fig 8a b The overexpression of circSMARCA5 was confirmed by in situhybridization and qPCR analysis Fig 8c along with decreased SMARCA5 protein levels and increased γH2AXlevels Fig 8d In addition qPCR analysis demonstratedthat circSMARCA5 can be detected in the bloodsuggesting that circSMARCA5 is a secretory moleculeCollectively these data demonstrate that circSMARCA5could serve as a potential therapeutic target to restoresensitivity to cisplatin therapy in breast cancerDiscussionPrevious studies have indicated that circRNAs have multiple functions in cancer development and progression[] In this study we identified multiple expressedcircRNAs in breast cancer samples and observed averagehigher abundance of circRNAs over their host genes inperipheral blood than tissues which might contribute tothe exploration of diagnostic biomarkerfor breastcancer We then identified that circSMARCA5 is significantly decreased in breast cancer tissues using RNAseqMore importantly we define a critical role for circSMARCA5 in the regulation of DNA damage repaircapacity and the drug sensitivity of breast cancer cellsin vitro and in vivo through the negative regulation ofits parent gene SMARCA5 These findings are of highclinical relevance because chemotherapy with cisplatinand bleomycin remains the standard of care in breastcancer [] Hence the restoration of circSMARCA5levels provides an approach to overcome treatmentresistance in breast cancer patientsSMARCA5 also known as SNF2H is a member of theSWISNF chromatinremodeling complex During DNAdamage repair processes SMARCA5 is recruited toDNA damage sites where it induces the ubiquitinationand phosphorylation of histone H2A which facilitateschromatin remodeling and DNA damage repair [ ]In this study we show that circSMARCA5 expressionresulted in the downregulation of SMARCA5 and theeffect of circSMARCA5 overexpression on DNA repaircapacity was reversed by concomitant SMARCA5 overexpression suggesting that the effect of circSMARCA5on DNA repair capacity is mediated through SMARCA5circRNAs exert functions in various ways such as forming an Rloop with DNA to regulate splicing and transcriptional pausing [] For example circSEPALLATA3regulates the splicing of its parent mRNA through Rloop formation [] In addition circRNAs are a novelclass of ceRNAs that sponge miRNAs thus positivelyregulating gene expression [ ] Additionally circRNAs such as exonintron circRNAs regulate geneexpression through specific RNARNA interactions withU1 snRNA [] Furthermore circRNAs also exertfunctions by binding to proteins and regulating theiractivities [] We identified one mechanism by whichcircSMARCA5 regulates the drug sensitivity of breastcancer cells to cisplatin and bleomycin through thedownregulation of SMARC5 circSMARCA5 is recruitedto its parent gene locus leading to Rloop formationtranscriptiontruncatedÎSMARCA5 protein upregulation and decreased SMARCA5 expression This regulatory mechanism has alsobeen verified in cervical cancer Hela cells Figure S14However our evidence demonstrates that circSMARCA5has no significant effect on the proliferation migrationand apoptosis of breast cancer cells suggesting that thismolecule functions in a celltype and contextdependentmanner Notablythatnonfunctionalterminationweprovideevidence 0cXu Molecular Cancer Page of Fig circSMARCA5 can form an Rloop with its parent gene a Secondary structure prediction for circSMARCA5 using the Mfold program Thesequence KEY shared by the minimum free energy structure and the thermodynamic ensemble structure is marked by red b Thethiophosphorus nucleic acid analog ANT complementary to KEY and its mutant ANTmut were synthesized in vitro c Dotblot verifying theinteraction between circSMARCA and ANT or ANTmut d DRIPqPCR analysis on exon or exon sequences of SMARCA5 to detect theassociation of circSMARCA5 in MCF7 cells overexpressing ANT or ANTmut RNase Htreated andor DRIPqPCR analysis of the exonsequence as a control indicates P e Western blot analysis shows that transfection of ANT into circSMARCA5overexpressing cells canrestore SMARCA5 protein levels but ANTmut cannot f g Dotblot analysis quantifying Rloop strength between the SMARCA5 locus andcircSMARCA5 or circSMARCA5mut guanine converted to cytosine of the KEY sequence h DRIPqPCR in MCF7 cells transfected withcircSMARCA5 or circSMARCA5mut RNase Htreated genomic DNA and qPCR of exon1314 were treated as controls indicates P iWestern blot analysis shows that overexpression of circSMARCA5 to MCF7 cells can decrease SMARCA5 protein levels but circSMARCA5mutcannot 0cXu Molecular Cancer Page of Fig circSMARCA5 decreases DNA repair capacity a circSMARCA5 increases sensitivity to cisplatin or bleomycin in MCF7 cells MCF7 cellsstably expressing control vector or pLCDHcircSMARCA5 were treated with cisplatin or bleomycin for h and CCK8 was used to measure cellviability b Relative colony formation units of MCF7 cells stably expressing control vector or pLCDHcircSMARCA5 treated with μM cisplatin or μgml bleomycin After hours the drugs were replaced by fresh medium The number of colonies was quantified c d e Single cell gelelectrophoresis SCGE assay indicating that circSMARCA5 overexpression inhibits cell recovery from DNA damage MCF7 cells stably expressingcontrol vector or pLCDHcircSMARCA5 treated with μM cisplatin or μgml bleomycin After incubation for h the cells were recoveredwith fresh medium for or hours and then collected for SCGE analysis c immunofluorescence assay using an antiγH2AX antibody d andwestern blot assay with the indicated antibodies e f RTqPCR assay showing the relative levels of several key cell cycle genes in MCF7 cellsstably expressing control vector or pLCDHcircSMARCA5 treated with DMSO or μM cisplatin for h and replaced with fresh medium for h indicates P g SCGE assay showing that the cotransfection of ANT in circSMARCA5overexpressing cells can restore the DNA repaircapacity | 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 |
cancer is still one of the most prevalent and highmortality diseases summing more than million deaths in This has motivated researchers to study the application of machine learningbased solutionsfor cancer detection to accelerate its diagnosis and help its prevention Among several approaches one is toautomatically classify tumor samples through their gene expression analysisMethodsstomach breast and lung To do so we have adopted a previously described methodology with which we comparethe performance of different autoencoders AEs used as a deep neural network weight initialization technique Ourexperiments consist in assessing two different approaches when training the classification model fixing theweights after pretraining the AEs or allowing finetuning of the entire network and two different strategies forembedding the AEs into the classification network namely by only importing the encoding layers or by inserting thecomplete AE We then study how varying the number of layers in the first strategy the AEs latent vector dimensionand the imputation technique in the data preprocessing step impacts the networks overall classification performanceFinally with the goal of assessing how well does this pipeline generalize we apply the same methodology to twoadditional datasets that include features extracted from images of malaria thin blood smears and breast masses cellnuclei We also discard the possibility of overfitting by using heldout test sets in the images datasetsResults The methodology attained good overall results for both RNASeq and image extracted data Weoutperformed the established baseline for all the considered datasets achieving an average F1 score of Continued on next pageCorrespondence mafaldafferreirafeuppt1Faculty of Engineering University of Porto Rua Dr Roberto Frias sn Porto Portugal2INESC TEC Institute for Systems and Computer Engineering Technology andScience Porto Portugal 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 give appropriatecredit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes weremade The images or other third party material in this are included in the s Creative Commons licence unlessindicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The CreativeCommons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Continued from previous pageand and an MCC of and for the RNASeq when detecting thyroid cancer the Malaria and theWisconsin Breast Cancer data respectivelyConclusions We observed that the approach of finetuning the weights of the top layers imported from the AEreached higher results for all the presented experiences and all the considered datasets We outperformed all theprevious reported results when comparing to the established baselinesKeywords Cancer Classification Deep learning Autoencoders Gene expression analysisBackgroundCancer is a label for a group of diseases that is characterized by abnormal and continuous cell growth with thepotential to spread through its surrounding tissues andother body parts [] During cancer was the secondleading cause of death globally accountable for milliondeaths where around were in developing countries[] Throughout the years and given the evolution of techniques technology and treatments in medicine cancersurvival rates have been improving [] However there arestill some types that have survival rates of under suchas pancreatic esophagus and liver cancers Its prevalencemakes it more crucial to correctly and accurately classify such diseases For tackling this need many researchgroups have been trying to help on accelerating cancerdiagnosis by experimenting and studying the applicationof machine learning algorithms to this problem []When automatically classifying tumor samples oneapproach is to analyze the samples derived molecularinformation which is its gene expression signatures Geneexpression is the phenotypic manifestation of a gene enes by the processes of genetic transcription and translation [] By studying it this gene map can help to betterunderstand cancers molecular basis which can have adirect influence on this diseases life cycle prognosis diagnosis and treatment There are two main cancer genomicsprojects The Cancer Genome Atlas TCGA [] andThe International Cancer Genome Consortium ICGC[] that aim to translate gene expression systematizing thousands of samples across different types of cancersWith this elevated number of features each representing a particular gene one may find genomewide geneexpression assays datasets in these projects However thistype of data presents some challenges because of alow number of samples an unbalanced class distribution with few examples of healthy samples and a high potential of underlying noise and errors due toeventual technical and biological covariates [] This difficulty in gathering data accurately is underlying for everydataset creation The equipment used to collect the datahas intrinsic errors associated mechanical of acquisitionand others hence the dataset will reflect these errorsSeveral authors have chosen the previously mentionedapproach of analyzing the gene expression of tumor samples Many of the developed methodologies in this scopeuse straightforward supervised training especially whenusing deep neural networks DNNs relying on theirdepth to produce the best results Gao [] proposedDeepCC a supervised deep cancer subtype classificationframework based on deep learning of functional spectra quantifying activities of biological pathways robust tomissing data The authors conducted two studies eachwith a different cancer detection colorectal and breastcancer data The authors claimed that the describedmethod achieved overall higher sensitivity specificity andaccuracy compared with other classical machine learningmethods widely used for this kind of task namely randomforests support vector machine SVM gradient boostingmachine and multinomial logistic regression algorithmswith an accuracy higher than Sun [] proposed Genome Deep Learning GDLa methodology aiming to study the relationship betweengenomic variations and traits based on DNNs Thisstudy analyzed over six thousand samples of Whole ExonSequencing WES mutations files from different cancer types from TCGA and nearly two thousand healthyWES samples from the one thousand genomes projectsThe main goal of GDL was to distinguish cancerous fromhealthy samples The authors built models to identify each type of cancer separately a totalspecific modelable to detect healthy and cancerous samples and a mixedmodel to distinguish between all types of cancerbasedon GDL All the experiments were evaluated througha three performance metrics accuracy sensitivityand specificity and b Receiver Operating Characteristic curves with the respective Area Under the CurveROCAUC This methodology achieved a mean accuracy of on the specific models on mixturemodels and on total specific models for canceridentificationIn [] Kim compared the performances of a neural network a linear SVM a radial basisfunctionkernel SVM a knearest neighbors and arandom forest when identifying types of cancers and 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of healthy tissues The classifiers were trained with RNAseq and scRNAseq data from TCGA where they selectedup to the most significant genes expressed for eachof the cancer variations To determine the optimal number of genes for each classifiers binary classification taskthe methods mentioned above were trained with different sizes of gene expression datasets from to genes When learning with genes the neural network the linear SVM and the radial basis functionkernelSVM models achieved their best performance with a witha Matthews Correlation Coefficient MCC of and respectively The knearest neighbors and random forest models achieved an MCC of and accordingly when using genes Furthermore theauthors identified classes with an accuracy of over and achieved a mean MCC of and a mean accuracy of with the neural network classifierHowever many DNNs besides the known challenges regarding their training setting [] have a highertendency to overfit which one can detect when applying the same architecture to unseen data or to a heldouttest Thus our motivation focuses on exploring unsupervised pretraining methods based on a lowerdimensionallatent representation with the usage of an autoencoderAE This approach is grounded in the hypothesis thata there is unessential information in high dimensionality datasets and b the acquisition and processing errorspotentially present in the dataset are discarded contributing to a lower probability of overfitting [] Furthermorepretraining AEs and using the learned weights as priorsof the supervised classification task not just improves themodel initialization but also often leads to better generalization and performance [] This may be one of thereasons why AEs are found to be the most predominantstrategy when analyzing RNASeq data []To support our motivation and choices we presentsome works that include unsupervised training in theirmethodologies In [] the authors designed a solution by combining a Multilayer Perceptron and StackedDenoising Autoencoder MLPSAE aiming to predicthow good genetic variants can be a factor in gene expression changes This model is composed of layers inputtwo hidden layers from the AEs and output and trained itto minimize the chosen loss function the Mean SquaredError MSE The authors started by training the AEs witha stochastic gradient descent algorithm to later use themon the multilayer perceptron training phase as weight initialization crossvalidation was used to select the bestmodel The performance of the chosen model was compared with the Lasso and Random Forest methods andevaluated on predicting gene expression values for a different dataset The authors concluded that their approach outperformed both the Lasso and Random Forest algorithms with an MSE of versus and respectively and was able to capture the change ingene expression quantificationThe authors in [] described a study of four different methods of unsupervised feature learning PrincipalComponent Analysis PCA Kernel Principal Component Analysis KPCA Denoising AE DAE and StackedDenoising AE combined with distinct sampling methods when tackling a classification task The authorsfocused on assessing how influential the input nodes areon the reconstructed data of the AEs output when feeding these combinations to a shallow artificial networktrained to distinguish papillary thyroid carcinoma fromhealthy samples The authors highlighted two differentresults in their 5fold cross validation experiment thecombination of a SMOTE [] with Tomek links and aKPCA was the one with the best overall performancewith a mean F1 score of while the usage of a DAEachieved a mean F1 score of In [] presented a stacked sparse autoencoder SSAEsemisupervised deep learning pipeline applied to cancer detection using RNASeq data By employing layerwise pretraining and a sparsity penalty this approachhelps to capture more significant information from theknown high dimensionality of RNASeq datasets usingthe filtered information to the sequent classification taskThe SSAE model was tested on three different TCGARNASeq datasets corresponding to lung stomach andbreast cancers with healthy and cancerous samplesand compared it to four others classification methodsan SVM a Random Forest a neural network supervisedlearning only and a vanilla AE The authors performed5fold cross validation and evaluated the models performance through four metrics accuracy precision recalland F1 score The results show that the semisuperviseddeep learning approach achieved superior performanceover the other considered methods with an average F1score of across the three used datasetsThe authors in [] developed a methodology for detecting papillary thyroid carcinoma They analyzed how theusage of AEs as a weight initialization method affectedthe performance of a DNN Six types of AEs were considered Basic AE Denoising AE Sparse AE DenoisingSparse AE Deep AE and Deep Sparse Denoising AEBefore being integrated into the classifier architecture allAEs were trained to minimize the reconstruction errorSubsequently they were used to initialize the weights ofthe first layers of classification neural network meaningthat the AE layers become the top layers of the wholeclassification architecture using two different strategieswhen importing the weights just the encoding layersand all the pretrained AE Moreover in the training phase the authors studied two different approacheswhen building the classifier a fixing the weights ofthe AE and b allowing subsequent finetuning of all 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of the networks weights The authors used stratified 5foldcrossvalidation and evaluated the model through distinct metrics Loss Accuracy Precision Recall and F1score The authors reported that the overall best resultwas achieved through a combination of Denoising AEfollowed by its complete import into the classification network and by allowing subsequent finetuning throughsupervised training yielding an F1 score of in which the main goalIn [] the authors present a transfer learning methodologyis to explore whetherleveraging the information extracted from a large RNASeq data repository with multiple cancer types leadsto extract important latent features that can help complex and specific prediction tasks such as identifyingbreast cancer neoplasia The authors used the TCGAPanCancer dataset which is composed of approximately RNASeq gene expression examples of distincttumor types This data was split into two sets breast cancer and nonbreast cancer data The nonbreast data isfirstly used to train the three selected architectures forthis study a sparse AE a deep sparse AE and a deepsparse denoising AE models Then the breast data isused to finetune the resulting AEs After pretrainingthese models the authors aim to predict the breast tumorintrinsicsubtypes which is given by the PAM50 subtype information included in the clinical data included inthe PanCancer data The extracted features from the AEbased architectures are then fed as input to three differentmachine learning classifiers namely Logistic RegressionSupport Vector Machine and a shallow Neural NetworkTo assess the deep AEs performance as feature extractionmethods the authors compared them to other classical feature extraction methods combining them with theclassification algorithms previously mentioned ANOVAMutual Information ChiSquared and PCA A 10foldcross validation was performed and all the combinationswere compared through the accuracy metric The resultsshowed the deep sparse denoising AE performs best whenusing the AE extracted features where the combinationwith a shallow neural network leads to the best overall of ±In [] Ferreira used the same methodologydescribed in [] to discriminate different types of cancerinstead of distinguishing cancerous samples from healthyones In this case they aimed to identify thyroid skin andstomach cancer correctly Given that a Denoising AE wasthe AE that lead to the best results in previous studiesthe authors chose to single it out instead of the original The rest of the experiments remained the same strategies for importing the pretrained AE into the top layersof the classifier two approaches when training the classifier to detect different types of cancer same evaluation ofthe obtained results Although in a different domain thebest outcome was reached with a combination of the samestrategy and the same approach in the previous work []with an F1 score of when identifying thyroid cancerMethodsWe extend the previously described work in [] byassembling three different types of experiments dividedinto two main parts where we use three different AEsand five types of cancer samples In the first one we analyze the performance of a deep neural network DNNusing the same pipeline to identify different types of cancer In the second part we choose one of the used AEsto assess how the variance of its latent vector dimension impacts the essential information capture and therefore possibly influencing the classifiers performance and different data imputation strategies can influence theoverall performance in the classification task Moreoverwe study if the network architecture is correlated withits overall performance and how the model reacts whentraining with a different data type dataset We built thispipeline in Python using the Numpy [] and Pandas []packages for the data preprocessing step the Keras deeplearning library [] running on top of TensorFlow andthe ScikitLearn [] package to train and evaluate themodels and the Matplotlib [] library for visualizationAdditionally we used an NVIDIA GeForce RTX TiGPU on a Ubuntu operating systemThis section is anized as follows The data subsection describes the used data and its inherent preprocessing Autoencoders subsection overviews the AEs considered to this study Methodology subsection outlinesthe pipeline for each of the referred experiments Evaluation subsection details how we evaluate the results toprovide statistical evidence Finally Baseline subsectionpresents the established baseline results for all the useddatasetsThe dataIn our experiments we use two different types of datawhich are described in the subsections that followRNASeq dataWe used five different RNASeq datasets from The Cancer Genomes Atlas TCGA [] each representing a typeTable Five instances of the thyroid RNASeq dataset we have usedTPTEP1 AKR1C6PUBE2Q2P2 HMGB1P1 LOC155060 ZZZ3 NANANANANANANANANANA NA NA NA NA NAThe first line the header contains the genes names and the column valuesrepresent its expression samplewise except for the first column which is thesample ID NA stands for missing value for a particular gene and sample 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of of cancer thyroid skin stomach breast and lung Onecan find a sample of the described data in Table The datasets were downloaded from the cBioPortal []which gathers cancerrelated data from different projectsincluding TCGA To train DNNs we need as many data aswe can get Ergo our first criterion was to choose cancertypes that had the highest number of examples Additionally we decided to gice priority to cancer types withhigh mortality and high incidence rates We use the samethyroid skin and stomach datasets presented in []alongside the lung and breast datasets The data filteringprocess in the cBioPortal comprised searching with thekeywords PanCancer sorting the obtained results fromhighest to lowest RNASeq examples and finally selectingthe thyroid skin stomach breast and lung datasetsAll five datasets are composed of approximately thousand features Each column feature in these datasetsrepresents a specific gene and the cell values for each column are the expression of that gene in a particular sampleAll the RNASeq data were normalized according to thedistribution based on all samples The expression distribution of a gene is estimated by calculating the mean andvariance of all samples with expression values and discarding zeros and nonnumeric values such as NA Nullor NaN which are substituted by NA [] With the fivedatasets we gathered examples of thyroid cancer of skin cancer of stomach cancer of breast cancer and of lung cancer We would like to emphasizethat this dataset is only a toy dataset since the data doesnot fairly reflect the immense difficulty associated withidentifying cancer in a real scenarioThe preprocessing pipeline was executed for each RNASeq dataset separately Firstly we removed the columnsthat had only one value throughout all samples Whena value is constant for all the examples there is noentropic value with no value variation one cannot inferany information In total and columns were removed on the thyroid skin stomach breast and lung datasets respectively By default weattributed the remaining missing values represented byNA in the dataset as observable in Table with the meanvalue of the column where the missing value is [] Further normalization was not applied in the data Finally weadded the Label column to link the instances to their typeof cancer when training the classifierSince we aim to distinguish several cancer variations wetest all cancers against each other assigning the positivevalue one to the class of interest and zero to the remainingones When detecting thyroid cancer all thyroid examplesare labeled as one and the skin stomach breast and lunginstances as zero and henceforwardAfter processing all the datasets it is improbable thatthe preprocessing phase removed the same columns in allof them To guarantee the same features describe all thesamples we intersect all the datasets and use the resultas our final dataset Also given that the breast cancerdatasets had almost the double of instances we applydownsampling and randomly select breast cancerexamples to keep the final dataset as evenly distributedfor all the cancers as possible In the end the resultingdataset has approximately instances and more than thousand genesData of features extracted from imagesWe use two datasets of two different diseases composedof features extracted from images malaria and breast cancer Since we aim to evaluate how well this methodologygeneralizes by using distinct types of data we are nowable to gather evidence supporting this premiseThe malaria dataset was created by the FraunhoferAICOS institution through the MalariaScope project[] Their main goal is to develop lowcost solutions thatcan provide fast reliable and accurate results on detecting such disease particularly in developing countries In[] the authors thoroughly describe the feature extraction process from thin blood smear images exclusivelyacquired with smartphones The resulting dataset is composed of samples and features These featureswere normalized between [ ] via scaling and groupedinto three main groups geometry color and textureFrom all the examples approximately contain malariaparasites Due to the high unbalance between Malariaand NonMalaria labels we performed downsampling onthe NonMalaria class where we randomly selected examples We decided to choose instead of dueto a wide variety of nonparasite artifacts Once the samples were selected and similarly to the preprocessing stepof the RNASeq data we verify if there are features withconstant values and remove them if that is the case Ourworking malaria dataset has instances negativeand positive and feature columnsThe Wisconsin Breast Cancer dataset [] from the UCIMachine Learning Repository is composed of examples and features These features are computed from afine needle aspirate digitized image of a breast mass anddescribe the cell nuclei characteristics present in thoseimages such as texture area concavity and symmetryFrom the examples approximately are benignsamples and are malign ones No under or oversampling techniques were applied since we do not find it to beneeded As performed in the malaria data we checked ifthere were columns with constant values for which therewere not The data was used as is with the proportionsand characteristics described aboveAutoencodersAn autoencoder AE [] is an unsupervised featurelearning neural network that aims to copy its input based 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of on a lower dimensional representation This type of architecture is able to extract features by reducing the dimension of its hidden layer [] which helps the AE to focuson capturing the essential features that best represent thedataLet the encoding and decoding functions of the AE be fand g parameterized on θe and θd respectively where θ θeª θd L being the loss function and J the cost function tobe minimized When learning the AE aims to find value θthatargminθJθ X LX gθdfθe Xpenalizing the reconstruction of the input given by ËX fθe X the more distinct ËX is the bigger the appliedgθdpenalty When training an AE we use Mean Squared ErrorMSE as the loss function and the Rectified Linear Unitsactivation function ReLU [] for all its layers Currentlyusing ReLU as activation is the default recommendationwhen training neural networks [] Similarly using MSEas the loss function is a fairly common practice present inthe literature when training AEs [ ]We use the AEs as a weight initialization technique[] since evidence supports that using unsupervised pretraining guides the learning towards basins of attractionof minima that support better generalization from thetraining dataset [] Thus we pretrained them beforeimporting the encoding part or all their layers to theclassification neural networkBasic autoencoder AEThe simplest AE has only one hidden layer This type ofAE learns through the optimization cost function presented in Eq With the combination of linear activationsReLU and the MSE loss function these AEs behave similarly to the Principle Component Analysis PCA method when trained with an MSE an AE learns the principalsubspace of the training data consequentially []Denoising autoencoder DAEA Denoising AE DAE [] aims not just to reproduce theinput but also to keep its information intact to undo theeffect of an intentional corruption process applied to theoriginal data Its cost function can be described byFig Overall pipeline of our experiments This figure illustrates the chosen metodology for our work Firstly we pretrain the autoencoders AEsbefore embedding them to the top layers of the classification network fullfilling either Strategy import only the encoding layers from the AE orStrategy import the complete AE Each of the full assembled architectures is then trained to detect one of the cancer types in the input dataThe training process can follow two different approaches regarding the imported weights of the AEs A fixing them or B allowing subsequentfinetune I represents the input layer E the encoding layerËI the output layer of the AE at the classification region of the network D represents thefully connected layer and O the output of the classifer 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of θfθe ËXJθ X LX gθdargminwhere ËX is a copy of the input X intentionally corruptedby a sort of noise [] To simulate a form of BernoulliNoise [] we apply a Dropout layer immediately after theinput layer where of the connections are randomly cutSparse autoencoderSimilarly to a DAE a Sparse AE SAE learning processalso has two main goals minimizing the reconstruction error when aiming to copy the input data and applying a sparsity penatly represented by 01 to theparameters involved in the encoding partJθ X LX gθdfθe X λ · 01θeargminθAlthough it also tries to reproduce X an SAE canaddress unique statistical features of the dataset it hasbeen trained on [ ] To deliver that sparsity elementwe use an L1 penalty with a λ of MethodologyWe have adopted the methodology described in []which was also used in [] Our experiments consist ofan analysis of the performance of a DNN trained to classify different cancer types studying how three differentfactors may impact the network performance The top layers where we use three different AEs asweight initialization The dimension of the latent vector of the AEs thatmeans the encoding layer size The imputation technique to replace missing datawhen preprocessing the datasetsBesides the top layers imported from the AE the classification part of the full architecture is composed of aBatch Normalization layer [] followed by two FullyConnected layers with a ReLU [] activation Since weaim to detect one type of cancer at the time the last layer the predictive one is a single neuron layer with aSigmoid nonlinearity [] This activation considers thatif the probability of the classification is lower than the sample is classified as negative that is not having thedisease otherwise the sample is classified as positiveTo assess the following experiments we decided to onlyuse the AE that achieved the best results in the firstexperiments For points and we try three different dimensions and For the data imputationstudy we use three strategies replacing the data witha the mean column value used as default a constantvalue in this case zero and b with the most frequentvalueFurthermore we want to study if when using Strategy importing the complete AE into the classification network the model yields better results just because it hasone more layer and therefore more parameters to trainTo observe if the classifier is better only by being deeperwe pretrained the AE and at the embedding step forStrategy we add a decoder layer with all its weightsrandomized guaranteeing that there are no discrepanciesconcerning the networks topological complexity for bothstrategiesFinally we want to assess how the pipeline behaveswhen dealing with different data types besides RNAseq entries Hence we apply the same methodologyto the image extracted features datasets described inThe data section to assess if the model can adapt andgeneralize well to these data characteristicsFor all these we follow the same pipeline see Fig Foreach experience we start by pretraining a different AE tominimize the reconstruction error before importing theminto the top of the classification architecture When doingso we choose one of the two strategies considered for thisstudy add just the encoding layers or add all thepretrained AE After the embedding of the AE to the toplayers we consider two different approaches in the training process A fixing the imported weights of the AElayers and B by allowing them to be finetuned duringthe model training for the classification taskWith the complete architectures AE as the top part ofthe classification network assembled we train each oneto distinguish¢ The RNASeq input data as one of cancers namely¢ The malaria input data as Malaria or NonMalaria¢ The breast masses input data as Malign or Benignthyroid skin stomach breast and lungEvaluationWe use stratified 10fold crossvalidation to ensure andprovide statistical evidence The AEs are trained during epochs and the classifier during with a batchsize of The classification model is trained with thebinary crossentropy loss function [] and with an Adamoptimizer [] Furthermore we assess the overall performance of the model in the training and validation setsby analyzing five more metrics Accuracy Matthews Correlation Coefficient MCC [] Precision Recall and F1score and provide the Receiving Operator Curve with therespective Area Under the Curve ROCAUC and thePrecisionRecall CurveFurthermore to study how the model generalizes tounseen data during the training phase we evaluate theperformance of the best architecture combination on aheldout test set for the Malaria and the Wisconsin BreastCancer datasets For both and separately we use a ratio ofone third to create two new splits Therefore 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Table Baseline results for cancer detection using a Fully Connected Neural Network the classification architecture without the AEas top layersThyroidSkinStom | 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 |
Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efï¬cacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the ï¬nal counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artiï¬cialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunoï¬uorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image ï¬ltering edge detection image expansion and contraction techniquesusing pythons CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and speciï¬city ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and speciï¬cityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the seeds shed from the primary tumor andor metastatic lesions and rooted in a new soiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic Whats more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with ï¬ow cytometry which is still thegold standard of CTCdetection Racila Howeverthis method that lack of the cancer speciï¬c markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunoï¬uorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes ï¬laments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identiï¬cation is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identiï¬cation and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a ï¬uorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression proï¬le Ligthart redeï¬ned the CTCs by computer algorithms after the manualcounting The stricter deï¬nition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classiï¬er was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classiï¬cation methods stillremain subjective as the rules are set artiï¬cially The ï¬xedconditions may not identify the morphologically heterogeneousCTCs integrally Whats more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artiï¬cial intelligence AI recent yearsmachine learning become an exciting ï¬eld for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the worlds ï¬rst internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood ï¬ow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors diagnosis This artiï¬cial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the ï¬eldof medical research speciï¬cally medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identiï¬cation Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artiï¬cial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The ï¬rst ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical leveltesting data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overï¬ttingEnrichment and imFISH Identiï¬cation ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was ï¬rst centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared ï¬xed and dried cells were used to perform the imFISHThe slides were ï¬xed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at ¦C The probe was eluted and the slides were washedtwice in SSC Then the CD45 ï¬uorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at ¦C After incubation CD45 ï¬uorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a ï¬uorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentï¬uorescent colors Usually manual counting is the goldstandard but its a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We ï¬rst selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image ï¬ltering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was deï¬ned as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs Identiï¬cationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artiï¬cial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasï¬rst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the ï¬rst ï¬ve layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe ï¬vefold cross validation was used to prevent overï¬ttingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The ï¬nal hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear AC The imFISH result of CEP8 CD45 and DAPI D The merge of panelsAC E The CTCs were identiï¬ed by CV segmentation method and marked in red box ac The CEP8 signal points were identiï¬ed by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workï¬ow is shown belowSp The grid was deï¬ned on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function softmaxReLU tanh kernel regularizer type l1 l2regularization factor The range of possible values were deï¬ned of eachdimension Allestablishing the best onethe possibleconï¬gurations weresearched forEvaluation Criteria for Classiï¬cationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassiï¬cation model such as sensitivity Se or recall speciï¬citySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 precision recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The ï¬rst ï¬ve layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix deï¬nitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artiï¬cial error and save costs we performedthe traditional image identiï¬cation method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was deï¬ned as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1AC the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identiï¬cation and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and speciï¬city were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identiï¬cation of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole ï¬owchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand speciï¬city were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and speciï¬city in CTC countingisslightly worse than the ï¬rst method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesnt cover the whole ï¬lm but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The ï¬owchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspeciï¬city in CTC counting We had changed the ï¬lter sizeand number in all convolution layers in order to ï¬nd thebest CNN parameters We found diï¬erent ï¬lter size andnumber will inï¬uence the results largely We changed ï¬lternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about We tried toincrease the ï¬lter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the ï¬lter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classiï¬cation Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more inï¬uence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened theï¬uorescence images according to the ï¬gures quality In order toimprove the eï¬ciency we used the machine instead of ngmanual screening First the pythons package was used to mage segmentation The obtained recognition sensitivity andspeciï¬city are and respectively In addition therecognition sensitivity and speciï¬city can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participantsof kin Written informed consent was obtained from theindividuals and minors legal guardiannext of kin for thepublication of any potentially identiï¬able images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the ï¬nal version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientiï¬c Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers Basel 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"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 |
"clinical development of immune checkpoint inhibitors ICIs therapy has ushered in a new era of antitumor therapy with sustained responses and significant survival advantages observed in multiple tumors mostpatients do not benefit Therefore more and more attention has been paid to the identification and developmentof predictive biomarkers for the response of ICIs and more indepth and comprehensive understanding has beencontinuously explored in recent years Predictive markers of ICIs efficacy have been gradually explored from theexpression of intermolecular interactions within tumor cells to the expression of various molecules and cells intumor microenvironment and been extended to the exploration of circulating and host systemic markers With thedevelopment of highthroughput sequencing and microarray technology a variety of biomarker strategies havebeen deeply explored and gradually achieved the process from the identification of single marker to thedevelopment of multifactorial synergistic predictive markers Comprehensive predictivemodels developed byintegrating different types of data based on different components of tumorhost interactions is the direction offuture research and will have a profound impact in the field of precision immunooncology In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs and discuss their future directions in achievingprecision immunooncologyKeywords Neoplasm Immune checkpoint inhibitor Predictive biomarker Tumor mutation burden Programmeddeath ligand1BackgroundImmune checkpoint inhibitors ICIs therapy has usheredin a new era of antitumor therapy with sustained responses and significant survival advantages observed inmultiple tumors Antiprogrammed cell death1programmed cell deathligand PD1PDL1 antibody hasbeen approved for secondline or firstline treatment in avariety of malignant neoplasms including melanoma lungcancer renal cell carcinoma RCC head and neck squamous cell carcinoma HNSCC and gastroesophageal Correspondence cuijwjlueducnCancer Center the First Hospital of Jilin University Xinmin StreetChangchun Jilin Chinacancer [ ] However despite the breakthrough in clinical treatment with ICIs most patients do not benefitPembrolizumab or nivolumab has an objective responserate ORR of in firstline melanoma and insecondline nonsmall cell lung cancer NSCLC []Therefore in recent years more and more attentions havebeen paid to the identification and development of predictive biomarkers for the efficacy of ICIs and more indepth and comprehensive understanding has also beenobtained in recent yearsincluding new data on biomarkers of tumor genome and neoantigen tumor immune microenvironmentbiopsybiomarkers hostrelated factors and all of which havephenotypeliquid 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 0cBai Biomarker Research Page of technologyimmunohistochemicalmade many new advances in the corresponding fieldsWith the development and continuous improvement ofmultiplexhighthroughput sequencing and microarray technology a variety of biomarker strategies have emerged and graduallyrealized the process from the identification of singlemarker to the development of multifactorial synergisticpredictive markers The development of predictive biomarkers contributes to revealing the therapeutic mechanisms of ICIs and the interaction mechanisms betweentumor and host immunity achieving decisionmaking ofindividualized antitumorimmunotherapy monitoringefficacy and disease development guiding clinical trial design as well as for further understanding of drug resistance mechanisms and tumor prognosis In this review wedeeply analyze the exploration course and research progress of predictive biomarkers as an adjunctive tool totumor immunotherapy in effectively identifying the efficacy of ICIs It should be pointed out here that when reading and collating we try to read and include all therelevant s In the process of selecting s we include the authoritative s published in highleveljournals or the latest research results and objectively describe and analyze their roles in this field as well as discuss the reasons that different research results may beinvolvedAdvances of multiple predictive biomarkers toICIs efficacyi Tumor genome and neoantigen biomarkersTumor mutation burdenSignificant correlations between high tumor mutationburden TMB and response to ICIs have been reported inseveral cancer types [] including urothelial carcinoma[] small cell lung cancer SCLC [] NSCLC []melanoma [] and human papilloma virus HPVnegative HNSCC [] A metaanalysis of cancer typesshowed that the mean response rate was positively correlated with log TMB [] The National ComprehensiveCancer Network NCCN guidelines have adopted TMBas the recommended test for patients with NSCLC receiving immunotherapy Although the results in some clinicalstudies of RCC [] HPVpositive HNSCC [] and melanoma receiving antiPD1 after recurrence [] showedthat TMB alone also did not clearly distinguish respondersand predict OS it is still exciting that multiple studies inthe American Society of Clinical Oncology ASCOmeeting have confirmed the predictive value of TMB inimmunization or combination therapy KEYNOTE061study [ ] CONDOR study [] EAGLE study []EPOC1704 study [] etc consolidating its status ofTMB as an independent predictor And in April theUS Food and Drug Administration FDA prioritized theapproval of TMB as a companion diagnostic biomarkerfor pembrolizumabNonetheless the cutoff values of TMB were defineddifferently across studies and assay platforms such asatezolizumab mtMb in urothelial cancer pembrolizumab mtMb in NSCLC and atezolizumab¥ ¥ or ¥ mtMb in NSCLC [] andnivolumab plus ipilimumab ¥ mtMb in NSCLC [] which needs further study to confirm the optimalcutoff value in different tumors Moreover the NGSpanels have approved by the FDA that can be used to estimate TMB include the MSKIMPACT and FoundationOne CDx panel the detection results of which arehighly consistent with whole exome sequencing WES[ ] and other solutions are under development Astudy detecting TMB cutoff value at mtMb in NSCLC patients with the FoundationOne platformcontaining a gene panel found that compared withTMBL patients overall survival OS and DCR was significantly improved in TMBH patients treated withantiPD1L1 drug [] Both WES and targeted NGS a422cancergene panel performed in patients withNSCLC treated with antiPD1L1 demonstrated thatTMBH population has a significantly better durableclinical benefitDCB and progressionfree survivalPFS [] These findings demonstrate the feasibility ofcomprehensive genomic profiling CGP but the designof optimal next generation sequencing NGS panel thatis more accurate comprehensive and costeffective isstill not clear In addition given that bTMB was identified as a predictor of PFS but failed to differentiate patients with OS benefits researchers consider the need toexplore other more precise factors eg allele frequencyAF A study that developed a new bTMB algorithm intwo independent cohorts POPLAR and OAK showedthat modified bTMB low AF bTMB LAFbTMB mutation counts with an AF was significantly associated with favorable HR 95CI p PFS HR 95CI p andORR p after immunotherapy but required tobe prospectively validated [] Finally static biomarkersare insufficient to accurately predict response due to thecomplexity of tumorimmune interactions A recent analysis of tumor genomewide dynamic detection in pretreatment and ontreatment melanomasfound thatpretreatment TMB was only associated with OS in untreated patients while early 4week ontreatment changein TMB ÎTMB was strongly associated with antiPD1response and OS in the entire cohort [] The detectionof ÎTMB is helpful for early evaluating the response totherapy of patient but its clinical usability limited by thedifficulty in obtaining tissue samples and high price whileliquid biopsy discussed below might better 0cBai Biomarker Research Page of In addition epigenetic changes are associated withTMB The latest study investigated the association between TMB and DNA methylation DNAm to explorepotential complimentary biomarkers for NSCLC immunotherapies The results showed that high TMBNSCLCs had more DNAm aberrance and copy numbervariations CNVs showing certain value in predictingefficacy such as HOX gene methylation status and TMB[] Thus the correlated exploration of epigenetics hasattracted more attention in recent years and liquidbiopsybased epigenetic studies may become a future research direction Exploration in Chinese NSCLC patientsshowed that NSCLCs with high TMB had DNAm aberrance and CNVs Some insertion and deletion indelmutations can lead to frameshifts and more immunogenic neoantigens [] In the pancancer analysis of cancer types evaluated in The Cancer Genome AtlasTCGA RCC had the highest indel mutation load andframeshift indel mutations were found to produce threetimes more candidate neoantigens per mutation thannonsynonymousnsSNVs[] Somatic copy number alterations SCNAs are another feature of the genomic landscape of tumors andpancancer TCGA analysis revealed an inverse correlation between SCNAs atthe singlearm or wholechromosomelevel and immune infiltration in tumortypes tested [] and this result was subsequently replicated in a larger study of TCGA []single nucleotide variantsDNA damage response pathwaysGenetic variation involved in DNA mismatch repairMMR pathway can lead to microsatellite instabilityMSI a specific type of high TMB tumors and increased numbers of CD8 tumor infiltrating lymphocytesTILs PD1TILs and indoleamine 23dioxygenaseIDO tumor cells have been shown in MMR deficiencydMMR colorectal cancer [] Recently five clinical trials Keynote016 including multipletumor types have shown that patients with dMMRMSIH can achieve durable responses to pembrolizmabBased on this pembrolizumab is approved by the USFDA for the treatment of any advanced solid tumor withdMMRMSIH and nivolumab in combination with ipilimumab has also shown promising response in dMMRMSIH colorectal cancer [] In addition dMMR canalso cause mutations in the DNA polymerase gene epsilondelta POLEPOLD1increasing the mutationload and neoantigen load Analysis of POLEPOLD1mutations in patients with different cancer typesshowed that patients with these mutations had significantly higher TMB and OS Therefore it may be an infordependentInidentifying patients who benefitaddition pathways of base excision repairBERand prognostic markerfrom ICIs []risk factorhomologous recombination repair HRR MMR in theDNA damage response DDR signaling network contribute more significantly to TMB or neoantigens whichhave the highest levels when comutated [] It hadbeen identified that comutations in the DDR pathwaysof HRR and MMR or HRR and BER defined as comutare associated with increased levels of TMB neoantigenload and immune gene expression signatures Comutpatients showed a higher ORR and longer PFS or OS indicating that comut can be used as predictors of response to ICIs and provide a potentially convenientmethod for future clinical practice []Specific mutated gene pathways in tumor cellsIt is worth noting that alterations of signaling pathwaysin tumor cells affect the responsiveness to immunotherapy Patients with mutations in the interferon IFNγpathway genes IFNGR12 JAK12 and IRF1 are poorlyresponsive to ICIs treatment and confer resistance []A study found that in patients receiving immunotherapytumor cells can downregulate or alter IFNγ signalingpathways such as lossoffunction alleles of genes encoding for JAK12 and changes in STAT1 to escape the influence of IFNγ [] resulting in poor efficacy andresistance Recent studies suggest that inactivating mutations in a mammalian analog of the chromatin remodeling SWISNF complex and unique genes of the PBAFcomplex Pbrm1 Arid2 and Brd7 lead to sensitivitiesto ICIs [ ] Loss of function of the PBAF complexincreased chromatin accessibility to transcription regulator elements of IFNγinducible genes within tumorcells and subsequently increased production of CXCL9CXCL10 chemokines leading to more efficient recruitment of effector T cells into tumors [] In human cancers expression of Arid2 and Pbrm1 are related toexpression of T cell cytotoxicity genes which confirmedin Pbrm1deficient murine melanomas with strongly infiltrated by cytotoxic T cells and responsive to immunotherapy [ ]In addition doublestranded RNAdsRNA editing enzyme adenosine deaminase acting onRNA ADAR1 protein can block the IFNγ signalingpathway and lead to poor ICIs efficacy and resistanceLoss of function of ADAR1 in tumor cells can reduce AtoI editing of interferoninducible RNA species and leadto dsRNA ligand sensing by PKR and melanomadifferentiationassociated protein MDA5 This resultsin growth inhibition and tumor inflammation respectively and profoundly sensitizes tumors to immunotherapy [] Finally demethylation positively regulates thetranscriptional activity of some immunerelated genesincluding PDL1 and IFN signaling pathway genes sensitizingto anticytotoxic Tlymphocyteassociatedprotein4 CTLA4 therapy []it 0cBai Biomarker Research Page of In addition to the IFNγrelated signaling pathway alterations in other tumor genome such as tumor oncogenes and suppressor genes pathways and pathwaysrelated to tumor cell proliferation and infiltration canalso affect immunotherapy efficacy Epidermal growthfactor receptor EGFR and anaplastic lymphoma kinaseALK mutations have been shown to be associated withreduced response rates to ICIs and low TMB and therefore the FDA does not recommend firstline ICIstreatment in patients with EGRF or ALK positive tumors[ ] certain types of mutations in MDM2MDM4and ARID1A can predict nonresponse to ICIs in highTMB tumors [] NSCLC with KRAS and STK11 comutated was associated with reduced response andshorter survival in three independent cohorts of patientstreated with antiPD1 therapy [] and STK11 deficiency was an independent indicator of poor antiPD1response in NSCLC with KRAS mutant however at the American Association for Cancer Research AACRmeeting of patients in the Keynote042 studyNCT02220894 update data were tested for STK11 andKEAP1 and the results showed that patients could benefit from pembrolizumab regardless of STK11 and KEAP1status but patients with STK11 mutations did not respond well to chemotherapy but given that only ofall patients had mutation detection the results may beaffected in initial data from studies using targeted NGSpanels suggested that duration of ICIstreatment was associated with certain BRAF and MET alterations butnot TMB status [] NOTCH signaling pathway is associated with the occurrence development and prognosisof tumors especially with the biological function of cancer stem cells Recent breakthrough findings have distinguished deleterious NOTCH mutation showing that itcan be used as a potential predictor of favorable ICI response in NSCLC potentially via greater transcription ofgenes related to DNA damage response and immune activation [] Another tumorspecific inheritance thatmay influence ICIs efficacy is the aberrant expression ofendogenous retroviruses ERVs Pancancer analysisidentified a positive correlation of transcript expressionof ERVs with Tcell activity in various tumors [] andpatient prognosis [] Furthermore with the improvement of precision detection technology the accurateanalysis of negative mutation sites helps to identify thepossibly effective ones For example the analysis of studydata of secondline PD1L1 inhibitor therapy found thatthe mPFS of patients with KRAS G12C or G12V was significantly better than that of patients with KRAS mutations at other sites []In addition several pancancer biomarkers are recentlyapproved by the FDA For example given the effectiveORR of and a disease control rate DCR of in secondline cholangiocarcinoma patients treated withanalysispemigatinib a new targeted therapy the recent FDA approval of pemigatinib for the treatment of previouslytreated patients with locally advanced or metastatic cholangiocarcinoma with fibroblast growth factor receptor FGFR2 fusion or rearrangement and the comprehensive genomicassay FoundationOne CDxdeveloped by Foundation Medicine as a companiondiagnostic Also exciting is the recent FDA approval ofthe targeted anticancer drug capmatinib for the treatment of metastatic NSCLC with MET exon skippingMETex14 mutations including firstline patients andpreviously treated patients also using FoundationOneCDx as a companion diagnostic to help detect specificmutations present in tumor tissueimmunogenicity ofNeoantigen loadNeoantigen load the number of mutations actually targeted by T cells may be directly related to the responseto ICIs [] A retrospective study showed thatclonal neoantigen burden was associated with the longerOS in primary lung adenocarcinomas p []Traditionallycomputational neoantigen predictionshave focused on major histocompatibility complexMHC binding of peptides based on anchor residueidentities however neoantigen loads identified by thismethod are generally not superior to overall TMB inpredicting ICIs efficacy or survival [] In recent practice this neoantigen can be assessed by the difference inpredicted MHCI binding affinity between the wildtypepeptide and the corresponding mutant peptide knownas the differential agretopicity index DAI reflectingclinically relevanttumor peptide[] A high DAI value indicates that the mutant peptidesignificantly increases binding affinity to MHC compared to the wildtype sequence and can generate moreimmune responses Studies on previously published cohorts treated with three ICIs have shown that DAI outperforms TMB and the traditionally defined neoantigenload in predicting survival [ ] In additionlowneoantigen intratumour heterogeneity might also be important for ICIs response Analysis of the lung adenocarcinoma TCGA database found that combining highmutational load and low intratumoral neoantigen heterogeneity was significantly associated with OSand longer lasting clinical benefit than either variablealone [] Anotherreported method for assessingneoantigen foreignness is based on sequence homologyof experimentally validated immunogenic microbial epitopes in the Immune Epitope Database IEDB [] butit does not account for all possible human leukocyteantigen HLA contexts In addition the detection forneoantigen can be reflected from different levels such aspeptides or genomes A study developed the Neopepseealgorithm using a machine learning approach incorporating 0cBai Biomarker Research Page of integration of nine immunogenicity features and gene mutation expression levels [] and its application to melanoma and leukemia patients could improve the sensitivityand specificity of neoantigen prediction Recently it has alsobeen shown that promoter hypermethylation of neoantigengenes may be an important mechanism for immune editingand tumor immune evasion [] indicating that combineddetection of tumor genome and epigenetics may providemore information for immunotherapy efficacyii Tumor immune microenvironment phenotypebiomarkerscells is also considered separately as one of the biomarkersto distinguish the benefit population called immune positive score IPS Herbst [] showed that response toatezolizumab treatment was significantly associated withhigh levels of PDL1 expression on the surface of TILs before treatment but not with PDL1 expression on tumorcells p Finally other inhibitory immune pathways may affect the response to ICIs therapy including Tcelllymphocyte activationgene3 LAG3 and Vdomain Ig suppressor of Tcell activation VISTA which can be used as potential biomarkers for ICIs responseimmunoglobulin3 TIM3PDL1 expressionGiven that multiple studies in a variety of tumors havedemonstrated a positive correlation between PDL1 expression and response to ICIs or OS even in firstlinecombination therapy [] pembrolizumab is currently approved by the FDA for use in patients with PDL1 PDL1 ¥ of tumor cells in firstline treatmentand ¥ in secondline treatment NSCLC and PDL1immunohistochemistry IHC as a companion diagnosticfor antiPD1 therapy in NSCLC patients [ ] However some studies have not detected a significant correlation between PDL1 expression and response to ICIs[ ] and PDL1 negative patients can still benefitclinically with treatment with ICI or combination treatment with ICIs [] with ORRs ranging from to Therefore PDL1 cannot yet be a comprehensive and independent biomarker in clinical practice in assessing efficacy with following challenges still existing FirstlyPDL1 assay and antibody are not standardized []Secondly PDL1 expression is temporally and spatiallyheterogeneous [] A study of metastatic NSCLCtreated with ICIs showed that PDL1 varies substantiallyacross different anatomic sites and during clinicalcourse being highest in adrenal liver and lymph nodemetastases and lower in bone and brain metastases Andthe predictive value of PDL1 at different biopsy sites forthe benefit of ICIs in NSCLC may vary higher PDL1 inlung or distant metastasis specimens was significantly associated with higher response rate PFS and OS whilePDL1 in lymph node metastasis biopsy was not associated with either response or survival [] Thirdly positive score and cutoff value of PDL1 expression is notstandardized [] At present PDL1 positive scoremainly focuses on the PDL1 expression level of tumorcells that is tumor proportion score TPS But PDL1is also expressed on immune cells such as lymphocytesand macrophages and stromal cells thus the investigators introduce the concept of combined positive scoreCPS which is the proportion score of the sum of PDL1 expressed by tumor cells and tumorassociated immune cells In addition PDL1 expression on immuneresponseto ICIsimmunetreatmentBiomarkers of tumorinfiltrating immune cellsOverall immune status of tumor microenvironmentThe pattern of tumor immune infiltration can be broadlyclassified into immuneinflamed immuneexcluded andimmunedesert [] Immuneinflamed is characterizedby the presence of CD8 and CD4 T cells in the tumorparenchyma accompanied by the expression of immunecheckpoint molecules [] indicating a potential antitumor[]immuneexcluded is characterized by the presence ofdifferent immune cell types in the aggressive margin orstroma of tumor but cannot infiltration into tumor parenchyma [ ] Analysis of pretreatment samples forantiPD1PDL1 revealed a relatively high abundance ofCD8T cells at the invasive margin in responders andserial sampling during treatment showed an increasedinfiltration of CD8T cells into tumor parenchyma []while immunedesert phenotype is characterized by theabsence of abundant T cells in the parenchyma orstroma of tumors and poor response to ICItreatment[] Recentlyimmunoscore has been proposed as avalid marker for characterizing the immune status oftumor microenvironment TME classifying tumors aswell as predicting treatment response and prognosis[] which involves the density of two lymphocyte populations CD8 and memory [CD45RO] T cells in thecenter and invading margin of tumor [] Mlecnik et al[] evaluated immunoscore in specimens of stageIIV colorectal tumor and confirmed that it was significantly associated with PFS DFS and OS and multivariate analysis also showed the superiority of immunoscorein predicting disease recurrence and survival The valueof immunoscore to predicting ICIs efficacy is being validated internationally in clinical trials of melanoma andNSCLC []A wider assessment of active immune responses withinTME by immune gene expression profiling might effectively predict clinical benefit to ICIs strategies Analysisof total RNA and genes that were substantially differentbetween the patient groups in pretreatment tumor biopsies revealed atleast a 25fold increase in the 0cBai Biomarker Research Page of expression of immunerelated genes in clinically active patientsincluding cytotoxic T cell markers egCD8A perforin granzyme B Th1 cytokines or chemokines MHCII and other immunerelated genes egNKG7 IDO1 [] Ascierto [] screened morethan immunerelated genes in patients with recurrent breast cancer years after treatment and thosewithout recurrence more than years later and foundthat five genes IGK GBP1 STAT1 IGLL5 and OCLNwere highly overexpressed in patients with recurrencefree survival In addition IFNγinduced immune genesignatures may be effective biomarkers for predicting theclinical benefit of treatment with ICIs The study developed IFNγ scores combining multiple immune variablesbased on gene signatures which were then extendedto gene signatures in a validation set of melanomapatients including genes encoding IFNγ granzymes AB perforin IDO1 and other immunerelated genesBoth gene scores showed significant associations withbest overall response rate and PFS Optimized cutoffvalues for IFNγ scores based on receiver operatingcharacteristic curve ROC curve can achieve a positivepredictive value of for responders and a negativepredictive value of for nonresponders []Immune cells with specific phenotypes in TMEThe phenotype of TILs also influences the efficacy ofICIs The study used singlecell mRNA sequencingscRNAseq data analysis to identify two major CD8Tcell phenotypes within melanoma memorylike andexhausted [] the proportion of which is strongly correlated with response to ICIs The research furtherfound that the transcription factor TCF7 is selectivelyexpressed in memorylike T cells so the ratio ofCD8TCF7 to CD8TCF7TILs is strongly correlatedwith improved response and survival in melanoma patients treated with antiPD1 [] Balatoni []found that of immune cells in TME were positivelyassociated with OS after treatment including CD4 andCD8 T cells FOXP3 T cells CD20 B cells CD134and CD137 cells and NKp46 cells and different immune cells at different sites were differently associatedwith clinical outcomes Researchers found that only asmall proportion of CD8 TILsin tumors couldrecognize tumor mutationassociated antigens while another population bystander cells was insensitive anddifferential CD39 expression was the key molecule thatdistinguished the two populations [] Analysis of peripheral blood from a patient with colorectal cancer whoresponded rapidly to pembrolizumab treatment showedhigh expression of CD39 on CD8 TILs indicating thatCD39CD8TIL may be a promising predictive biomarker [] The fact of very low level of CD39 expression on CD8TILs in of EGFRmutant NSCLC isconsistent with their low response rate to antiPD1immunotherapyIn addition a study showed that Fc domain glycan ofthe drug and Fcγ receptor FcγR expressed by the hostbone marrow cells could determine the ability of PD1tumorassociated macrophages TAMs to capture antiPD1 drugs from the surface of T cells which leads toPD1 inhibitor resistance [] and the association ofTAMs and poor antiPD1 response was reported inmelanoma cohorts [] antiPD1 response was associated with an increase in CD8T cells and natural killercells NK cells and a decrease in macrophages [] andhigh intratumoral myeloid markers were associated witha nearly 6fold decrease in mPFS after antiPDL1 therapy in RCC emphasizing the inhibitory role of myeloidcells in response to ICIs [] In conclusion immunecells in TME show a great promise in the developmentof predictive biomarkers for ICIsimmunerepertoireDiversity of immune repertoires in TMEEffective T cell responses involve the activation and expansion of specific antigenreactive T cell clones so diversity ofin intratumoral orperipheral may correlate with ICIs responses and can bequantified as richness and clonality [] However theresults seem to be complex with some studies finding apositive correlation between TIL clonality and the response to ICIs before [] or after [] treatment whileothers showing that only an increase in TIL clonalityduring treatment is associated with the response to antiPD1 [ ] others show that intratumoral T cellclonality is not associated with survival while peripheralT cell clonality is inversely associated with PFS and OS[] Tumeh [] further investigated whetherbaseline TILs have a narrow T cell receptor TCR repertoire focusing on tumorspecific immune responsesand whether this narrow TCR repertoire correlates withpembrolizumab responses They found that respondingpatient had more restricted usage of the TCR beta chainie a more clonal less diverse population than patientswith progressive disease and showed a 10times increasein these clones after treatmentimplying a tumorspecific response to treatment in these patients Notablybaseline TCR clonality was not highly correlated withTIL density suggesting that some patients with restricted TCR clonality specific for tumor antigens maystill benefit from antiPD1 therapy even though TILdensity is low Recently researchers have proposed theimmune repertoire IRIndex the average frequency ofshared TCR clones in T clones in TILs and peripheralPD1CD8 T cells They found that neoantigenstimulated TCR agreed with IRIndex and patients withhigh IRindex had better immune activation and highergene expression profiles GEPs score subsequently they 0cBai Biomarker Research Page of confirmed the predictive value of IRindex to ICIs efficacy DCRPFS But considering that it is difficult tosort out PD1CD8 T cells in tumor tissue based ontwo separate patient cohorts a research confirmed thatTCR repertoire diversity and clonality of peripheral PD1CD8T cells may serve as noninvasive predictors ofclinical outcomes after ICIs in patients with NSCLC[] The viewpoints of T cell diversity and TCR clonality as markers of ICIs efficacy need to be further validated in a large patient populationiiiLiquid biopsy biomarkersPeripheral blood cell biomarkersPeripheral blood is a noninvasive source to explore potential biomarkers for ICIs and although associationswith clinical benefit and survival have been observed itseffectiveness has not been validated in prospective studies Analysis of melanoma treated with ipilimumabshowed that improved OS and PFS were associated withbaseline values of peripheral blood components including low absolute neutrophil countlow neutrophiltolymphocyte ratio NLR low absolute monocyte countlow frequency of myelogenous suppressor cells high frequency of FoxP3 Treg cells high lymphocyte frequencyhigh eosinophil count and clinical benefit also associated with the dynamic changes of blood markers duringincluding decreased FoxP3Treg concentratreatmenttions and increased lymphocyte and eosinophil counts[] Reports in patients with melanoma treated withpembrolizumab and in patients with NSCLC treatedwith nivolumab have shown that NLR is associated withworse tumor response [ ] Multivariate analysis inmelanoma patients treated with antiPD1 antibodiesshowed that NLR was the only factor associated withworse ORR and shorter PFS indicating that NLR is astrong predictor of worse outcome in patients treatedwith ICI [] Low baseline lactate dehydrogenase LDHlevels high relativeabsolute eosinophil counts and relative lymphocyte counts were associated with prolongedOS in antiPD1 and CTLA4 treated melanoma [] Given that previous studies have proposed the importance of baseline derived NLR dNLR and LDHlevels as prognostic markers a recent study proposed acomposite prognost | Thyroid_Cancer |
"analyse the quality of information included in websites aimed at the public on COVID19Methods Yahoo Google and Bing search engines were browsed using selected keywords on COVID19ï¬rst websites from each search engine for each keyword were evaluated Validated tools wereThe used to assess readability [Flesch Reading Ease Score FRES] usability and reliability LIDA tool andquality DISCERN instrument Nonparametric tests were used for statistical analysesResults Eightyfour eligible sites were analysed The median FRES score was range 00 Themedian LIDA usability and reliability scores were range 00 and 37range14 00 respectively Alow overall LIDA score was recorded for n of the websites The median DISCERN score 14 was found in websites The DISCERNwas range The DISCERN score of score was significantly associated with LIDA usability and reliability scores p and the FRES scorep Conclusion The majority of websites on COVID19 for the public had moderate to low scores with regardsto readability usability reliability and qualityPractice Implications Prompt strategies should be implemented to standardize online health informationon COVID19 during this pandemic to ensure the general public has access to good quality reliableinformationï¾ Elsevier BV All rights reserved Introductionfor The coronavirus COVID19 pandemic has become the greatestglobal health crisis of the st century [] During this pandemicthe demand information on COVID19 has skyrocketedInformation such as latest news updates on the pandemic itssymptoms prevention and mechanism of transmission are highlysought by the public [] On the other hand free access toinformation especially through social media which is accessed bythe majority [] has led to an increase in misinformation and panicassociated with COVID19 [] Although high quality healthinformation is known to be related to lower stress levels andbetter psychological health [] previous studies have shown thatonline information on many medical disorders to be of substandard quality []A previous study done on websites related to COVID19 hasreported substandard quality information that could potentiallymislead the public [] However this study has used a limitedsearch strategy and had not assessed some important areasincluding usability and reliability of the information Thereforethis topic remains a knowledge gap in COVID19 research []Therefore we conducted this study to analyse the current COVID websites targeting the general public in terms of qualityusability readability and reliability using a wide search strategyand validated instruments MethodsAbbreviations USA United States of America FRES Flesch reading ease scoreHONcode Health on the net Code of Conduct SPSS Statistical package for socialsciences NICE National Institute for Health and Care Excellence WHO WorldHealth anization Corresponding author at Department of Surgery Faculty of MedicineUniversity of Colombo PO Box Kynsey Road Colombo Western ProvinceSri LankaEmail addresses ravindrijayasinghegmailcom R Jayasingheranasigmcgmailcom S Ranasinghe umeshejayagmailcom U Jayarajahsanjeewasrgcmbaclk S SeneviratneYahoo Google and Bing were searched using the keywordssevere acute respiratory synnovel coronavirusSARSCoV2 and coronavirus The searchdrome coronavirus2 COVID19was performed during the ï¬rst week of May The details ofthe search strategy and the piloting process are provided in thesupplementary material File S1 []Two independent investigators with previous experience ofconducting similar studies assessed the selected websites []Prior to the assessment a pilot run was conducted to ensure101016jpec20200800107383991ï¾ Elsevier BV All rights reservedPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxxxxxuniformity and accuracy The information on symptoms investigations public health measures and available treatmentmodalities were collected The accuracy of the content wasassessed using the national institute for health and care excellenceNICE guidelines on COVID19 [] A rating was given as all ornone based on congruence with the guidelinesValidated instruments were used to assess the quality ofwebsites Readability was assessed using the Flesch Reading EaseScore FRES [] The LIDA Instrument 2007Version12 wasused to analyse the content and the design of the websites usingthe usability and reliability domains [] The quality of thecontent was assessed using the DISCERN questionnaire which has questions in two separate groups [] The detailed assessmentcriteria and the scoring system is included in the supplementarymaterial File S1A website was classiï¬ed as governmental if it was maintainedby the countrys public health authority If managed by privateinstitutions nongovernmental anizations or voluntary institutions independent from the government they were consideredas nongovernmental The online healthrelated websites arestandardized in terms of their credibility and reliability by onlinecertiï¬cation sites We chose the Health on the Net code of conductHONcode which is the oldest and widely used out of the qualityevaluation tools available []Data analysis was performed using SPSS Version20 softwareand the associations were determined with non parametric testsA pvalue of was considered statistically significant ResultsOf the retrieved websites were excluded and thein the analysis Theremaining websites were characteristics of the websites are mentioned in Table included Half were governmental websites and only n were HON accredited websites The median FRES was range 00 10th12th grade readability level which is classiï¬ed asfairly difï¬cult to read Only three websites had a readabilityscore of above equivalent to 7th grade which is therecommended standardThe overall median LIDA score was range whilethe median LIDA usability and reliability scores were range 00 and range 00 respectively The median DISCERNscore was range which classiï¬es websites as being offair quality Excellent Good Fair Poor Very poor However the top websitesTable A were of excellent qualityTable Website characteristicsWebsite Characteristics Frequency PercentageUsability Governmental websites Notforproï¬t and private websites HONcode accredited Readability score of above equivalent to 7th grade Readability Date of publication stated References mentioned Disclosure statement by authors Infographics Moderate Low Moderate 00 Low score Moderate 00 Low Used Textonly Reliability Table Correlation between DISCERN scores and other factorsDISCERN SCORELow Mean High 00 Mean Range 00 00 00 00 Range 00 00 00 00 N P valueP0001P0001P0001P P P LIDA Usability LIDA Reliability LIDA Overall FRES Score Government HON Certiï¬cation No N No Yes Yes Signiï¬cant correlations were observed between the DISCERNscore and the overall LIDA score as well as LIDA usability andreliability scores Table p HONcode certiï¬ed websitessites obtained significantly higher DISCERN scores p Pertaining to the currency of information only publishers stated the date of the publication Most websites n did not declare the sources of evidence This was furtherestablished by the median reliability score of Nevertheless the authors have included a disclosure statement in mostn websiteslowFigures A1 and A2 summarize the rating of websites onindividual criteria assessed by the DISCERN tool The speciï¬cinformation provided regarding COVID19 is shown in Fig More than half of the websites failed to discuss the treatmentoptions available n beneï¬ts or risks n and effects of no treatment n Furthermore potentialcomplications and prognosis were stated only in and websites respectively Discussion and conclusion DiscussionThis study has shown that still most of the websites on onlinehealth information on COVID19 are of suboptimal quality exceptfor a few credible sources of goodquality health informationNevertheless the websites ranked among the top according tothe DISCERN score Table A2 had high scores indicating thepotential for publishing credible highquality information onlinewhich would beneï¬t the publicin turn causes panic which ranges Misinformation is a major concern during this pandemic aspeople fail to spend adequate time to critically analyse the onlineinformation This fromhoarding medical supplies to panic shopping and using drugswithout prescription with negative social and medical consequences [] Therefore measures implemented to ensure the qualityand accuracy of online information by the responsible authoritiesmay help negate these adverse consequencesinformation Stating the methods of content production with names of thecontributing authors may help increase the credibility of onlinehealth information while displaying the date of the publicationprovides an idea of the currency of the information Absence ofin over half of the websites was a majorsuch drawback especially for COVID19 where new information isgenerated almost daily Health authorities should therefore ensurethat the patient information websites provide the above information and certify websites based on such details so that the publiccan get information from trusted sources []Most users of the worldwide web only have an average level ofeducation and reading skills [] Guidance from the NationalPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxxxxx Fig Website characteristics evaluated outside the DISCERN toolInstitute of Health NIH had shown that the readability should bebelow the level of seventh grade for the lay public to adequatelyunderstand the content [] However the median readabilitylevel was found to be equivalent to 10th12th grade readabilitySuch complexities with the readability of information mayincrease the risk of misunderstandings or misinterpretation Usingshort sentences in writing using the active voice using 12point orlarger font size using illustrations and nontextual media asappropriate and accompanying explanations with examples wouldbe helpful to overcome this problem []So far only a limited number of studies have been done to assessthe quality of health information websites related to COVID19 Thestudy by CuanBaltazar et al prior to February reported poorquality information with approximately of included websiteswith low DISCERN scores [] Our study done three months latershows similar results with only a minimal improvement in thequality of information Furthermore the Cuan Baltazar study hadseveral limitations which includes the limited search strategy andnoninclusion of key quality parameters including readabilityFurthermore out of the sites they had includedwere online news sites that are not considered as patientinformation websites In that study the HONcode seal waspresent only in n websites whereas in our study ofthe sites were HONcode certiï¬edThere were several limitations in this study Although mostpopular search engines were used in this study under defaultsettings they may produce variable results depending on manyfactors including geographical location and popularity of websitesat a given point of time The algorithms unique to those searchengines are subjected to constant change and therefore the exactresults of our study may not be reproducible However we believethe general patterns observed in our study are validproviding health information to the general public are to be ofsubstandard quality Practice implicationsTo improve the credibility of the content the websites shouldstate methods of content production and display the date of thepublication to give an idea about the currency of the informationTo improve the readability of the content the websites shouldincorporate more nontextual media write in short sentencesusing the activevoice and use larger font sizes The patientinformation websites should display scores of reliability qualityand readability as a guidance for its users Furthermore it is vitalfor medical regulatory authorities and the government to imposeregulations to ensure quality and to prevent the spread ofmisinformationAvailability of data and materialsOn reasonable request from the corresponding author the dataused in the above study can be made availableEthics approval and consent to participateUnnecessary in this type of studyInformed consent and patient detailsNot applicable in this type of studyFundingNone ConclusionCRediT authorship contribution statementThis study has shown the quality readability usability andreliability of the information on COVID19 on majority of websitesRavindri Jayasinghe Conceptualization Methodology Datacuration Writing original draft Visualization InvestigationPlease cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0cModelGPEC No of Pages R Jayasinghe et al Patient Education and Counseling xxx xxxxxxValidation Formal analysis Resources Sonali RanasingheConceptualization Methodology Data curation Writing originaldraft Visualization Investigation Validation Formal analysisResources Umesh Jayarajah Conceptualization MethodologyData curation Writing original draft Visualization InvestigationValidation Formal analysis Resources Sanjeewa SeneviratneConceptualization Methodology Writing review editingSupervision Project administrationDeclaration of Competing InterestThe authors report no declarations of interestAcknowledgementsNone declaredAppendix A Supplementary dataSupplementary material related to this can be found in101016jversion at online the pec202008001References[] IJ Bes do Nascimento N Cacic HM Abdulazeem Novel Coronavirusinfection COVID19 in humans a scoping review and metaanalysis JClinical Med [] HT Le DN Nguyen AS Beydoun XTT Le TT Nguyen QT Pham NTK Ta QT Nguyen AN Nguyen MT Hoang Demand for health information onCOVID19 among Vietnamese Int J Environ Res Public Health [] C Wang R Pan X Wan Y Tan L Xu RS McIntyre FN Choo B Tran R Ho VK Sharma A longitudinal study on the mental health of general populationduring the COVID19 epidemic in China Brain Behav Immun [] CS Ho CY Chee RC Ho Mental health strategies to combat the psychologicalimpact of COVID19 beyond paranoia and panic Ann Acad Med Singapore [] C Wang R Pan X Wan Y Tan L Xu CS Ho RC Ho Immediate psychologicalresponses and associated factors during the initial stage of the coronavirus disease COVID19 epidemic among the general population inChina Int J Environ Res Public Health [] RH Waidyasekera U Jayarajah DN Samarasekera Quality and scientiï¬caccuracy of patientoriented information on the internet on minimallyinvasive surgery for colorectal cancer Health Policy Technol [] R Jayasinghe S Ranasinghe U Jayarajah S Seneviratne Quality of patientoriented webbased information on oesophageal cancer J Cancer Educ In press[] JY CuanBaltazar MJ MuñozPerez C RobledoVega MF PÃrezZepeda ESotoVega Misinformation of COVID19 on the internet Infodemiology studyJMIR Public Health Surveill 2020e18444[] BX Tran GH Ha LH Nguyen GT Vu MT Hoang HT Le CA Latkin CS HoR Ho Studies of novel coronavirus disease COVID19 pandemic a globalanalysis of literature Int J Environ Res Public Health [] G Eysenbach C Köhler How do consumers search for and appraise healthinformation on the world wide web Qualitative study using focus groupsusability tests and indepth interviews Brit Med J [] AS Prasanth U Jayarajah R Mohanappirian SA Seneviratne Assessment ofthe quality of patientoriented information over internet on testicular cancerBMC Cancer [] V Udayanga U Jayarajah SD Colonne SA Seneviratne Quality of thepatientoriented information on thyroid cancer in the internet Health PolicyTechnol [] National Institute for Health and Care Excellence Coronavirus COVID19 Accessed April wwwniceukcovid19[] Readable The Flesch Reading Ease and FleschKincaid Grade Level Accessed February readablecomblogtheï¬eschreadingeaseandï¬eschkincaidgradelevel[] Minervation The Minervation Validation Instrument for Healthcare WebsitesLIDA Tool Accessed February httpwwwminervationcomwpcontentuploads201104MinervationLIDAinstrumentv12pdf[] Minervation Is the Lida Website Assessment Tool Valid Accessed February httpwwwminervationcomdoeslidawork[] Discern Online The DISCERN Instrument Accessed February httpwwwdiscernukdiscern_instrumentphp[] E Fahy R Hardikar A Fox S Mackay Quality of patient health information onthe Internet reviewing a complex and evolving landscape Australas Med J [] J Kluger As Disinfectant Use Soars to Fight Coronavirus So Do AccidentalPoisonings Accessed April timecom5824316coronavirusdisinfectantpoisoning[] BX Tran AK Dang PK Thai HT Le XTT Le TTT Do TH Nguyen HQPham HT Phan GT Vu Coverage of health information by different sourcesin communities implication for COVID19 epidemic response Int J EnvironRes Public Health [] National Institutes of Health How to Write EasyToRead Health Materials Accessed April wwwscribdcomdocument261199628HowtoWriteEasyToReadHealthMaterialsMedlinePlus[] DM DAlessandro P Kingsley J JohnsonWest The readability of pediatricpatient education materials on the world wide web Arch Pediatr AdolescMed Please cite this in press as R Jayasinghe et al Quality of online information for the general public on COVID19 Patient Educ Couns 101016jpec202008001 0c" | Thyroid_Cancer |
This letter summarizes recommendations from the interdisciplinary working group of renal tumors IAGN of the German Cancer Society for the systemic treatment of advancedmetastatic renal cell carcinoma in the context of the current SARSCoV2 pandemicBackgroundDuring the SARSCoV2 pandemic oncologists faced several new challenges Jones et a0al Hanna et a0al Lai et a0al Data on SARSCoV2 in cancer patients suggested outstanding rates of severe clinical courses and mortality up to upon SARSCoV2 infection although methodological limitations of these observations are of concern Desai et a0al Liang et a0al Yang et a0al Zhang et a0al However oncological therapies might raise SARSCoV2related morbidity and mortality Desai et a0al Liang et a0al Yang et a0al Zhang et a0al Checkpoint inhibitors CPI could Jens Bedke and Viktor Gr¼nwald shared senior authorviktruenwaldukessendejensbedkemedunituebingende Jens Bedke Viktor Gr¼nwald Department of a0Hematology Hemostasis Oncology and a0Stem Cell Transplantation Hannover Medical School Hannover GermanyImmunonclogical COoperative Group ICOG of a0the a0Comprehensive Cancer Center Lower Saxoney Hannover CCCN Hannover Germany Genitourinary Oncology Department of a0Urology University Hospital Dresden Dresden Germany Department of a0Urology University Medicine Greifswald Greifswald Germanynegatively interfere with the pathogenesis of SARSCoV2 and an overlap of symptoms of SARSCoV2 and cancer treatment related adverse events can be assumed which could result in a diagnostic challenge Zhang et a0al Bersanelli Rotz et a0al In particular interstitial pneumonia from SARSCoV2 cannot be differentiated from CPIassociated pneumonitis with certainty using CT images and may present with overlapping features Zhang et a0al Bersanelli Pneumonitis in CPItreated patients are rare with an incidence von but accounts with treatmentrelated death rate of Wang et a0al Naidoo et a0al Effects of immune suppression due to supportive measure has also to be considered although only preliminary data exist on effects of steroids in cancer patients during the SARSCoV2 pandemic Yang et a0al UrologieAltstadtquartier Magdeburg Magdeburg Germany Department of a0Radiation Oncology West German Cancer Center University Hospital Essen of a0the a0University DuisburgEssen Essen Germany Urology and a0Urooncology Practice Berlin Vice Chairman duo Deutsche UroOnkologen Berlin Germany Department of a0Urology Eberhard Karls University Tuebingen HoppeSeylerStr a0T¼bingen GermanyInterdisciplinary Genitourinary Oncology WestGerman Cancer Center Essen Clinic for a0Medical Oncology and a0Clinic for a0Urology OZ1 University Hospital Essen A¶R Room Hufelandstrae a0Essen Germany Vol01234567891 0c Journal of Cancer Research and Clinical Oncology As a result use of CPI in advancedmetastatic renal cell carcinoma patients mRCC may be associated with a specific risk and requires a critical riskbenefit assessment Therefore choosing the appropriate therapeutic regimen in mRCC is more challenging than everSeveral recommendations focus on reducing the risk of SARSCoV2 exposures for patients as well as for medical staff as part of oncological routine and addresses mainly questions of resource allocation Discussion about treatmentspecific considerations remains scare Herein we report a consensus of the interdisciplinary working group on renal tumors IAGN of the German cancer society towards treatment of mRCCDuring the process of treatment evaluation the assessment of treatment indication must be made on an individual basis However once selecting treatment patient and tumorspecific parameters patient´s comorbidity and the availability of local caregiving facilities should be taken into account In our opinion two major scenarios according to systemic treatment have to be considered initiation or change medication of a systemic treatment upon progressing diseases and measures taken during ongoing systemic treatmentConsidering urgency once a0initiating treatment upon a0advanced or a0metastatic RCC For systemic treatment the appropriate treatment should be selected based on patientspecific factors that take the overall picture into account when searching for treatment Escudier et a0al The indication for the systemic treatment of mRCC should be made strictly taking active surveillance and deferred medical treatment into consideration Escudier et a0al This should minimize patient exposure to the atrisk medical care environment as well potentially towards particular treatmentassociated risk in terms of SARSCoV2 infection Patients who do not require urgent systemic treatment should therefore primarily be offered active surveillance and deferred medical treatment No validated tool that identifies indolent disease is established However various parameters are used by clinicians to identify the appropriate patient population for active surveillance Table a0 represents a number of such variables which are used by the authorsAlso considering the CARMENAdiscussion on palliative nephrectomy in asymptomatic synchronous metastasized patients cytoreductive nephrectomy should be considered critically respecting the fact of reported nosocomial SARSCoV2 infection in cancer patients as well as a surprising high rate of SARSCoV2 diseases among cancer patients receiving surgery Liang et a0al Zhang et a0al Table Clinical parameters to estimate the urgency of treatment need in patients with metastasized renal cell carcinoma UnfavorableParametersHigh tumor load polytopic Patientrelatedbone metastases andor multiple an systemsECOG Risk factors for secondary complicationsbFavorableOligometastasis glandular involvementa an system involved asymptomaticECOG Clear cell RCC TumorrelatedSarcomatoid subtype medullary subtype nonclear cell subtypesIntermediate and poor riskFavorable riskPrognosis according to the IMDCa Pancreas thyroid gland saliva adrenal glandsab Impending complications in the event of further progression eg bronchial compression pathological fracture cross section vascular erosionocclusionHow should I treat a a0patient with a0favorable riskSuitable systemic treatment is selected based on its marketing authorization principle recommendations as well as on published data The strengths and weaknesses of a treatment regime should be weighed against its suitability for the best possible application In order to minimize the treatment risk and if the clinical course is indolent the necessity for systemic therapy is low and active surveillance is our preferred option Restaging is recommended in a0months time Based on these considerations the feasibility of an active monitoring strategy or initiation of a therapeutic measure should be evaluatedIn light of CheckMate214 JAVELINRenal and Keynote426 studies no significant overall survival OS benefit was gained by any of the immune combinations for patients with a favorable risk profile HR for OS ipilimumabnivolumab vs sunitinib CI axitinibpembrolizumab vs sunitinib CI axitinibavelumab vs sunitinib CI Motzer et a0al KeytrudaEMEA Choueiri et a0al A major limitation of these data are the short followup duration which limits the data interpretation However at the current state there is no signal that a specific combination is superior in OS expectations when compared to single agent sunitinib This is supported by molecular findings wherein IMDC favorable risk is associated with proangiogenic dependency McDermott et a0al However a proportion of patients exert an inflamed tumor type which may identify a patient population with potential clinical benefit from 0cJournal of Cancer Research and Clinical Oncology Table Summary of the treatment recommendations for firstline treatment of metastatic RCC during the SARSCoV2 pandemicIMDC risk groupsFavorableIntermediateUnfavorable SelectionActive surveillanceAxitinib avelumabAxitinib pembrolizumabAxitinib avelumabAxitinib pembrolizumabIpilimumab nivolumabOptionTKIActive surveillance restaging in a0months cabozantinibIpilimumab nivolumabCabozantinibThe list is shown in alphabetical order When selecting treatment the individual patient and tumor characteristics listed in Table a0 should be consideredCPI treatment Today there is no clinical test available to identify such patients The best treatment strategy in these patients remains has not been defined and data is scarce The identification of indolent disease and the absence of a survival signal for combinations in this patient population should caution treatment intensification and puts active monitoring and single agent TKI as preferred treatment options in the treatment algorithm of this patient cohortHow should I treat a a0patient at a0intermediate or a0poor riskIn cases who exert favorable clinical parameters depicted Table a0 patients with intermediate risk may receive active monitoring and deferred medical treatment as an alternative strategy to upfront medical treatment However most patients require systemic treatment and clinical benefits outweighs risk of immunerelated adverse eventsSARS CoV2Patients with an intermediate or unfavorable risk profile had longer OS when taking immune combinations than patients taking TKI alone which is why they should be used preferentially [HR for OS ipilimumabnivolumab vs sunitinib CI axitinibpembrolizumab intermediate prognosis CI poor prognosis CI axitinibavelumab intermediate prognosis CI poor prognosis CI Cabozantinib vs sunitinib ] Motzer et a0al KeytrudaEMEA2020 Choueiri et a0al To minimize the risk of immunemediated adverse events as well as high dose steroids application due to immune related adverse events a careful patients based valuing is demanded to choose the best treatment regimen Table a0 Overall if clinical parameters in this cohort are favorable the TKI cabozantinib reflects a therapeutic option Otherwise an immune combination represents the preferred form of treatment in particular in poor prognosis patients wherein the clear harm of the mRCC and the oncological efficacy overweights the risk of pandemic associated concernsWhat to a0conclude in a0SARSCoV pandemic for a0mRCC Although precise guidelines according to mRCC treatment reflects best efficacy and QoL data within the SARSCoV2 pandemic a patientcentered treatment choice which is adapted to the local pandemic situation is warranted s Table a0 Reflecting SARSCoV2related comorbidity patient´s and tumor characteristic´s adverse events and hospitalization rates seem to be useful parameters to adjust riskbenefit ratio pandemicadapted mRCC treatment choice However expanding realworld register data will answer the question which concerns where the right one over or undertreatmentAcknowledgements Open Access funding provided by Projekt DEALCompliance with ethical standards Conflict of interest PI Receipt of grantsresearch supports Astra Zeneca BMS Bayer Lilly Merck Novartis EISAI Pfizer MSD AstraZeneca Roche Ipsen Receipt of honoraria or consultation fees Arbeitsgemeinschaft interdiziplin¤re Medizin Altana Health Service AstaZeneca Budosy BMS Bayer DKGOnkoweb ESAI EUSA IDInstitut Ipsen Impulze MedKom Acadamy Merck Medac MediSeminar MSD MTEAcadamy New Concept Oncology NGAAcadamy Novartis Pfizer Roche Roche StreamedUP Solution Acadamy Onkowissen Think Wired Participation in a company sponsored speakers bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense BMS Byer DKG EUSA Ipsen Novartis Merck PharmaMar CG Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees Astellas Astra Zeneca BMS EISAI IPSEN MSD Merck Serono Pfizer Participation in a company sponsored speakers bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Not applicable NK Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees Astellas BMS EISAI EUSA IPSEN Jansen MSD Novartis Pfizer Participation in a company sponsored speakers bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Aristo BMS Janssen EUSA Pharma MSD TG Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees Astra Zeneca BMS EISAI IPSEN MSD Merck Serono Novartis 0c Journal of Cancer Research and Clinical OncologySanofiAventis Pfizer Roche Participation in a company sponsored speakers bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Not applicable MJ Receipt of grantsresearch supports Not applicable Receipt of honoraria or consultation fees BMS Bayer EISAI EUSA Hexal Jansen Merck Serono Medac MSD Pfizer Roche Participation in a company sponsored speakers bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Astellas Bayer Pfizer JB Receipt of grantsresearch supports inst Eisai Ipsen MSD Novartis Roche Pfizer Receipt of honoraria or consultation fees AstraZeneca Astellas BMS Eisai Ipsen MSD Novartis Roche EUSA Pharma Nektar Pfizer Participation in a company sponsored speakers bureau Not applicable Stock shareholder Not applicable Spousepartner Not applicable Other support please specify Travel Expense Inst BMS Ipsen Pfizer Roche VG Receipt of grantsresearch supports Astra Zeneca BMS MSD Ipsen Novartis Pfizer Receipt of honoraria or consultation fees Astra Zeneca BMS MSD Ipsen Novartis pfizer Merck Serno EUSAPharm EISAI Roche Bayer JanssenVilag Lilly PharmaMar Participation in a company sponsored speakers bureau Not applicable Stock shareholder Astra Zeneca BMS MSD Spousepartner Not applicable Other support please specify Travel Expense Astra Zeneca BMS Ipsen Pfizer BayerOpen Access This article 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 article are included in the articles Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the articles 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 sesby40ReferencesBersanelli M Controversies about COVID19 and anticancer treatment with immune checkpoint inhibitors Immunotherapy https doi102217imt20200067Choueiri TK Hessel C Halabi S Sanford B Michaelson MD Hahn O et a0al Cabozantinib versus sunitinib as initial therapy for metastatic renal cell carcinoma of intermediate or poor risk Alliance A031203 CABOSUN randomised trial PROGRESSIONfree survival by independent review and overall survival update Eur J Cancer [Internet] Choueiri TK Motzer RJ Rini BI Haanen J Campbell MT Venugopal B et a0al Updated efficacy results from the JAVELIN Renal trial firstline avelumab plus axitinib versus sunitinib in patients with advanced renal cell carcinoma Ann Oncol https doi101016jannon c202004010Desai A Sachdeva S Parekh T Desai R COVID19 and cancer lessons from a pooled metaanalysis JCO Glob Oncol Escudier B Porta C Schmidinger M RiouxLeclercq N Bex A Khoo V et a0al Renal cell carcinoma ESMO clinical practice guidelines for diagnosis treatment and followup Ann Oncol [Internet] Hanna TP Evans GA Booth CM Cancer COVID19 and the precautionary principle prioritizing treatment during a global pandemic Nat Rev Clin Oncol Jones D Neal RD Duffy SRG Scott SE Whitaker KL Brain K Comment Impact of the COVID19 pandemic on the symptomatic diagnosis of cancer the view from primary 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followup of a randomized phase clinical trial J Immunother Cancer 82e000891 https doi101136jitc202000089 Rotz SJ Leino D Szabo S Mangino JL Turpin BK Pressey JG Severe cytokine release syndrome in a patient receiving PD1directed therapy Pediatr Blood Cancer https doi101002pbc26642 Wang DY Salem JE Cohen JV Chandra S Menzer C Ye F et a0al Fatal toxic effects associated with immune checkpoint inhibitors a systematic review and metaanalysis JAMA Oncol Yang F Shi S Zhu J Shi J Dai K Chen X Clinical characteristics and outcomes of cancer patients with COVID19 J Med Virol https doi101002jmv25972 Zhang L Zhu F Xie L Wang C Wang J Chen R et a0al Clinical characteristics of COVID19infected cancer patients a retrospective case study in three hospitals within Wuhan China Annal oncol KeytrudaEMEAHC003820 R0081 https wwwemaeurop aeuendocum entsprodu ctinfor matio nkeytr udaeparprodu ctprodu ctinf ormat ion_enpdf Accessed May Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 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 |
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 |
"Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens Cyclic GMPAMP synthase cGAS and its downstream effector stimulator of interferongenes STING are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype I interferons IFNs and other inflammatory cytokines Accumulating evidence suggests that the activation ofthe cGASSTING axis is critical for antitumor immunity The downstream cytokines regulated by cGASSTINGespecially type I IFNs serve as bridges connecting innate immunity with adaptive immunity Accordingly a growingnumber of studies have focused on the synthesis and screening of STING pathway agonists However chronicSTING activation may lead to a protumor phenotype in certain malignancies Hence the cGASSTING signalingpathway must be orchestrated properly when STING agonists are used alone or in combination In this review wediscuss the dichotomous roles of the cGASSTING pathway in tumor development and the latest advances in theuse of STING agonistsKeywords cGASSTING Innate immunity Type I interferon STING agonists Antitumor response CancerdevelopmentIntroductionThe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses[]Protection againston patternrecognition receptors PRRs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens Correspondence zqliucsueducn Juyan Zheng and Junluan Mo contributed equally to this work1Department of Clinical Pharmacology Hunan Key Laboratory ofPharmacogenetics and National Clinical Research Center for GeriatricDisorders Xiangya Hospital Central South University Changsha Peoples Republic of China2Institute of Clinical Pharmacology Engineering Research Center for appliedTechnology of Pharmacogenomics of Ministry of Education Central SouthUniversity Changsha Peoples Republic of ChinaFull list of author information is available at the end of the adaptive immunity [] Abnormal RNA or DNA RNADNA hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns PAMPs [ ] Cells associated with innate immunity recognize different microbial PAMPsthrough specific PRRs thereby playing key roles in hostresistance to microbial infection [] The pathways governing RNA recognition such as retinoid acid induciblegene I RIGIlike receptors have been reviewed elsewhere and will not be covered herein In the case of DNArecognition one of the best known PRRs is Tolllike receptor TLR9 which senses extracellular CpG hypomethylated DNA that has entered the cytosol through thephagosomelysosome system [] In addition the AIM2like receptor AIM2 inflammasome can be triggered afterthe entry of doublestranded DNA dsDNA into 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 0cZheng Molecular Cancer Page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as IL1and IL18 and the activation of gasdermin D leading topyroptosis [] Nevertheless the most notable PRR iscGAS a direct cytosolic dsDNA sensor which was identified by Dr Chens group in [] Once cGAS bindsto dsDNA the cGASSTING pathway is activated to further induce the expression of type I IFNs and other inflammatory cytokinesthus triggering innate immuneresponses [] Mounting evidence suggests that cGASSTING signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis Hence in this review we summarize themechanism of cGASSTING activation and elaboratefindings regarding its dual effects on tumor developmentCurrent advances in the use of STING agonists as a novelstrategy for antitumor therapy are also reviewedInsights into the cGASSTING signal transductioncascadecGAS is an innate immune sensor that identifies variouscytosolic dsDNAincluding DNA with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderivedDNA and selfDNA Table In the cytoplasm cGAS isactivated by interacting with dsDNA in a sequence[]independent butStructural and biochemical analyses have revealed thatthe Cterminal lobe of cGAS contains a conserved zinclengthdependent mannerionbinding module that mediates DNA binding andcGAS dimerization [ ] DNA ligands promotecGAS activation primarily by inducing conformationalchanges around the catalytic site and in the DNAbinding structures of cGASthe GScontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cGAS activation byDNA [] In addition to the primary DNAbinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops [] The proximity ofthe two DNAbinding sites in cGAS leads to a cGASDNA complex assembly in which two cGAS moleculesembrace two molecules of dsDNA [ ] The cGASdimers are anized in headtohead alignment nextto the DNA [] and thus form stable ladderlike networks between one long curved dsDNA helix or two independent dsDNA strands [ ] In this way eachindividual cGASdsDNA complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsDNA as more likely to activate cGAS [] In additionlong DNA is more efficient than short DNA in drivingthe liquidliquid phase separation of cGAS and the formation ofcriticallydependent on the concentration of cGAS and DNA inthe cytoplasm [] cGAS and dsDNA are spatially concentratedcGASdimerization and activation [] Once cGAS andcGAS liquidlike dropletsin liquiddropletsistofacilitateTable Classification of the cytosolic dsDNA that activates the cGASSTING signaling axisClassificationSelfDNASource of dsDNAMicronucleiPossible mechanismsRupture of the micronuclei membrane leads to exposureof chromatin DNA that is recognized by cGAS whichactivates the cGASSTING pathwayReferences[]MitochondrionNuclear RNAPathogenderived DNADNA virusHSV1 HSV2 KSHV adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus RetrovirusHIV SIV murine leukemia virusRNA virusWest Nile virus dengue virus VSVSARSCOV2BacteriaListeria monocytogenes Mycobacteriumtuberculosis Listeria Shigella FrancisellaChlamydia and NeisseriaMitochondrial stress induces mtDNA leakage into thecytosol thus activating the STING pathway and inducingproduction of cytokinesFacilitated by endogenous retroelements nuclear RNAcan be reversely transcribed into DNA that activatescGASSTING signaling[][]DNA viruses invade host cells and release pathogenderivedDNA to induce STING activation[]DNA intermediates generated from reverse transcription maybe recognized by cGAS to stimulate downstream STINGsignaling[]Infection with RNA viruses might cause cellular damage andcell death which results in the release of cellular DNA andfurther activation of the cGASSTING axis SARSCoV2 bindingto ACE2 can lead to excessive angiotensin II signaling thatactivates the STING pathway in mice[]Bacteria produce CDNs such as cyclic diGMP and cyclicdiAMP which can directly bind to and activate STING[ ]HSV1 herpes simplex virus HSV2 herpes simplex virus KSHV Kaposi sarcomaassociated herpesvirus HIV human immunodeficiency virus SIV simianimmunodeficiency virus VSV vesicular stomatitis virus CDNs cyclic dinucleotides and SARSCOV2 severe acute respiratory syndrome coronavirus 0cZheng Molecular Cancer Page of dsDNA interacts structural switches rearrange the catalytic pocket to enable cGAS to catalyze the synthesis of²²cyclic GMPAMP ²²cGAMP with ATP andGTP as substrates The first step in this process is theformation of a linear dinucleotide ²pppG ²²pAwith ATP serving as the donor and ²OH on GTP serving as the acceptor Then the intermediate product flipsover in the catalytic pocket placing GTP at the donorposition and AMP at the acceptor position to form asecond ²² phosphodiester bond [ ] Notablyalthough dsRNA or singlestrand DNA ssDNA is ableto bind to cGAS neither can rearrange the catalyticpocket which may explain the exclusive activation ofcGAS by dsDNA Ultimately cGAMP acts as a secondmessenger to bind to and activate STING a small endoplasmic reticulum ERlocated protein KD withfour putative transmembrane domains [ ] Normally in a resting state STING is retained in the ER byinteracting with the Ca2 sensor stromalinteractionmolecule STIM1 [] The cytosolic ligandbindingdomain LBD of STING exists as the most functionalunit capable of integrating with ²² cGAMP or CDNscyclic dinucleotides such as cdiAMP cdiGMP or ²²cGAMP from bacteria Upon interaction the obviousclosure of the ligand binding pocket in the LBD is observed which is related to the activation of STING []Next STING transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe ER to the perinuclear area facilitated by cytoplasmiccoat protein complex II COPII and ADPribosylationfactor ARF GTPases [ ] In the Golgi STING ispalmitoylated atCys88 andCys91 a posttranslational modification necessary forSTING activation [] Modified STING recruits thekinase TANKbinding kinase TBK1 in turn the Cterminal domains of STING are phosphorylated byTBK1 and then phosphorylated STING recruits interferon regulatory factor IRF3 which is also phosphorylated by TBK1 and dimerizes ultimately dimerizedIRF3 enters the nucleus and exerts its function in thetranscription of type I IFNs and interferonstimulatedgenes ISGs [] In parallel STING can also bind toand stimulate IκB kinase IKK to mediate the production of nuclear factorκB NFκBdriven inflammatorygenes Upon signal transduction termination STING istransferred to endolysosomes for degradation [] Considering that cGAMP can be transferred through gapjunctions or delivered in viralexosome packages cGASSTING signaling may be activated in the cytoplasmwithout dsDNA [ ] Moreover newly produced typeI IFNs activate heterodimer interferon receptors IFNAR1 and IFNAR2 through paracrine signaling and thusinduce the transcription of ISGs [ ] In summaryonce virusderived DNA and selfDNA are located intwo cysteine residuesthe cytoplasm they can be sensed by cGAS and a cGASdsDNA complex is formed to catalyze the synthesis of ²²cGAMP with ATP and GTP Then ²²cGAMP and bacteriaderived CDNs induce STING activation and mediate the release of downstream type IIFNs TNFα and IL6 which are prerequisites for antimicrobial defense and antitumor effects The wholeprocess shows that the dsDNAcGASSTING axis canlead to the activation of both innate and adaptive immunity Fig The antitumor functions of the cGASSTINGsignaling pathwayRecent evidence has revealed the close association of thecGASSTING pathway with cancer development Thissignaling pathway is generally regarded as a potent regulator of cancer immunity A STINGmediated immunesupportive microenvironment can hamper malignancyoccurrence []stressbyTumor cell cytosolic dsDNA induces STING activationUnder normal circumstances DNA is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity [] However DNA leaks aberrantly in tumorcells [ ] Cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism [] Under these intense states nuclear andmitochondrial DNA is fragile and easily damaged whichleads to eventual DNA leakage in the forms of micronuclei chromatin fragments andor free telomeric DNA[ ] Chromosomal instability CIN is the primary source of cytoplasmic DNA in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance [] Excessive proliferation of cancer cells results in unstable genomes [] usuallychromosomal missegregation during mitosis Due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner [] The vulnerable membraneof micronuclei easily exposes the inner DNA to the cytoplasm and activates the cGASSTING signaling axis [] Exogenous stimuli such as chemotherapy and irradiation can also cause DNA damage In addition to leakednuclear DNA oxidative stressinduced mitochondrialDNA leakage is another crucial initiator of STING pathway activation Several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death Therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial DNA escape [ ] Other sourcessuch as apoptotic cellderived DNA exosomal DNAExoDNA and transposable elements have also beencharacterized 0cZheng Molecular Cancer Page of Fig The cGASSTING DNA sensing signaling pathway Various DNA derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic DNA sensor cGAS cGAS catalyzes the synthesis of ²²cGAMP in the presence of ATP and GTP then ²²cGAMP bindsto the ER adaptor STING which also can be activated by CDNs derived from bacteria Upon activation STING translocates from ER to Golgicompartments where it activates TBK1 and IKK which phosphorylate IRF3 and IκBα respectively Then IRF3 and IκBα dimerize and enter nucleusinitiating the transcription of Type I IFN TNF and IL6 The primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cGASSTING activation intumor cells [ ]Type I IFNs mediators of STING and adaptive antitumoreffectscGASSTING signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner On the one hand DNA damage can provokeacute STING signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype SASP which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] In contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein BCL2associated X BAX and downregulating the BCL2 apoptosis[] On the other hand STINGsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type I IFNs to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination []STING activation in nonmalignant cells causes tumorsuppressive effects as well STING signaling protectsagainst colitisassociated carcinomas CACs induced byazoxymethane AOM and dextran sulfate sodiumDSS which induce DNA damage in intestinal epithelialcells and further trigger STING activation Downstreamcytokines of STING signaling such as IL1 and IL18prevent neoplastic transformation by facilitating woundrepair More importantly STING signaling can also provoke cytotoxic T cell responses to control tumorigenesis[] Necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor ATFlike BATF3drivenlineage of dendritic cells DCs [] BATF3 DCs take intumorassociated antigens and migrate towardsthe 0cZheng Molecular Cancer Page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific CD8 T cellsThen CD8 T cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells In turn damaged cancercells release more antigens that are further captured byDCs the whole process forms a positive feedback loopcalled the cancerimmunity cycle [] Tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and T cell priming and activation withtumor antigenspecific T cell priming and activationrelying on DCs and type I IFN release [] The involvement of type I IFNs in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate PRR pathways as potential immunomodulators Mice lacking TLR9 myeloid differentiationprimary response gene MyD88 cytosolic RNA sensor MAVS or the purinergic receptor P2X7R maintainintact antitumor immunity responses whereas mice deficient in STING or IRF3 present with impaired CD8 Tcell priming and activation [ ] In fact dying tumorcells can release multiple damageassociated molecularpatterns DAMPs to trigger innate immune responsesin DCs among these released stimuli tumor cellderivedDNA is a pivotal inducer In general the phagocytosis ofapoptotic cells causesimmune silence because ofDNasebased degradation [] Nevertheless tumor cellreleased DNA can be preserved in the DC endolysosomal compartment through an unknown mechanism [] cGAS recognizes DNA invading the cytoplasm andinduces the activation of STING cascades excretion oftype I IFNs and expression of ISGs Additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial DNAmight be packaged in exosomes Exosomal DNAExoDNA animates STING signaling once it is absorbedby tumorinfiltrating DCs [] Finallytumor cellderived cGAMP can also be transferred to host DCs bythe folate transporter SLC19A1 and then directly bindsto STING activating it in DCs [] A recent study moredirectly demonstrated that cellautonomous STING promoted the maintenance of stem celllike CD8 T cellsand augmented antitumor T cell responses and mechanistically cGASSTINGmediated type I interferon signaling reinforced the stem celllike CD8 T celldifferentiation program mainly by restraining Akt activity []Immune cellderived type I IFNs have crucial functions in antitumor immunity control On the one handtype I IFNs boost cross presentation by various mechanisms first they stimulate the maturation of DCs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of MHC I molecules on the cellsurface [ ] finally they accelerate DC migrationtowardslymph nodes where they can crossprimetumorspecific CD8 T cells [] On the other handtype I IFNs drive the expression of multiple chemokinessuch as CXCL9 and CXCL10 both of which are necessary for cytotoxic T lymphocyte CTL transfer and infiltration [] Similarly type I IFNs restrain the defaultimmune suppressive action of regulatory T Treg cellsby downregulating phosphodiesterase PDE4 and upregulating cyclic AMP cAMP [] Consequently typeI IFNs serve as bridges linking the cGASSTING pathway with CD8 T cellmediated antitumor immunityThe antitumor mechanisms of the cGASSTING signaling axis are illustrated in Fig Indeed previous studies revealed that STING activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [] Additionally STING expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent STING expression [] Lower STING expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] Consistentlycompared with that in the MCFG10A mammary epithelial cell line lower STING expression was detected inmalignant breast cancer cellincluding MCF7HBL100 and T47D cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] Collectivelythat cGASSTING signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinesSTING pathway agonists as cancer therapeuticsThe immunostimulatory potential of the cGASSTINGpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmentTME can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectorT cells Recent drug research has focused on the development of STING agonists because of their potential inanticancer therapy [ ] To date various kinds ofSTING agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates DMXAA and its analogsand small molecular agonists In addition some conventional antitumor therapeutics can also indirectly activateSTING such as chemotherapy radiotherapy RT andtargeted therapy [] In addition STING agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination STING 0cZheng Molecular Cancer Page of Fig The antitumor immunity effect of the cGASSTING pathway DNA damage leads to the formation of dsDNA in tumor cells upon itsstimulation STING signaling is activated and promotes the release of Type I IFN which is crucial for DC maturation STING signaling activation inDCs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cGAMP gap junctions Then DCs migrate towards the tumordraining lymph node and crossprime tumor specific CD8 T cells withthe help of Type I IFNs Finally T cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowCyclic dinucleotides CDNsCDNs constitute a main type of STING agonist whichmainly originate from bacteria The known naturalCDNs consist of exogenous cyclic diGMP cdiGMPcdiAMP ²²cGAMP and endogenous ²²cGAMPAmong these cdiGMP cdiAMP and ²²cGAMPare synthesized by bacteria and identified as secondarymessengers that mediate STING signal transduction inprokaryotic cells while ²²cGAMP functions as theinitiator of STING in mammalian cells [] The antitumor potential of these natural dinucleotides was firstproven by the finding that cdiGMP could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomaH508 cells was inhibited with μM cdiGMP []Intraperitoneal ip injection of highdose cdiGMPdirectly activated caspase3 and triggered T1 tumoripcell apoptosis in vitro nmol of cdiGMP reduced thegrowth of T1 tumor cells in vitro by and nmreduced it by while lowdose cdiGMP nmol accelerated the adaptive T cell response by converting a subgroup of myeloidderived suppressor cellsMDSCs into immune stimulatory cells producing IL12injection of ²²cGAMP [] Consistentlymgkg expedited dramatic leukemic elimination in ElTCL1 transgenic mice bearing chronic lymphocyticleukemia CLL and promoted tumor shrinkage of multiple myeloma in vivo [] From the perspective of endogenous CDNs ²²cGAMP mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing CT26 colon adenocarcinomain a dosagedependent manner relying on DC activationand T cell crosspriming [] More recently OhkuriT further demonstrated that intratumoral it injection of ²²cGAMP μg25 μLdose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0cZheng Molecular Cancer Page of cancer T1luc squamous cell carcinoma mSCC1colon cancer CT26 and melanoma B16F10 mousemodels [] Notably the it injection of ²²cGAMPinhibited not only tumor growth but also lung metastases in mice bearing B16F10 cellderived tumors suggesting that cGAMPinduced CD8 Tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth []termedvaccineSTINGVAXConsidering the superior properties of STING signaling in activating adaptive immunityit is rational toutilize STING agonists such as CDNs as cancer vaccineadjuvants to increase tumor immunogenicity [] Fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor GMCSF and bacteriaderived or synthetic CDNs Theyobserved that after it injection of STINGVAX with μg of CDNs per vaccine dose the volume of B16melanoma tumors was dramatically reduced in a dosedependent manner Compared to mice receiving GMCSF cancer vaccine alone STINGVAXtreated mice hadmore infiltrating CD8 IFNγ T cells in the tumormicroenvironment The in vivo antitumor effect of STINGVAX was also verified in models of colon carcinomaCT26 pancreatic carcinoma Panc02 and upper aerodigestive squamous cell carcinoma SCCFVII []Although natural CDNs are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting First native CDNs are easily degraded by enzymes inside the cellor in the bloodstream Second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede CDNs from penetrating cell membranes to activate cytosolic STING leading to low bioavailability andpoor retention of the CDNs in specific cells and tissuesThird unintentional toxicities and narrow therapeuticwindows are also unavoidable Thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening [] Regarding agonistdelivery Smith reported that biopolymer implantscodelivering cdiGMP μg and chimeric antigen receptor T CART cells resulted in significant tumor regression in mice bearing pancreatic tumors[]Moreoveriv administration of cdiGMPYSK05Lip equivalent to μg of cdiGMP aYSK05liposome delivery system encapsulating cdiGMP led to a tremendous decrease in metastatic lesionsin a B16F10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced [] Chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cGAMP cGAMPNP could activate the STING axis more effectively than solublecGAMP and converted the immunosuppressive TME toa tumoricidal state in a transplanted B16F10 cell melanoma model and in a genetically engineered triplenegative breast cancer model [] Moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of STING agonistsIntroduction of a dinucleotide cyclasecoding gene intothe Escherichia coli Nissle strain was an attempt at realizing this effect however advancements to the systemare needed []tobysnakeApartdigestionresistancecompoundatoms The modifiedfrom improving delivery methods CDNswith superior properties are currently being synthesized and tested For instance to prevent enzymatichydrolysis of cGAMP the nonbridging oxygen atomsin cGAMP phosphodiester linkages were replaced by²²sulfurcGsAsMP showed resistance against degradation byENPP1 a major ²²cGAMP hydrolasetherebyleading to a longer halflife and sustained high affinity for human STING hSTING[] Syntheticdithio mixedlinkage CDNs with both Rp Rp R Rand Rp Sp R S dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforSTING A novel superior modified product ML RRS2 CDA also termed ADUS100 had the potencyto activate all hSTING variants and mouse STINGmSTING ADUS100 had higher efficiency in activating STING signaling than endogenous or exogenous CDNs mainly because of its enhanced stabilityand lipophilicity Its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding B16 melanoma T1 breast cancer and CT26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of ADUS100 three mg doseswhen tumor volumes reached mm3 [] TheremarkableforhSTING laid the foundation for its clinical use Related clinicaltrials of ADUS100 are outlined inTable In addition to ADUS100 some other novelSTING agonists have been well designed IACS8779and IACS8803 are two highly potent ²²thiophosphate CDN analogs that induced striking systemicantitumorin a B16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with ADUS100or cGAMP [] The characteristics and preclinicalapplications of all these mentioned CNDs are summarized in Table Because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of CDNs have beenand high affinityresponsescurativeeffect 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonistsClassificationCharacteristicsApplicationmodelsNatural CDNagonistscdiGMPPoor membrane permeabilitysuitable for various codeliverytechnologiesColon cancer H508cells T1 metastaticbreast cancerTreatmentinformation μM nmol ip nmol ip nmol ip²²cGAMP²²cGAMPHigher binding affinity formSTING than for hSTINGHigher affinity for hSTING thanits lineage isomers binds tovarious STING nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembraneChronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipCT26 colonadenocarcinoma mgkgbreast cancer T1lucsquamous cellcarcinomasmSCC1 μg25 μLdose it μg25 μLdose itcolon cancer CT26 μg25 μLdose itmelanoma B16F10 μg25 μLdose itTherapeutic effects References[ ][][ ]Inhibitsproliferation tumorregression tumorregressionAccelerates TcellresponseLeukemiaeliminationSuppressesgrowthRestrainstumorigenesisImproves survivalrateDelays tumrowthDelays tumrowthDelays tumrowthDelays tumrowthSTINGVAXSyntheticCDNagonistsPotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercGAMPNPsBiopolymer scaffolds cdiGMP and CAR T cellscdiGMPYSK05Lip²²cGsAsMPADUS100IACS8779IACS8803NonCDNagonistsFAALiposomal nanops NPsdeliver cGAMP intracellularlymore effectively than realizedwith soluble cGAMPEradicates tumors moreeffectively than systemicdeliveryYSK05 is a lipid that can efficientlydeliver cdiGMP to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapeMore resistant to degradation byENPP1 tenfold more potent atinducing IFN secretion potentialuse as a cancer vaccine adjuvantImproves stability and lipophilicityhigher affinity for hSTING thannatural CDN agonists capable toactivate all hSTING variants andmSTINGStimulates a superior systemicantitumor response thanADUS100 and cGAMPCauses hemorrhagic necrosisfailed in a phase I clinical trialdue to species specificity μg CDNs itReduces tumorvolume[]B16 melanomacolon carcinomaCT26pancreaticcarcinoma Panc02B16F10 melanomaivTNBCCreates atumoricidal state[]Pancreatic cancer μg cdiGMPTumor regression[]B16F10 mousemelanoma μg cdiGMP ivDecreasesmetastasisTHP1 monocytesB16 melanomathree mg doses it T1 breast cancerthree mg doses itMC26 colon cancerthree mg doses itDurable tumorregressionDurable tumorregressionDurable tumorregression[][][]B16 melanoma μg on day and posttumor implantationAntitumorresponse[]Murine colontumorsExtensive tumorrejection[ ]DMXAAFirst discovered as a vascularRat mammary mgkg ipHigh anticancer[ 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonists ContinuedClassificationCharacteristicsApplicationmodelsTreatmentinformationInduces proinflammatory cytokinesin a STINGdependent mannerHuman fibroblastsAntiviral activity[]Selectively induces STINGdependentsynthesis and secretion of bioactiveIFNs no evidence of binding directlyto STINGActivates STING | 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 |
" 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 |
Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tients clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum the appendix and the stomach [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton Competing Interests The authors have declared that no competing interests exist Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s Fig Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon cm heterogeneously with a contiguous enhancing mass pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement Fig Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after carcinoid epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol [] Gut P Czarnywojtek A Fischbach J B Ëaczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al Scintigraphic imaging of body neuroendocrine tumors Radiographics [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer 0c' | 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 |
"evaluate the clinicopathologic characteristics of Lymph Node metastasisbetween investing layer of Cervical fascia and deep fascia of infrahyoid strap Muscles LNCM in papillary thyroidcarcinoma PTCMethods Retrospective review of patients with PTC who underwent thyroidectomy and central compartment neckdissection CND from January to January was performed in two tertiary referral academic medicalcenters A total of consecutive patients with PTC who underwent thyroidectomy and CND were included inthe retrospective review The LNCM was resected as a separate specimen by the surgeon and the clinicopathologiccharacteristics of the patients were recorded Multivariate logistic regression analysis was performed to identify riskfactors for LNCM metastasisResults Of PTC patients patients had lymph nodes in the LNCM Among them caseswere confirmed to be positive in the LNCM In total the metastasis rate of LNCM in PTC patients was Univariate analysis revealed that the metastasis of LNCM were more likely to have a primary site in theinferior pole extrathyroidal extension ETE central cervical metastasis level III and level IV metastasis Multivariateanalysis further showed tumor location in the inferior pole ETE level III and level IV metastasis conferred asignificantly increased odds ratio for LNCM metastasisConclusion Attention should be paid to the lymph tissue in the LNCM for PTC patients especially in presence of aprimary site in the inferior pole ETE level III and level IV metastasisKeywords Thyroid carcinoma Surgery Central compartment neck dissection Recurrence Suprasternal spaceIntroductionPatients with papillary thyroid carcinoma PTC have afavorable prognosis with central neck locoregional recurrence varying from to [] The goal of a prophylacticor therapeutic central compartment neck dissection pCNDor tCND is to decrease the incidence of local recurrenceby removing all lymphatic tissue within the levels VI and Correspondence wugaosongwhueducn1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei Peoples Republic of ChinaFull list of author information is available at the end of the VII compartments which are generally the first and themost commonly involved with metastasis [] For patientswithout evidence of lymph node metastasis on preoperativeevaluation the additive value of a pCND at the time ofthyroidectomy is controversial Some authors advocatepCND considering high rate of occult metastaticnodal disease in cN0 PTC [] while other authors considerthat there is no highlevel evidence in favor of pCND []The performance of pCND is dependent on the weightgiven to the risks and benefits of pCND [] Consideringthe oncologic benefits of CND and the risks of a repeat 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 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of neck operation performing pCND is recommended toevery patient in China [ ]Although American Thyroid Association ATA guideline has defined the boundary of central neck compartment there is also significant variability in terms of theextent of CND In routine clinical practice CND canrange from sampling a few nodes in the paratrachealregion to a complete clearance from left carotid artery toright carotid artery and down to and including the uppermediastinum [] Owing to the variant extent of CNDsome central compartments are easily to be neglectedFor thyroid carcinoma patients with specific clinicopathologic characteristics incomplete lymph node dissectionmay result in increased recurrence reoperation andreoperationassociated complications [] Lymph Nodebetween investing layer of Cervical fascia and deep fasciainfrahyoid strap Muscles LNCM has not beenofreported The LNCM compartment is defined as followssuperiorly by the hyoid bonelaterally by the carotidarteries anteriorly by the investing layer of cervicalfascia and posteriorly by the deep fascia of infrahyoidstrap muscles LNCM space includes suprasternal spaceand intrainfrahyoid strap muscle spaceAnatomically LNCM is located anterior to the strapmuscles We consider that what is special about the concept of the LNCM is that it is belong to level VI but is aneasily overlooked anatomical area by a strap musculatureinvolving the sternohyoid and sternothyroid musclesduring selective or modified neck dissection Although themetastasis in LNCM was seldom it did occur in somePTC patients with regional recurrence As part of LCNMsuprasternal space metastasis for thyroid cancer wereinvestigated in three studies [] Thus we routinelydetected the suprasternal space and intrainfrahyoid strapmuscle space Fig This study was performed to identify the clinicopathologic characteristics and indication forlymph node metastasis dissection in the LNCMMaterials and methodsPatientsA retrospective review from the clinical and histopathologydatabase of two tertiary referral academic medical centersTongji Hospital of Huazhong University of Science andTechnology and Zhongnan Hospital of Wuhan Universityfrom January to January were conducted In theinstitutions preoperative examinations consisted of athorough physical examination neck ultrasound a clinicalevaluation of thyroid nodules and neck lymph nodes Fineneedle aspiration cytology FNAC were performed in patients who were suspected thyroid nodules or lymph nodeWith a pathological confirmation of PTC all the patientsreceived a thyroidectomy with CND Accordingly a pCNDwas performed for cN0 patients and a therapeutic CNDwas performed for cN1 patients The inclusion criteria forthe patients were as follows the clinical history completely recorded the LNCM was resected as a separatespecimen by the surgeon PTC patients who underwentthyroidectomy plus CND with or without lateral neckdissection A total of consecutive PTC patients wereenrolled The medical ethic committee of ZhongnanHospital of Wuhan University approved the procedure andinformed written consent was obtained from all patientsSurgical approachAll the operations were performed by the same seniorsurgeon Gaosong Wu with the patients under generalFig Four subdivisions Level VI 1st Level VI 2nd Level VI 3rd and Level VI 4th of central neck compartment are divided by deep fascia ofinfrahyoid muscles pretracheal visceral fascial and right recurrent laryngeal nerve 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of anesthesia Thyroidectomy was performed with a standard technique of fine capsular en bloc dissection andresection from inferior pole to superior pole []Intraoperative neuromonitoring was employed for all ofthe thyroidectomies [] Superior parathyroid glandswere identified and preserved in situ inferior parathyroidglands were protected in situ or autotransplanted in thesternocleidomastoid muscle according to three certaintypes based on their blood supply and location [ ]After the incision of the investing layer of cervicalfascia the interval between sternohyoid and sternothyroid muscles and the space anterior to the sternohyoidmuscle above the clavicle and the sternum weredetected If there was fibrofatty tissue Instead of the enbloc removal of the entire centrallymph nodes theLNCM was resected as a separate specimen if occurredThe presence or absence of lymph node metastasis wasdefined according to postoperative pathological reportsWhile dissecting paratracheal lymph nodes intraoperative neuromonitoring was employed to detect RLN fromdistally to proximally minimizing morbidity from injuryto RLN during compartment nodal dissection LNCMand other compartment lymphatic tissue were processedfor routine hematoxylin and eosine HE separatelyThe pathologic results were independently determinedby two qualified pathologists without any prior knowledge of the patients clinical dataData collection and statistics analysisTo determine the relation between LNCM metastasis andclinicopathologic factors such as age sex primary tumorsite lateral cervical lymph node metastasis level VI metastasis the chisquare test and Fishers exact test were usedas appropriate Multivariate logistic regression analysiswas performed to identify risk factors for LNCM metastasis of PTC P was considered statistically significantAll calculations were performed using SPSS for windows Postthyroidectomy hypocalcemia lasting for morethan months was considered as permanent VCP All patients were followed up every months postoperativelyResultsPatients detected with LNCMAfter reviewing patients who underwent thyroidectomy plus CND with or without lateral neck dissectionfrom January to January patients were detected with LNCM and patients wereabsent of LNCM The average tumor size of LNCM was cm and the mean number of lymph nodes sampledfrom LNCM was ranging from to Table showsthe comparison of clinicopathologic characteristics between the present LNCM group and the absent groupIn univariate analysis Hashimotos disease p multifocality leisions p the tumor located ininferior portion p extrathyroidal extensionETE p central cervical metastasis p level III and level IV metastasis p were significantly associated with high prevalence of LNCMPatients with metastatic LNCMAmong a total of patients with LNCM metastaticLNCM was found in patients Table compares the clinicopathologic characteristics between themetastatic LNCM group and the nonmetastatic LNCMgroup Three hundred eightythree patients were confirmed free of LNCM metastasis of themwith clinically negative node performed pCND and of them with clinically positive performed tCND All thepatients in the metastatic LNCM group performedtCND Lateral neck dissection was performed in cases in the metastatic LNCM group and cases in the nonmetastatic group all lateral neckdissection wastherapeutically performed Univariateanalysis was performed for the patients with and patients without metastatic LNCM Age at diagnosisgender and tumor size coexistent thyroid disease tumorfocality and level II metastasis were not correlated withLNCM metastasis Univariate analysis identified tumorlocated in the inferior pole central cervical metastasisETE level III and level IV metastasis as significant predictors of LNCM metastasis in our study population Multivariate analysis further showed that tumor location ETElevel III and level IV metastasis conferred a significantlyincreased odds ratio for LNCM metastasis Table ComplicationsThe median followup time was months range of patients had voice changes all of whomrecovered within months Temporary vocal cord paralysis was confirmed in patients by laryngoscope andthirteen permanent hypocalcemia was observed aftersurgeryDiscussionIn order to achieve the best chance of cure and effectivedisease control thoroughness of dissection has to betaken into account We prospectively performed comprehensive CND for PTC patients who underwent thyroidectomy and CND In addition data were analyzed for PTC patients to investigate the clinicopathologic characteristics for LNCM metastasis The occurrence rate ofLNCM was and of the patients harbored metastatic LNCM In total the positiverate of the LNCM was In this studymultivariate analysis revealed that a primary site in the inferior pole ETE level III and level IV metastasis were ofhigher LNCM metastasis rate which was consistent withthe findings by the previous report oflymph node 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for patients who had lymph nodes in LNCM compared tothose who did notVariablesAge mean ± SDAbsent n Present n P value¤ GenderFemale MaleTumor size cm ¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimotos diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes No LNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid Musclesmetastasis between sternocleidomastoid and sternohyoid muscle []Several studies have emphasized the importance of similar compartment in neck dissection for thyroid carcinomaSun pioneered the confirmation of the significantinvolvement of lymph node metastasis between sternocleidomastoid and sternohyoid muscle LNSS in lateralneck dissection [] which anatomically classified as part ofthe space of Burns They concluded that the positive rate ofLNSS was in clinically nodepositive cN PTCwhich was correlated with a primary site in the inferiorpole the lateral nodal metastasis level III and level IV nodalmetastasis [] Then Homma [] reported two casesof PTC patients with level III and IV lymph node metastases as well as metastasis in the suprasternal space Yu et al[] investigated the clinical significance of the suprasternalspace lymph node SSLN in pathological nodepositivepN PTC patients They concluded that metastasis rate ofSSLN was and the high SSLN metastasis of PTC wascorrelated with primary cancer site in the inferior thyroidpole strap muscle invasion level IV metastasis and LNSSmetastasis In our experience LNCM was rarely occurredin the central neck compartment and the positiveLNCM in PTC patients was infrequent as well Notably among the patients with pN PTC themetastasis rate of LNCM was which was muchlower than the metastasis incidence of SSLN described by Yu [] According to their results onefifth patients with pN PTC were performed incompleteCND and remained metastatic lymph nodes 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for positive LNCMVariablesAge mean ± SDMetastasis n Nonmetastasis n ¤ GenderFemale MaleTumor size cm ¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimotos diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes NoLNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid MusclesP value The total number of lymph nodes in the central neckcan range from to [] There is no consensus on thenumber of nodes removed or examined that would constitute an adequate dissection Aimed to allow surgeons tomore accurately report the extent of lymphadenectomyTable Multivariate analysis of predictors for LNCM positivityVariablesTumor locationp valueCentral cervical metastasisExtrathyroidal extensionLevel III metastasisLevel IV metastasisOR CI LNCM Lymph Node between superficial layer of deep Cervical fascia and deepfascia of infrahyoid Musclesvisceralwe divide the central neck compartment into four subdivisions by deep fascia of infrahyoid strap muscles pretrachealfascial and right RLN Fig Theproposed LNCM compartment is bounded superiorly bythe hyoid bone laterally by the carotid arteries anteriorlyby the investing layer of the cervical fascia and posteriorlyby the deep fascia of infrahyoid strap muscles which is defined as Level VI 1st In the current study suprasternalspace composed part of the LNCM Fig Compared toSSLN reported by Yu [] LNCM encompasseslymph nodes in the suprasternal space and lymph nodesbetween sternohyoid and sternothyroid muscles Figs and LNCM can fall under the normal subdivisions ofthe central compartment Subdivisions can actually recordthe extent of the CND which is able to provide detailedinformation for the possible second operation 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Fig LNCM coverage area in vivo Lymph nodes between sternohyoid and sternothyroid muscles a and lymph nodes in the suprasternal space bIncluding LNCM as an anatomical part of the centralneck allows for removal of previously unrecognized micrometastatic disease in of PTC patients with the inferiorportion lesions ETElevel III and level IV metastasisDissection of the LNCM space is less invasive and easy toachieve and is not timeconsuming It is at the entrance ofcentral neck compartment which is easy to expose and haslow risk of damaging RLN or parathyroid With the application ofintraoperative neuromonitoring and in situpreservation or autotransplantation of parathyroid theoccurrence of vocal cord paralysis and permanenthypoparathyroidism in the current study were lowerin this study [ ] Therefore in cases where LNCMspace metastasis is suspected or preoperative ultrasoundand CT suggests LNCM metastasis greater attentionshould be paid to the nodal tissue in the LNCM space inthyroid carcinoma patients These patients might benefitfrom a reduced risk of regional recurrence central neckreoperative morbidity and improved decision making inrelation to the use of radioiodine ablationThere are severallimitations in the present studyThe retrospective design is a limitation of the studyAnd this was two tertiary referral centers retrospective review and routine prophylactic nodal surgerywas offered in China however it is not standard elsewhere in the world which is a major limitation Aprospective randomized trial with a long time followup period may help to further evaluate the clinicalsignificance of LNCM in PTC patientsConclusionsIn summary additional dissection of nodes in theLNCM were accessible and might not increase morbidity Therefore attention should be paid to the lymphtissue between investing layer of cervical fascia and deepfascia ofinfrahyoid strap muscles for PTC patientsespecially in presence of inferior portion lesions ETElevel III and level IV metastasisAcknowledgementsThe authors thank the studied patients for their willingness to cooperatewith our studyAuthors contributionsGaosong Wu Study concepts and design Qianqian Yuan Study designmanuscript preparation and editing Jinxuan Hou Data analysis andmanuscript editing Yiqin Liao Data acquisition Lewei Zheng Manuscriptpreparation Fang Lu Data acquisition Kun Wang Quality control of dataand algorithms The authors read and approved the final manuscriptFundingThe authors have no support or funding to reportAvailability of data and materialsNot applicableEthics approval and consent to participateThis research was comprised of human participants and was approved byMedical Ethics Committee of Wuhan University Zhongnan Hospital Informedconsent was obtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei Peoples Republic of China 2Department of Thyroid and Breast Surgery Tongji Hospital of TongjiMedical College of Huazhong University of Science and Technology Jiefang Avenue Wuhan Hubei Peoples Republic of China Received April Accepted August ReferencesLang BH Ng SH Lau LL Cowling BJ Wong KP Wan KY A systematic reviewand metaanalysis of prophylactic central neck dissection on shorttermlocoregional recurrence in papillary thyroid carcinoma after totalthyroidectomy Thyroid So YK Seo MY Son Y Prophylactic central lymph node dissection forclinically nodenegative papillary thyroid microcarcinoma influence onserum thyroglobulin level recurrence rate and postoperative complicationsSurgery Hughes DT Rosen JE Evans DB Grubbs E Wang TS Solórzano CCProphylactic central compartment neck dissection in papillary thyroid 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Cancer and effect on Locoregional recurrence Ann Surg Oncol McHenry CR Is prophylactic central compartment neck dissection indicatedfor clinically nodenegative papillary thyroid Cancer the answer isdependent on how the data are interpreted and the weight given to therisks and benefits Ann Surg Oncol Selberherr A Riss P Scheuba C Niederle B Prophylactic firststep centralneck dissection level does not increase morbidity after Totalthyroidectomy Ann Surg Oncol Hartl DM Leboulleux S Al Ghuzlan A Baudin E Chami L Schlumberger M Optimization of staging of the neck with prophylactic central andlateral neck dissection for papillary thyroid carcinoma Ann Surg McAlister ED Goldstein DP Rotstein LE Redefining classification ofcentral neck dissection in differentiated thyroid cancer Head Neck Miller JE AlAttar NC Brown OH Shaughness GG Rosculet NP Avram AM Location and causation of residual lymph node metastasis aftersurgical treatment of regionally advanced differentiated thyroid CancerThyroid Sun G Wang Y Zhu Y Wang Y Xu K Wei W Lymph node metastasisbetween sternocleidomastoid and sternohyoid muscle in clinically nodepositive papillary thyroid carcinoma Head Neck Homma A Hatakeyama H Mizumachi T Furusawa J Kano S Sakashita T Lymph node metastasis in the suprasternal space from thyroidpapillary cancer Int Cancer Conf J Yu S Ge J Sun B Wei Z Lei S Lymph node metastasis in suprasternal spacein pathological nodepositive papillary thyroid carcinoma Eur J Surg Oncol Wu G Kong D Thyroidectomy with Wu Gaosong's ProcedureVideoEndocrinologr httpsdoi101089ve20150050 Wu G Wang K Intraoperative Neuromonitoring and Protection of theSuperior Laryngeal Nerve with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160070 Wu G Cui Q Wang K Carbon Nanops for Identifying Lymph Nodesand Protecting Parathyroid Glands in Thyroid Lobectomy with IpsilateralCentral Compartment Lymph Nodes Dissection VideoEndocrinology httpsdoi101089ve20160064Kong D Cui Q Gaosong W A Novel Classification of Parathyroid Glands andTheir Preservation in Thyroidectomy VideoEndocrinology httpsdoi101089ve20170093 Wang K Wu G Intraoperative Neuromonitoring in Selective Neck Dissectionfor Thyroid Cancer SND IIa Vb with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160082 Yuan Q Wu G Hou J Liao X Liao Y Chiang F Correlation betweenelectrophysiological changes and outcomes of vocal cord function in recurrent laryngeal nerves with visual integrity during thyroidectomyThyroid Cui Q Li Z Kong D Wang K Wu G A prospective cohort study of novelfunctional types of parathyroid glands in thyroidectomy Medicine 9552e5810Tavares MR Cruz JA Waisberg DR Toledo SP Takeda FR Cernea CR et alLymph node distribution in the central compartment of the neck ananatomic study Head Neck Wang K Cai H Kong D Cui Q Zhang D Wu G The identificationpreservation and classification of the external branch of the superiorlaryngeal nerve in thyroidectomy World J Surg Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
"Mareks disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Mareks disease p53 P53 antibodyBackgroundMareks disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Mareks disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the guardian of the genome [] andthe cellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame and untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and 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 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMareks disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE b Spleen lymphoid tumor cells with cytoplasm expression HE c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE d Liver Negative controls were incubated with PBS HE which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as thehotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequenceGTGGCCGTCTATAAGAAATCAGA3AAAAAGGGGGCGTGGTCAGT3p532Annealing temperature Size of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturers instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions for min followed by cycles of for s for s for s and a final Table Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Students Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe Peoples Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Mareks disease virus lytic replicationoncogenesis and control Expert Rev Vaccines Reddy MS Book Review Mareks Disease An Evolving Problem Vet PatholBoodhoo N Gurung A Sharif S Behboudi S Mareks disease in chickens areview with focus on immunology Vet Res Lane DP p53 guardian of the genome Nature Bertzbach LD Kheimar A Ali FAZ Kaufer BB Viral Factors Involved inMareks Disease Virus MDV Pathogenesis Curr Clin Microbiol Rep Levine AJ p53 the cellular gatekeeper for growth and division Cell Soussi T B¨gue A Kress M Stehelin D May P Nucleotide sequence of acDNA encoding the chicken p53 nuclear oncoprotein Nucleic Acids ResZilfou JT Lowe SW Tumor suppressive functions of p53 Review ColdSpring Harb Perspect Biol 20091a001883Stiewe T Haran TE How mutations shape p53 interactions with thegenome to promote tumorigenesis and drug resistance Drug Resist UpdatK¶bel M Piskorz AM Lee S Lui S LePage C Marass F Rosenfeld N MesMasson AM Brenton JD Optimized p53 immunohistochemistry is anaccurate predictor of TP53 mutation in ovarian carcinoma J Pathol Clin Res Zekiye H B¼lent T Taner E p53 antibody is it an indicator ofdedifferentiated thyroid cancer Ann Nucl Med Balogh GA Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther MedSabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Mareks diseasetumorderived cell lines Leuk Res Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | Thyroid_Cancer |
progression of breast cancerare greatly affected by the immune environment Neutrophils are the most abundantleucocytes in circulation and act as the spearhead in ammationincluding inbreast cancer Circulating neutrophils are closely related to the prognosis of breastcancer patients and tumorltrating neutrophils have varied functions at differentstages of breast cancer such as antitumor or tumorpromoting neutrophils which aretermed N1 and N2 neutrophils respectively In this review we will discuss the utilityof circulating neutrophils for predicting prognosis and therapeutic efï¬cacy and theunderlying mechanisms of their chemotaxis the dynamic regulation of their antitumoror protumor functions and their different spatial distributions in tumor microenvironmentFinally we also discuss the possibility of targeting neutrophils as a therapeutic strategyin breast cancerKeywords breast cancer immunotherapy neutrophils neutrophiltolymphocyte ratio tumor microenvironmentSpecialty sectionThis was submitted toCancer Immunity and ImmunotherapyINTRODUCTIONa section of the journalFrontiers in ImmunologyReceived May Accepted July Published August CitationZhang W Shen Y Huang H Pan SJiang J Chen W Zhang T Zhang Cand Ni C A Rosetta Stone forBreast Cancer Prognostic Value andDynamic Regulation of Neutrophil inTumor MicroenvironmentFront Immunol 103389ï¬mmu202001779Breast cancer BC is the most common malignancy in women worldwide Although BCis classiï¬ed as a malignant disease with low immunogenicity recent evidence has revealeda promising outcome of therapies with blocking immune checkpoints in both early andadvanced stages The eï¬cacy of immunotherapy is closely related to the tumor immunemicroenvironment especially to ltrating immune cells To date macrophages and Tcells are the most wellstudied immune cells in BC whereas increasing evidence has indicatedthat neutrophils are also key in the oncogenesis and metastasis of BC in addition circulatingneutrophils have been reported to have great prognostic prediction value Neutrophils are themost abundant leucocytes in blood and usually act as the ï¬rst line of host defense against pathogens However due to their short life span an average of h in blood it is diï¬cult to employthis subset of cells for experiments which has resulted in a poor understanding of their role in solidtumors In addition some contradictory results reported in vitro studies or animal experimentshave suggested a dual eï¬ect of neutrophils in tumor developmentFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerNeutrophils can present both antitumorigenic N1 andprotumorigenic N2 phenotypesin various cancers orspeciï¬c circumstances The term neutrophil in several studiesalso includes both mature neutrophils and myeloidderivedsuppressor cells MDSCs MDSCs are described as a subsetof neutrophils with immunosuppressive functions that expressCD11b and Gr1 and can be divided into monocyticM CD11bLy6C MDSCs and GPMN CD11bLy6GMDSCs and GPMN MDSCs usually share a commonset of markers and similar morphologicalfeatures withneutrophils To avoid confusion we mainly focus on the biologicalfunction of mature neutrophils and related therapeutic strategiesfor targeting them in BC We provide a comprehensive reviewofthe prognostic value of circulating neutrophils and themechanisms of how tumorassociated neutrophils TANs exertantitumor or tumorpromoting functions in BC and in theend we also discuss the potential of targeting neutrophils as atherapeutic strategy in cancerPROGNOSTIC VALUE OF THENEUTROPHILTOLYMPHOCYTE RATIONLRTumors can be thought of as wounds that will not heal andare characterized by chronic ammation Neutrophils are themost rapidly responding immune cells to ammation andmany studies have found that the NLR is closely related to theprognosis and treatment response in patients bearing BC A recent metaanalysis of studies including patientswith both early and advanced BC revealed that patients witha higher NLR before treatment had poorer diseasefree survivalDFS than those with a lower NLR before treatment but theNLR was not related to overall survival OS the subgroupanalysis found that the NLR was associated with prognosisonly in earlystage patients but not in patients with metastasisAbbreviations BC Breast cancer MDSCs Myeloidderived suppressor cellsTANs Tumorassociated neutrophils NLR Neutrophiltolymphocyte ratio DFSDiseasefree survival OS Overall survival ALC Absolute lymphocyte count NCTNeoadjuvant chemotherapy pCR Pathological complete response PLR Platelettolymphocyte ratio TAMs Tumorassociated macrophages CTCs Circulatingtumor cells NETs Neutrophil extracellular traps MPO MyeloperoxidaseGCSF Granulocyte colonystimulating factor ECs Endothelial cells PMNsPolymorphonuclear neutrophils ICAM1 Intercellular adhesion molecule MMP9 Matrix metalloproteinases9 ROS Reactive oxygen species HMGB1Highmobility group box TLR4 Tolllike receptor TNBC Triplenegativebreast cancer MES Macrophageenriched subtype NES Neutrophilenrichedsubtype H2O2 Hydrogen peroxide TNFα Tumor necrosis factorα HOCIHypochlorous acid TRPM2Transientcation channelsubfamily M member ADCC Antibodydependent cellular cytotoxicityNK Natural killer NE Neutrophil elastase NRP1 Neuropilin1 IRS1 Insulinreceptorsubstrate1 PI3K Phosphatidylinositol 3kinase VEGF Vascularendothelial growth factor TIMP1 Tissue inhibitor of matrix metalloproteaseTGF Transforming growth factor 27HC 27hydroxycholesterol PAD4Peptidyl arginine deiminase TINs Tumorltrating neutrophils CRTConventionalradiotherapy MRT Microbeam radiation therapy DAMPsDamageassociated molecular patterns ICB Immune checkpoint blockadeLDNs Lowdensity neutrophils HDNs Highdensity neutrophils NAMPTNicotinamideadeninedinucleotide GTX granulocyte transfusiontransferase NAD Nicotinamidereceptor potentialphosphoribosyl Since similar metaanalyses were not based on individualpatient data which may cause significant bias we reviewed andcompared the individual reports and found some issues worthdiscussing here Widmann ï¬rst reported the correlationbetween the NLR and BC prognosis in patients and itwas found that a higher NLR ¥ before treatment was anadverse factor for both short and longterm mortality Themajority of retrospective studies thereafter have drawn similars and the NLR was found to be consistentamong diï¬erent BC subtypes at baseline However aprospective substudy of GEICAM9906 which comprised patients did not ï¬nd any prognostic value of the NLR afteradjustment for clinicopathological factors in addition a highNLR was independently associated with worse DFS in only highrisk patients the hormone receptornegativeHER2 populationand in patients with ¥ lymph node metastases Anotherstudy with early BC patients also found that the NLR beforesurgery was not associated with DFS indicating that thepresurgery NLR may be valuable only in patients with a hightumor burdenseveralIn addition to the above studiesstudies alsoexplored the prognostic value of the NLR posttreatment or withcontinuous assessment A retrospective study comparing theabsolute lymphocyte count ALC and the NLR eight consecutivetimes before and after chemotherapy found that patients whodied had lower ALC and higher NLR values than patients whoremained alive throughout the treatment course additionallyamong the patients who died a steady increase in the NLRover the baseline measurement was observed at subsequenttime points Another retrospective study included BCpatients with DFS values of more than years and it interestinglyfound that NLR sampled during followup rather than beforeany treatment was an independent prognostic factor for laterecurrence However there is still no compelling explanationfor the abovementioned inconsistent results In addition sincelymphocytes are critical in cancer immune surveillance andneutrophils have been reported to play a protumor role in moststudies low lymphocytes and high neutrophils in circulationmay also suggest immunosuppression status and studiesfocused on the relationship between neoadjuvant chemotherapyNCT and the NLR might support the above hypothesis Acomprehensive review of the existing reports shows that moststudies have found that a low NLR indicates a higher NCTresponse and pathological complete response pCR rate in addition the NLR has showed predictive value not only inall molecular types of BC but also in both operable and locallyadvanced BC Interestingly although Suppan et aldid not ï¬nd a significant correlation between the initial NLR andprognosis the same cohort revealed a low NLR as a significantparameter for predicting chemotherapy response p A low NLR was also reported to be associated with a higherresponse rate to primary endocrine therapy for locally advancedor metastatic BC Although increasing evidence suggests a close associationbetween the NLR and prognosis in BC several issues remain thatmake clinical application diï¬cult One of the most importantreasons is the lack of a consensus cutoï¬ value As we listhere Table the cutoï¬ values for the NLR in the publishedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerTABLE Characteristics of the studies related to neutrophiltolymphocyte ratioReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSigniï¬cance of NLRNR ysHigh NLR indicates lowersurvival rate p Surgery NCT moSurgery ysNR moUnivariate analysis indicateshigh NLR related to lower RFSp and OS p High NLR indicates lower5year OSMultivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Adjuvanttranstuzumab moHigh pretreatment NLRindicates shorter DFSNoh KoreaKoh KoreaYao ChinaLuminal ABHER2enrichedTNBCERPRpositiveHER2enrichedLuminal ABERPRpositiveHER2enrichedTNBCPistelli ItalyTNBCUlas TurkeyHER2enrichedJia ChinaER PRpositiveHER2enrichedTNBCBozkurt TurkeyTNBCAsano JapanTNBCn NLR n ¤ NLR ¥ n n NLR ¤ n NLR n n NLR NLR n NLR ¤ n NLR n n NLR n NLR n n NLR n NLR ¤ n n NLR ¤ n NLR n n NLR n NLR n NCT surgery mo moSurgeryadjuvantchemotherapyandradiotherapyNCT ysMultivariate analysis indicateslow NLR is related to superiorDFS p and p Multivariate analysis indicateshigh pretreatment NLR iscorrelated with poor DFS p and OS p Univariate analysis indicateslow NLR is related to favorableprognosis in TNBC patientswho achieved pCR p hazard ratio High pretreatment NLRindicates poor allcausemortality with a multivariableHR of CIHigh NLR upon recurrenceindicates shorter OSrecurrence rates p Low NLR indicates higher OSp Rimando USANonmetastatic BC n NLR ¤ n NLR n Radiotherapychemotherapy moIwase JapanTNBCn NRL n NLR ChemotherapyNRNCT surgery moHernandez et alSpainMiyagawa JapanFerroni ItalyLuminal ABERPRpositiveHER2enrichedTNBCLocally Advanced orMetastatic BCLuminal ABHER2enrichedTNBCn NLR n NLR ¥ n n NLR ¤ n NLR n Qiu ChinaNonmetastaticTNBCn NLR n NLR ¥ n EribulinNRLow NLR indicates better PFSp NCTchemotherapyendocrinetherapytrastuzumabregimensSurgery NCTchemotherapy moHigh pretreatment NLRindicates worse DFS HR and OS HR moLow NLR indicates higher OSp and DFS p ContinuedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alTABLE ContinuedNeutrophils in Breast CancerReferencesCountry Study periodCancer typeMedianage ysNo patientslowhigh NLRTreatmentFollowupSigniï¬cance of NLRIimori JapanLuminal ABHER2enrichedTNBCMando Argentina Early stage BCLee KoreaTNBCXuan ChinaTNBCFujmoto JapanWith high counts oflymphocytesImamura JapanHER2enrichedn NLR n NLR ¥ n n NRL n NLR ¤ n NLR n n NLR n NLR ¥ 293n n NLR n NLR n n NLR n NLR ¥ n Endocrinetherapy moSurgery moNCTNRSurgeryNRNRSurgeryadjuvantchemotherapiesendocrinetherapiesTrastuzumabemtansineNRLow NLR indicates aprolongation of PFS p and OS p High NLR indicates lower DFSp Low NLR indicates superiorOS p and DFS p Low NLR indicates longer DFSp Low NLR indicates better RFSp Low NLR at baseline indicatesbetter PFS p andOS p NLR Neutrophiltolymphocyte ratio ER Estrogen receptor PR Progesterone receptor HER2 Human epidermal growth factor receptor Mo Months Ys Years DFS Diseasefreesurvival OS Overall survival PFS Progressionfree survival RFS Relapse free survival pCR Pathological complete response TNBC Triplenegative breast cancer NCT Neoadjuvantchemotherapy NR Not recordedstudies were between and In addition based on individualstudies the sensitivity of the NLR ï¬uctuates greatly and the speciï¬city is much lower Therefore some researchers have tried to determine a betteralternative parameter In addition to the NLR the platelettolymphocyte PLR ratio has also been investigated and comparedwith the NLR in BC A single central retrospective study with hormone receptornegative nonmetastatic BC patients reportedthat both elevated NLR and PLR were associated with poor OShowever the multivariate analysis revealed that only the NLR p but not the PLR p was a significant indicatorfor both DFS and OS Additionally since the absolutelymphocyte count has also been reported as a prognostic factorthe predictive values of the PLR and NLR were evaluated afteradjusting for the total lymphocyte count The results showed thatthe PLR was no longer a significant predictor for 5year mortalityand the NLR remained a significant predictor irrespective ofthe lymphocyte count Furthermore it was revealed thatthe combination of the NLR and PLR could further improvethe predictive value Two retrospective studies found that thehighest rate of pCR was in the group of patients withan NLRlowPLRlow proï¬le and the lowest rate was inthe group with an NLRhighPLRhigh proï¬le in additionwhen the cutoï¬ values for the NLR and PLR were applied thespeciï¬city of predicting a pCR increased from to Howeverthe causal relationship between the NLR andpoor prognosis in malignant disease has yet to be illuminatedAccording to an assessment with paired peripheral bloodand pancreatic cancer specimens Takakura found thata high NLR was associated with increased tumorassociatedTAMsanditseemsdecreased Thereforetumorassociatedmacrophageslymphocytes but was not significantly related to CD66bltrating neutrophilsthat anincrease in neutrophils in peripheral blood is not necessarilyrelated to the number of TANs Several basic studies havesuggested a unique mechanism ofthe protumor functionof circulating neutrophils protecting circulating tumor cellsCTCs Circulating neutrophils can cluster around tumor cellsand induce tumor cell aggregation aiding tumor cell survivalby hiding them from immune surveillance Neutrophilextracellular traps NETs are webs of decondensed chromatbers conjugated together with histones myeloperoxidaseMPO elastase and other cytoplasmic proteins Recentstudies also found that neutrophils could form many NETs bothin circulation and in tumor lesions and could coordinate withplatelets to capture CTCs and facilitate cancer metastasis In addition neutropenia is very common in cancer patientsundergoing chemotherapy and supportive treatment withgranulocyte colonystimulating factor GCSF can inducea neutrophilic response as a consequence neutrophils areprimed toward a proNETotic phenotype and may suppress thecytotoxic activity of T cells as well as impair immune surveillance On the other hand lymphocytes have the propensityto mount an adaptive antitumor response in malignant disease and decreased lymphocyte numbers are considered to berelated to an insuï¬cient immunologic reaction which mayincrease the risk of tumor relapse or metastasis Clearlya general association between prognosis and the NLR exists inBC but large prospective studies and rigorous research are stillrequired to determine its clinical signiï¬canceFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast CancerMECHANISM OF NEUTROPHILCHEMOTAXIS TO THE TUMORMICROENVIRONMENTNeutrophils are considered the main immune cells that provideprotection against invading pathogens which can be induced bytrauma infection and malignant disease The recruitmentof neutrophils is greatly dependent on certain chemokinesincluding interleukin IL8 also known as CXCL8 CXCL and CXCL2 IL8 is a proammatory cytokineand acknowledged as the most important chemoattractant forneutrophils in the tumor microenvironment IL8 mainlycomes from endothelial cells ECs and monocytes in the tumormicroenvironment upon certain stimulation such as physicalinjury hypoxia chemotherapy or radiotherapy and other celltypes including ï¬broblasts and keratinocytes can secrete IL8 aswell In addition to its chemotactic eï¬ect it was revealedthat IL8 could provoke neutrophils to release NETs to assistcancer cell migration By livecell ï¬uorescence microscopyGupta conï¬rmed that activated ECs could induceNETosis characterized by typical extracellular DNA latticeswhen cocultured with polymorphonuclear neutrophils PMNsand activated ECs In addition activated ECs produceother ammatory cytokines such as Pselectin Eselectinand intercellular adhesion molecule ICAM1 to facilitateneutrophil adhesion to ECs and migration Furthermoretumorpromoting neutrophils in BC cells are also characterizedby high expression of matrix metalloproteinases9 MMP9 which was found to cleave CXCL5 potentiating itsaction in neutrophil recruitment as a positive feedback functionin tumors IL17 was also found to control neutrophilrecruitmentin lung metastasis of BC in a mouse modelCD3CD4 and γδ T cells were the major sources of IL17 and it was interesting to ï¬nd that the absence of γδT cells or neutrophils markedly reduced pulmonary and lymphnode metastases without uencing primary tumor progressionwhich suggested a collaborative relationship between γδ T cellsand neutrophils in promoting BC lung metastasis Howeverin an orthotopic hepatocellular carcinoma model Soï¬a et alreported that TANs exert an overt antitumor role by suppressingγδ T17 cells via reactive oxygen species ROS contraryto the phenomenon that within the 4T1derived BC modelCD11bLy6G neutrophils that ltrate and surround livermetastases were found to be tumor promoting Thesecontroversial results suggest both promoting and suppressiveroles of TANs in diï¬erent circumstancesHighmobility group box HMGB1 usually acts as adamageassociated molecular pattern that is released by dyingcells or stressed cells to initiate ammation and was laterfound to be an important chemoattractant for neutrophils Epithelial cellderived HMGB1 was found to recruit neutrophilsto the necrotic site through its receptor RAGE Enrichmentof platelets has been reported in the microenvironment ofmultiple cancers including BC and ltrating plateletscould be activated by the large amounts of adenosine phosphatereleased by necrotic cells as a result of chemotherapy Activated plateletderived HMGB1 known as the major mediatorof injuryinduced thrombosis in vivo can also stimulateNETosis through Tolllike receptor TLR4 and RAGE onneutrophils and as a positive feedback mechanism releasedNETs strongly induce a prothrombotic state and activate platelets Meanwhile tumor cellderived exosomal HMGB1 wasalso found to activate neutrophils through the TLR4NFκB pathway which promotes its survival by increasing theautophagic response and polarizing TANs to a protumor type It is noteworthy that various reports imply the coreposition of the NFκB pathway in the activation and recruitmentof neutrophils In addition to HMGB1 tumor cellsincluding BC cells have been reported to secrete other peptidessuch as a2 isoform VATPase a2V to activate the NFκBpathway in neutrophils thereby promoting their recruitment andinhibiting their apoptosis Additionally breast involutionafter weaning is characterized by acute ammation and anincrease in estrogen It was found that estrogen could inducethe mammary ltration of neutrophils and upregulate theexpression of protumor cytokineschemokines such as COX2and MMPs in mammary ltrating neutrophils similarIn additionto lymphocytes and macrophagesneutrophils are more likely to localize in tumors of triplenegativebreast cancer TNBC than to tumors of other BC subtypes Recently Zhang identiï¬ed neutrophils and macrophages asthe most frequent ltrating immune cells in various BC murinemodels and BC could be classiï¬ed into a macrophageenrichedsubtype MES and a neutrophilenriched subtype NES It wasinteresting to ï¬nd that there were only a few neutrophils inthe MES but a large number of macrophages in the NES This mutual repelling phenomenon in the MES and NES mayresult in spatial segregation within the same tumor The authorsspeculated that a possible mechanism could be the factors derivedfrom macrophages that inhibit the IL8dependent chemotaxis ofneutrophils ANTITUMOR FUNCTION OF TANs IN BCThe polarization of neutrophils can be diï¬erentially regulatedin the tumor microenvironment In a mouse model Fridlender found that TANs from the early tumor stage were liketumorkilling cells which produce high levels of hydrogenperoxide H2O2 tumor necrosis factor TNFα and NOand that TANs are more likely to obtain a protumorigenicphenotype with tumor progression Although few studieshave directly compared the phenotype and function of TANsbetween early and latestage tumors there are still some clues tosupport this hypothesis A phenotypical and functional analysisof TANs in earlystage lung cancer found an activated phenotypeCD62lowCD54high that was able to stimulate T cell proliferationand IFNγ release which suggested a proammatory ratherthan immunosuppressive state of TANs in earlystage lungcancer MPO is an enzyme characteristic of mature N1type neutrophils which are able to convert H2O2 to cytotoxichypochlorous acid HOCI Recently a retrospectivestudy of BC cases revealed that MPOpositive neutrophilsFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerin additiondeï¬ned as ¥ cellstissue punch were found in of evaluablecases while the luminal ERPR and Her2 Her2enriched andtriplenegative types had positive rates of and respectivelyltrationby MPOpositive neutrophils was a significant independentfavorable indicator for both OS and DFS Notably almost all ofthe patients included in this study had earlystage disease T1 N01 and the data suggested that MPOpositiveneutrophils were much more abundant in BC cases with low Tand N stages than in advanced cases in univariate analysesIn addition a direct tumor killing function of neutrophils hasalso been reported One of the classical factors working againsttumor cells is ROS Recent research in mouse BC models revealedthat ROSmediated cell lysis was dependent on Ca2 channelsand mediated by transient receptor potential cation channelsubfamily M member TRPM2 expression on tumor cells Although TCGA analysis revealed a high expression ofTRPM2 in BC cells httpgepia2cancerpkucnindex activeNOX1 catalase and SOD were also increased in the membraneof cancer cells forming a complex mechanism by which tumorcell apoptosis induced by ROS is prevented In additiontumor cells are characterized by enhanced metabolic activity andhigh levels of intracellular ROS which indicates that directcytotoxic eï¬ects of neutrophilproduced ROS are not suï¬cientIn addition to the direct cytotoxic eï¬ect TANs containing ROShave been found to strongly suppress IL17producing γδ Tcells which are critical for shaping the immune suppressivemicroenvironment in various solid tumors and have alsobeen reported to promote BC cell extravasation and metastasis In addition neutrophils could also express Fc receptors andexert antibodydependent cellular cytotoxicity ADCC eï¬ectssimilar to those of T cells and macrophagesleading to atrogocytosis eï¬ect to destroy cancer cells However somestudies have indicated that neutrophils are more likely to bedistributed at the periphery of tumors at the initiation stage which may make controlling tumor growth with thesecellcell contactdependent mechanisms ineï¬ectivePROTUMOR EFFECTS OF TANsMore studies suggest that neutrophils facilitate tumor promotionand metastasis in BC than antitumor eï¬ect Overexpression ofthe chemokines CCL2 and CCL17 is a recognized feature ofN2 neutrophils Richmond found that exogenousCCL2 enhances the killing eï¬ect of neutrophils against BCcells in vitro while this antitumor activity was not observedin vivo Instead intranasal delivery of CCL2 to BALBc micemarkedly enhanced lung metastasis of BC cells and increasedthe recruitment of CD4 T cells and CD8 central memory Tcells CCL17 secretion from TANs was found to support tumrowth by recruiting CD4 Treg cells and macrophages In addition to recruiting immunesuppressive cells TANs werereported to promote the accumulation of BC cells in the lungand directly inhibit natural killer NK cellmediated clearanceof tumor cells Human NK cells can be divided intoCD56dim antitumor and CD56bright protumor subsets andCD56bright NK cells are enriched in the tumor microenvironmentand draining lymph nodes Early reports revealed thatROS and arginase1 from neutrophils impair the maturation andcytotoxic function of NK cells but CD56brightCD16 NKcell are resistant to neutrophilderived ROS perhaps due to theirhigh antioxidative capacity Meanwhile NK cells couldbe recruited by TANs via CCL2 and CCL5 which may explainthe preferential accumulation of CD56bright NK cells in tumormicroenvironments with high ROS levels Extracellular arginine is crucial to signal local CD8 cellsand increase their CD3ζ expression which is key for T cellsto survey antigens presented on MHC class I molecules andit was also found to be necessary for T cell activation andsurvival Tumor cellderived IL8 could lead to TANdegranulation resulting in arginase1 release and conversion ofextracellular arginine to ornithine and urea thereby dampeningthe survival and cytotoxic eï¬ect of CD8 T cells Neutrophil elastase NE is also released by TANs and canbe endocytosed by tumor cells via neuropilin1 NRP1 thisresults in the crosspresentation of PR1 which is an NEderivedHLAA2restricted peptide that may be an immunotherapeutictarget Besides upon endocytosis NE is to bind insulinreceptor substrate1 IRS1 which removes the inhibitory eï¬ectof IRS1 on phosphatidylinositol 3kinase PI3K to enhance theproliferation of cancer cells Recentleukocytesreports highlighted thethe important characteristics of malignantespeciallyneutrophils preferentially uptake tumor derived extracellularvesicles or named exosomes Hypercoagulability is oneoftumors andhas been reported associated with NETs Breast cancer cell4T1derived exosomes induced NETs formation in neutrophilsbesides tumorderived exosomes also interacted with NETsto significantly accelerate venous thrombosis in vivo Furthermore several reports also indicated the cancer derivedexosomes prolonged lifespan of neutrophils and also polarizedneutrophils toward protumor type increasingevidence hasIn addition to direct modulation ofthe protumormicroenvironmentfound thatneutrophils promote tumor cell migration and the formation ofa metastatic niche Tumor angiogenesis is regardedas a prerequisite for tumor metastasis and TANs have beenrecognized as an important source of vascular endothelialgrowth factor VEGF upon speciï¬c stimulation in the tumormicroenvironment Neutrophils were also found to beone of the main sources of MMP9 and the link betweenMMP9 and VEGF has been reported previously The absenceof MMP9 has been reported to have a similar function as theinhibition of VEGF signaling indicating that MMP9 serves asan angiogenic switch during tumorigenesis by inducing VEGFrelease from the matrix In addition Gabriele et alalso found that MMP9 was expressed by a small number ofcells in close proximity to the vasculature such as ltratingammatory cells rather than tumor cells In additionseveral serine proteases are also produced by TANs such asNE cathepsin G and proteinase3 which have been reported toactivate MMP2 to promote tumor invasion and proliferation In addition although neutrophils were reportedFrontiers in Immunology wwwfrontiersinAugust Volume 0cZhang et alNeutrophils in Breast Cancerto produce little tissue inhibitor of matrix metalloproteaseTIMP1 Wang observed that BC cells with CD90positiveexpression could induce the TIMP1 secretion by TANs and asa reciprocal eï¬ect TIMP1 induced EMT and metastasis in BC Other neutrophilderived cytokines such as IL1 IL6and IL17α have been reported to initiate EMT of cancer cells byactivating JAK2STAT3 and ERK signaling Inadditiontheprimaryto modulatingtumormicroenvironment neutrophils can also assist the formationof the cancer premetastatic niche in distant ans CTCsare precursors for metastatic lesion formationintravascularNETs were found to protect CTCs from attack by circulatingimmune cells and dysregulated NETs were found to induceammatory vascular injury EC shrinkage and tissue damage Moreover in vitro and in vivo experiments foundthat activated neutrophils promote the adherence of CTCs toECs and facilitate their lung and liver metastasis RecentlyAceto provided strong evidence that neutrophils escortCTCs in BC to assist metastasis With detection of cellsurface markers and Wright Giemsa staining they identiï¬ed thatmost CTCassociated white blood cells were N2like neutrophilsIn addition singlecell RNA sequencing revealed higher Ki67expression in disseminated tumor cells from CTC neutrophilclusters than in standalone CTCs In the same study TNFαoncostatin M IL1 and IL6 were frequently expressed byCTCassociated neutrophils and matched by the receptors oncorresponding CTCs on the other hand CTCs from the CTCneutrophil clusters expressed high gene levels encoding GCSFtransforming growth factor TGF3 and IL15 which havebeen reported to activate neutrophils illuminating amechanism of neutrophilCTC cluster formationIn addition to escorting CTCs in circulation several studieshave found that neutrophil accumulation is a prerequisitefor cancer metastasis For both orthotopic transplantationand spontaneous BC models neutrophils were suggestedto accumulate in the distant an before cancer cellsltration Obesity and elevated cholesterol arerisk factors for BC development and poor prognosis Interestingly 27hydroxycholesterol 27HC increased thenumber of polymorphonuclearneutrophils and γδ T cells atdistal metastatic sites and neutrophils were required for themetastatic eï¬ects of 27HC Egeblad developeda confocal intravital lung imaging system and found that NETswere formed early in the lung and continued to form forthe next few days after tail vein injection of BC cells Inaddition based on immunoï¬uorescence staining of humanprimary BC and matched metastatic lung lesions they foundthat the abundance of NETs was highest in TNBC but NETswere absent or very rare in luminal BC samples which mayexplain the higher metastatic ability of TNBCs than luminalBCs In ovarian cancer an ux of neutrophils in the omentumwas also observed before metastasis and blockade of NETformation with peptidyl arginine deiminase PAD4 an enzymethat is essentia | Thyroid_Cancer |
Breast cancer is a common malignancy in women Among breast cancer types triplenegative breast cancer TNBC tends to affect younger women is prone to axillary lymph node lung and bone metastases and has a high recurrence rate Due to a lack of classic biomarkers the currently available treatments are surgery and chemotherapy no targeted standard treatment options are available Therefore it is urgent to find a novel and effective therapeutic target As alteration of ion channels and transporters in normal mammary cells may affect cell growth resulting in the development and progression of TNBC ion channels and transporters may be promising new therapeutic targets for TNBC This review summarizes ion channels and transporters related to TNBC and may provide new tumor biomarkers and help in the development of novel targeted therapiesKeywords Triplenegative breast cancer Ion channels Ion transporters Pathological roles Targeted therapyBackgroundBreast cancer BC is the common malignancy in women its incidence is increased each year [] and it has become a significant threat to womens health [] BC is a heterogeneous disease that can be divided into multiple molecular subtypes based on estrogen receptor ER progesterone receptor PR and human epidermal growth factor receptor HER2 expression providing important prognostic and predictive information [] There are four BC subtypes depending on receptor status luminal A luminal B HER2overexpressing and triplenegative breast cancer TNBC Among them TNBC is defined as ER PR and HER2 negative and it tends to affect younger women a0years of age it is Correspondence onlyoneliuxuemei163com 0078029sinacom Chengli Lu and Zhiyuan Ma contributed equally and share first authorship Department of Thyroid and Breast Surgery Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province China Department of Gastroenterology Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province ChinaFull list of author information is available at the end of the prone to axillary lymph node lung bone metastases and has a high recurrence rate [ ] Lehmann et a0al classified TNBC into six subtypes based on gene cluster sequence expression basallike basallike immunomodulatory mesenchymal mesenchymal stemlike and luminal androgen receptor subtypes [] After analyzing the RNA and DNA profile of TNBC tumors Matthew et a0 al classified TNBC into four subtypes including luminal androgen receptor mesenchymal basallike immunesuppressed and basallike immuneactivated subtypes [] The two classification methods have similarities and both provide theoretical bases for exploring targeted therapies for TNBCAlthough TNBC is the BC subtype that responds best to chemotherapy its recurrence and metastasis rates are higher than those of other BC subtypes [] Furthermore due to the lack of classic biomarkers TNBC lacks standard treatments guided by tumor biology and only surgery and chemotherapy are currently available as treatments [] Previous studies have shown that ion channels and transporters play important regulatory roles in mammary physiology and the initiation and progression of BC 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 0cLu a0et a0al Cancer Cell Int Page of [] However the detailed functional role of ion channels and transporters in TNBC has not been clarified and summarized In recent studies upregulation of NaH exchanger has been shown to promote the proliferation migration and invasion of the TNBC cell line MDAMB231 [ ] In addition Ca2 channels such as mitochondrial calcium uniporter MCU can promote TNBC cell migration invasion and lung metastasis [] and Alvarez et a0al [] reported that the twopore domain potassium channel KCNK5 is associated with a poor prognosis in TNBC Therefore ion channels and transporters play important regulatory roles in the pathophysiology of TNBC but there is currently no relevant review on this topic Here we review the pathological roles of ion channels and transporters including AQPs Cl channels Ca2 channels K channels and acidbase transporters in the initiation and progression of TNBCAQP channelsAQPs which compose a family of transmembrane water channel proteins modulate the movement of water and small solutes into and out of cells and maintain suitable concentrations of water and solutes for cell survival [] At least AQP subtypes AQP012 have been identified in mammals and are divided into two families based on transfer specificity namely the classic watertransporting AQP family and the solute water and glyceroltransporting glycoprotein family [] AQP02 AQP4 to AQP68 are mainly waterselective AQP3 AQP7 AQP9 AQP10 and AQP12 also transport glycerol and possibly other small solutes AQPs also play roles in the transport of ammonia urea carbon dioxide metalloids nitric oxide and certain ions [] Expression of AQP1 AQP35 and AQP1012 has been detected in normal human mammary tissue and is closely related to milk secretion [ ] In addition deletion of CCAATenhancer binding protein a family of transcription factors isoforms results in changes in mammary ductal morphogenesis and changes in expression of transport proteins such as AQP5 suggesting that AQP5 may be involved in mammary development [] Recent studies have shown that AQPs play carcinogenic roles by promoting angiogenesis enhancing invasive and metastatic potential and enhancing the transport of reactive oxygen species ROS [ ] In femalespecific cancers such as BC AQP1 and are the most important AQPs and they are been reported to be upregulated []AQP1 the membrane protein was the first reported mammalian AQP and plays a significant role in tumor cell migration proliferation and angiogenesis [] Clinical studies have shown that patients with TNBC have higher levels of AQP1 expression and that upregulation correlates with a poor prognosis [ ] AQP1 expression is induced by hypoxia through the EBoxChoRE transcription element which is affected by increased glucose consumption and metabolism [] AQP1 expression has been detected only in a subgroup of CK14positive basallike breast cancer BLBC cases [] CK14 has been used as a marker of basal mammary epithelial cells with in vivo regenerative ability in studies on mammary gland progenitor and stem cells [] Therefore it is speculated that expression of AQP1 is related to the stem cell characteristics of BLBC cells Hu et a0al demonstrated that AQP1 upregulation promotes extravasation and increases migration in a0vivo and in a0vitro in the mouse TNBC cell line 4T1 suggesting that this aquaporin enhances the rate of cell migration by promoting water permeability in cell protrusions [] Thus upregulation of AQP1 can promote the proliferation migration and invasion in TNBC cells Moreover in a0 vivo experiments have shown that AQP1 deficiency can reduce tumor mass volume vessel density and lung metastases in MMTVPyVT mouse mammary tumor virusdriven polyoma virus middle T oncogene mice and inhibition of AQP1 function andor expression is predicted to attenuate angiogenesis via reduced migration and invasion of endothelial cells [] Recently Irene AbreuRodriguez et a0al [] revealed that AQP1 expression is also responsive to hypoxiainducible factor HIF which may play a role in the VEGFindependent signaling mechanism inducing angiogenesis in a hypoxic environment Helen et a0al [] also reported that the triterpenoid saponins bacopaside I and bacopaside II can synergistically reduce the transcriptional expression of AQP1 and inhibit proliferation migration and invasion in MDAMB231 cells Similarly ginsenoside Rg3 a compound with anticancer activity isolated from ginseng inhibits AQP1 to attenuate cell proliferation through a mechanism that involves downregulation of AQP1 to induce cell cycle arrest in G0G1 phase by inhibiting cyclin D and E and inhibition of chemoattractantinduced cell migration and invasion by blocking AQP1mediated water flux in MDAMB231 cells [] These findings indicate that AQP1 plays an important role in the development and progression of TNBCOverexpression of AQP3 has been detected in the membranes and cytoplasm of TNBC tumor cells and is significantly associated with poor prognosis [] XuChen Cao et a0 al [] found that the presence of fibroblast growth factor2 FGF2 induced cell migration and metastasis in MDAMB231 cells by increasing AQP3 expression Moreover FGF receptor kinase FGFRK inhibitors PI3K inhibitors and MEK12 inhibitors all inhibit AQP3 expression suggesting that FGF receptor kinases increase AQP3 expression and promote FGF2induced cell migration by initiating downstream PI3K and ERK pathways In addition CuSO4 a water transport 0cLu a0et a0al Cancer Cell Int Page of inhibitor of AQP3 inhibits migration in MDAMB231 cells AQP3 downregulation reduces the proliferation invasion and migration of MDAMB231 cells while increasing sensitivity to 5fluorouracil chemotherapy The mechanism may be related to a decrease in glycerol permeability caused by AQP3 downregulation [] Overall these findings demonstrate that AQP3 plays a pivotal role in the initiation and progression of TNBC and specific inhibitors of AQP3 in clinical applications may improve the therapeutic effect of TNBC patientsSimilarly overexpression of AQP5 in the membrane and cytoplasm of TNBC cells has been detected and is significantly associated with poor prognosis [] Moreover patients with higher Ki67 expression are more likely to have abnormal AQP5 protein expression than patients with lower Ki67 expression [] Ki67 is a widely accepted proliferation marker [ ] and it is speculated that upregulation of AQP5 may promote proliferation in TNBC cellsIn summary AQP1 AQP3 and AQP5 are significantly upregulated in TNBC this upregulation is related to a poor prognosis and can promote the proliferation migration and invasion of TNBC cells These AQPs are promising new targets for the diagnosis and treatment of TNBCCl channelsCFTRCFTR is a member of the ATPbinding cassette transporter family that localizes at the apical membranes of normal epithelial cells CFTR is mainly responsible for conducting HCO3 and Cl and promoting HCO3 secretion in many tissues including the airway intestines and pancreas [] However when the extracellular concentration of Cl is higher than a0mmolL the permeability of CFTR to Cl is much greater than that of CFTR to HCO3 thus CFTR mainly conducts Cl under physiological conditions [] CFTR can also transport two other anions glutathione and thiocyanate which are involved in airway inflammation and oxidative stress [ ] Interestingly Pierre et a0al [] reported that CFTR is required for the tightly connected functions of normal epithelial tissues loss of CFTR reduces epithelial resistance and epithelial integrity and this effect is not related to the anion channel function of CFTRCFTR has been reported to be associated with several cancers such as cervical cancer [] colorectal cancer [] prostate cancer [] and BC [] Significant downregulation of CFTR expression is observed in BC tissue compared to normal mammary tissue [] Zhang et a0al demonstrated that overexpression of CFTR inhibits EMT invasion and migration in MDAMB231 cells via a mechanism that involves CFTR inhibition of NFκB targeting of urokinasetype plasminogen activator [] In addition CFTR overexpression inhibits the EMT and the invasiveness of MDAMB231 cells and reduces lung metastasis in xenograft models Increasing evidence reveals that downregulation of CFTR occurs after treatment with EMTinducing factors such as TGF suggesting that as a downstream effector CFTR plays important roles in mediating various EMT effects [ ] Moreover hypermethylation of the cancer genome leads to activation of oncogenes or suppression of tumorsuppressor genes thereby resulting in tumorigenesis [] It has also been observed that the methylation level of CFTR in BC tissues is much higher than that in normal tissues and treatment with DNA methylation inhibitors in TNBC cell lines MDAMB231 and MDAMB435 can rescue CFTR mRNA indicating that CFTR methylation plays an important role in TNBC [] ÎF508 is the most common mutation in CFTR causing the protein to be retained and degraded in the endoplasmic reticulum due to misfolding [] It is worth noting that although there is no difference in the incidence of BC between ÎF508 carriers and noncarriers patients with ÎF508 CFTR mutations all have grade III cancer indicating that CFTR defects are associated with BC progression [] Therefore CFTR methylation or mutation need to be further investigated in the future which may provide novel therapeutic intervention for TNBCChloride channel The chloride channel ClC family also plays an indispensable role in the transport of Cl [] There are nine family members in humans which are divided into two categories based on their distribution and physiological function Cl channel proteins ClC1 ClC2 ClCKa and ClCKb which mainly exist in the plasma membrane and play roles in stabilizing membrane electric potential or mediating epithelial transport and ClH reverse transporter proteins ClC37 which mainly exist in the vascular intima of the endosomelysosomal pathway and are localized at the plasma membrane only to a limited extent due to protein degradation and hydrolase activity [ ] In recent years it has been discovered that ClC3 can transport one hydrogen ion in exchange for two chloride ions [] with important roles in cancers such as nasopharyngeal carcinoma [] and BCClC3 overexpression is observed in tissues and the TNBC cell line MDAMB231 [ ] Studies by Zhou et a0 al revealed that knockdown of ClC3 downregulates expression of cyclin D1 and cyclin E and increases levels of p21 indicating that knockdown of ClC3 can block the cell cycle of MDAMB231 cells at G0G1 phase inhibiting cell proliferation Moreover knockdown of ClC3 suppresses tumor growth in xenograft models and significantly reduces levels of pERK12 in MDAMB231 cells 0cLu a0et a0al Cancer Cell Int Page of This indicates that ClC3 can promote the progression of TNBC by acting on the ERK12 signaling pathway [] Nevertheless relative research on ClC3 in TNBC is still very limited and extensive work is needed in the furtherCa2 channelsCa2 is a key nutrient in milk that plays a vital role in the mineralization of bones and teeth and as a second messenger ionized Ca2 is a key regulator of proliferation migration cell cycle progression and apoptosis [] The level of Ca2 is very low in the cytoplasm a0molL whereas it is somewhat higher in anelles a0 molL and highest in the extracellular level milieu a0 molL Hence a small amount of Ca2 can significantly change intracellular levels to activate downstream signaling molecules including calmodulin nuclear factor of activated Tcells NFAT NFκB calmodulindependent protein kinase II calpain and others [ ] In nonexcitatory mammary cells calcium channels play important roles in lactation and the maintenance of normal physiological functions [ ]Continuous increases in intracellular Ca2 levels will drive expression of oncogenes resulting in tumor growth and development especially the metastatic behavior of cancer cells and conferring tumor cells with resistance to apoptosis [] Abnormal expression of several Ca2 transporters and ion channels such as calcium releaseactivated calcium modulator Orai1 has been observed in TNBC and may lead to oncogenic Ca2 signaling [] Interestingly specific changes in the expression and function of Ca2 channels are related to hormone receptor status and differ significantly among BC subtypes []Calcium modulator Ca2 influx mainly depends on storeoperated calcium channels SOCCs When the Ca2 concentration in the endoplasmic reticulum declines to a certain level the STIM stromal interaction molecule which is located on the endoplasmic reticulum membrane moves to a position close to the highly selective calcium channel protein Orai on the cell membrane Subsequently Orai is activated to cause Ca2 influx and storeoperated calcium entry SOCE is initiated thereby replenishing the calcium store Some researchers have proposed that the canonical transient receptor potential TRPC also participates in the above process though the mechanism remains controversial There are two different claims that both Orai and TRPC form independent channels activated by the STIM protein and that Orai and TRPC subunits form heterochannels triggered by STIM [] There are three Orai1 isomers Orai1 to Orai3 and two STIM homologs STIM1 and STIM2 SOCE has been found to be primarily mediated by Orai1 and STIM1 in TNBC [] Compared with that in nonmalignant breast epithelial cells expression of Orai1 and STIM1 is significantly higher in TNBC cell lines and is associated with a poor prognosis [ ] Liu et a0 al [] reported that hypoxia can induce expression of Orai1 Notch1 and Jagged1 and Orai1 is significantly downregulated after blockade of Notch signaling suggesting that hypoxia can increase Orai1 expression in TNBC by activating Notch signaling Notch1Orai1SOCENFAT4 axis Similarly Mognol et a0al [] found that Orai1 promotes the invasion and angiogenesis of TNBC cell lines and activates NFAT4 which can regulate genes involved in the cell cycle apoptosis angiogenesis and metastasis In addition Yang et a0 al [] demonstrated that Orai1 and STIM1 promote the migration and invasion of MDAMB231 cells both in a0vivo and in a0vitro and the authors proposed that these proteins may at least partially control cell migration by regulating focal adhesion turnover Furthermore treatment with TGF can reduce expression of STIM1 whereas blockade of SOCE can impair TGFinduced G0G1 cell cycle arrest and inhibit the proliferation of MDAMB231 cells [] Based on the above research Orai1 and STIM1 may be new therapeutic targets for TNBC Indeed some selective SOCE inhibitors have shown encouraging inhibitory effects on TNBC but they are still only in the preclinical trial stage For example phemindole a diindole derivative reduces SOCE by downregulating STIM1 expression significantly inhibits the proliferation and migration of MDAMB231 cells reduces the growth of solid tumors in mouse models and produces a targeted antitumor effect in TNBC [] In addition Miroslava Didiasova et a0al [] revealed that elevated cell surfaceassociated enolase1 ENO1 expression correlates with augmented MDAMB231 cell migratory and invasive properties Pharmacological blockade a selective SOCC inhibitor NS1643 or knockdown of STIM1 or Orai1 reduces ENO1dependent migration of MDAMB231 cells These results demonstrate the pivotal role of SOCE in the regulation of ENO1 exteriorization and thus in the modulation of TNBC cell migratory and invasive properties indicated that Orai1 and STIM1 might be promising threptic targets for TNBCSecretory pathway Ca2ATPaseThe secretory pathway Ca2ATPase SPCA can direct Ca2 and Mn2 from the cytoplasm to the Golgi and postGolgi vesicles Two isotypes SPCA1 and SPCA2 are known and the distribution and function of the two differ SPCA1 is commonly expressed in mammalian tissues expression of SPCA2 is limited to highly absorptive and secretory epithelial cells including mammary 0cLu a0et a0al Cancer Cell Int Page of and salivary gland cells [] SPCA1 is highly expressed in TNBC cell lines and SPCA2 is highly expressed in cell lines of other subtypes [] Interestingly based on clinical samples Desma et a0al reported SPCA1 levels to be significantly elevated in the basal subtype of BC compared with all other subtypes and it is worth noting that changes in its expression affect posttranslational modification and transport of certain proteins important for tumor progression without significantly changing cytosolic calcium signaling SPCA1 inhibition also decreased MDAMB231 cell proliferation [] Moreover SPCA1 is a key regulator of insulinlike growth factor receptor IGF1R processing in TNBC cells and promotes the production of functional IGF1R IGF1R activity is associated with poor prognosis suggesting that targeting SPCA1 is an alternative IGF1Rinhibiting strategy [ ] Overall upregulation of SPCA1 may promote the initiation and progression of TNBC The main mechanism reported to date involves SPCA1mediated increase in functional IGF1R expressionMitochondrial calcium uniporterUpregulation of MCU expression on the mitochondrial membrane is closely related to a poor prognosis in BC [] miR340 correlates negatively with the metastatic potential of TNBC cells [] it may directly inhibit MCU expression to reduce glycolysis and exercise capacity and knockdown or inhibition of MCU inhibits the growth invasion and metastasis of MDAMB231 cells [] Interestingly Anna et a0 al [] demonstrated that mitochondrial Ca2 uptake is required for TNBC progression in a0vivo and that absorption of Ca2 by mitochondria promotes the production of sustained mitochondrial reactive oxygen species activating the HIF1α signaling pathway and promoting tumor growth and metastasis In addition inhibiting or silencing MCU also block seruminduced migration of MDAMB231 cells and reduce serum or thapsigargininduced SOCE suggesting that MCU promotes TNBC cell migration by regulating SOCE [] The above results indicate that overexpression of MCU may play an important oncogenic role in the growth invasion and metastasis of TNBC cells However the precise mechanism is unclearOther promising calcium channel targets in TNBC include TRPV6 [] Overall calcium channels are promising targets for TNBC treatment but most compounds targeting these channels are only in the preclinical trial stage Thus further research is neededK channelsThrough the action of NaKATPase two K molecules are transported into a cell in exchange for three sodium molecules which increases the intracellular K concentration K channels on the cell membrane are numerous and in humans more than genes encode major K channel subunits [] K channels play key roles in maintaining acidbase balance by functioning in concert with the NaH exchanger and NaKATPase [] controlling electrical excitability of nerves and muscles and participating in energy metabolism and other physiological processes In addition K channels can help regulate cell proliferation and cell cycle progression and are involved in tumorigenesis [] Many studies have reported dysregulated K channel expression in human cancers including BC astrocytictype brain cancer and prostate cancer [ ] Tumorrelated K channels can be divided into four main categories according to their domain structures and activation mechanisms voltagegated potassium channels which are controlled by changes in membrane potential calciumactivated potassium channels which are activated by intracellular calcium inwardly rectifying potassium channels and twoporedomain potassium channels K2P KCNK [] However the carcinogenic mechanism of K channels remains rather clear Nuria et a0 al [] proposed that K channels may participate in and regulate tumor progression through permeationrelated mechanisms including changes in membrane potential Ca2 driving forces and cell volume regulation and nonconductive mechanisms dependent on proteinprotein interactionsThe Kv111 channel also known as human etheragogorelated gene hERG1 is not expressed in normal breast cells but is expressed in BC with a relationship with subtype Indeed TNBC exhibits lower expression of Kv111 compared with other subtypes [] Olivia Crociani et a0al [] showed that the mRNA levels of Kv111 change throughout the cell cycle peaking in G0G1 phase Moreover Lansu et a0al [] reported that stimulation of Kv111 led to inhibition of proliferation in MDAMB231 cells and that an agonist the diphenylurea derivative NS1643 caused a significant inhibition of cell proliferation This phenomenon can be linked to a rapid decrease in the cyclin E2 protein level which causes accumulation of cells in G0G1 phase and an increase in tumor suppressor proteins and markers for cellular senescence including p21 p16INK4a and galactosidase activity Therefore Kv111 inhibits TNBC cell proliferation by activating a cellular senescence program [] Breuer et a0 al confirmed that NS1643 reprograms the EMT by attenuating the Wntcatenin signaling pathway inhibits cancer cell stemness and significantly reduces the metastatic spread of breast tumors in a MDAMB231 mouse model [] Regardless cardiotoxicity is an important limiting factor for potential therapeutic molecules acting on Kv111 Although the activator is well tolerated 0cLu a0et a0al Cancer Cell Int Page of in BC potential effects include tachycardia [] Overall the potential benefits of Kv111 activators as anticancer drugs outweigh their side effectsIn addition many other channels are altered in TNBC For example some K2P channels with differential expression may serve as novel molecular markers associated with TNBC RNASeq analysis of K2P channels has shown that overexpression of KCNK5 KCNK9 and KCNK12 and low expression of KCNK6 and KCNK15 are related to TNBC [] The above findings indicate that K channels play an important role in TNBC and are expected to be diagnosis markersAcidbase transportersThe pH of milk is significantly lower than that of plasma indicating that there may be some acidbase transporters in the mammary gland that regulate the pH between the extracellular fluid and milk [] A uniform feature among solid tumors with high metabolic and proliferative rates is a significantly different pH from that of normal tissue [] Cancer cells can maintain a weakly acidic intracellular pH that is even more alkaline than that of normal cells suggesting that tumor cells have a powerful pH regulation system a0[]The NaH exchanger NHE a membrane transporter mainly catalyzes the exchange of intracellular H for extracellular Na in mammals thereby maintaining the pH balance inside and outside the cell [ ] There are subtypes of NHE with tissue and membranespecific expression patterns NHE15 are located on the plasma membrane and NHE69 are on intracellular anelle membranes NHE10 is only expressed in osteoclasts [] In addition NHE plays indispensable roles in maintaining normal mammary structure and physiological functions [] NHE1 SLC9A1 is universally expressed in epithelial cells and upregulated in BC tissues compared to normal tissues [] Studies have shown that hypoxia various growth factors and hormones among others can activate NHE1 and enhanced NHE1 activity can reduce extracellular pH and promote metastasis of MDAMB231 cells [] Furthermore it has been proposed that NHE1 promotes metastasis and remodeling of the extracellular matrix by acidifying the extracellular microenvironment [] In addition NHE1 knockdown reduces the migration invasion and growth of xenograft tumors of MDAMB231 cells increasing the susceptibility of these cells to paclitaxel [ ] Moreover knockdown of NHE1 or NBCn1 SLC4A7 in the MDAMB231 cell line significantly reduced the steadystate intracellular pH value after acid load the ability to restore pHi and the primary tumor growth of xenografts in a0 vivo but NBCn1 knockdown prolonged tumorfree survival and reduced cell proliferation [ ] It has been confirmed that NHE1 and NBCn1 promote the development of TNBC through different mechanisms There are two main NHE1 inhibitors amiloride and cariporide which are more effective than amiloride and highly selective [] Amiloride is a potassiumsparing diuretic and has blocking effects on a variety of ion channels such as NHE and the NaCa2 exchanger Cariporide is a highly specific and powerful NHE1 inhibitor that is relatively well tolerated in humans with heart disease [] Moreover a study has suggested that KR33028 a novel small molecule inhibitor of NHE1 produces a cellular phenotype comparable to that of NHE1 knockout cells and significantly decreases rates of migration invasion and colony growth in TNBC cell lines MDAMB231 MDAMB468 and Hs578T [] The above findings suggest that NHE1 may play an important role in the progression TNBCAdditionally other acidbase transporters are also altered in TNBC and are expected to emerge as new targets for TNBC treatment For instance NBCe1 SLC4A4 knockdown reduces cell proliferation invasion and migration in TNBC cells expressing high levels of NBCe1 [] The above findings all suggest that the acidbase transporters have essential functions in the occurrence and development of TNBC but further research is neededConclusionsDysfunction of ion channels and transporters in the mammary resulted in development and progression of TNBC Despite extensive work has been performed to investigate the expression pattern functional diversity regulatory mechanism and pathophysiology of different ion transporters in TNBC the systematic review is rare in this field Therefore this review focuses on different pathological function of multiple families in the development and progression of TBNC including the AQPs Cl channels Ca2 channels K channels and acidbase transporters Fig a0 Table a0 We hope that we can provide a basic systemics and summarised knowledge to this field advocating researchers play more attention on the pathophysiological role of ion channels and transporters in the development and progression of TNBC which may provide novel targets for the clinical diagnosis and treatment of TNBC 0cLu a0et a0al Cancer Cell Int Page of Fig Pathological roles of ion channels and transporters in triplenegative breast cancer cells Alteration and dysfunction of AQPs Cl channels Ca2 channels K channels NaHCO3 transporter and NaH exchanger results in abnormality of ion transport and disorder of multiple signaling pathway including WNT PI3K TGF Notch and VEGF etc eventually promoting TNBC cell proliferation migration and invasion but inhibiting apoptosis | Thyroid_Cancer |
increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no ï¬delity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizens involvement as volunteers in giving healthand disease data to assess the critical situation for diseases spread and prevention in neighbourhood effect scientiï¬canalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sectors increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe ï¬rst integral deï¬nition of public health was given byWinslow as science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individualsThe American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian uenza HPAI H5N1 in withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difï¬culties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman Hanaï¬Bojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconï¬rmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML ï¬les from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to beneï¬t public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no ï¬delityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in ï¬ghting cholera After years the maps wereidentiï¬ed as a communication tool in understanding andtracking of infectious diseases such as the uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classiï¬ed into four generaAlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenusSevere Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoVhadearlier demonstrated that coronaviruses can cause signiï¬cant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a spatial healthinformation infrastructure In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participants number and oftheir spread local and mass and thoroughnesstime and resource investment or King described for the people with the people or by the people about Citizen Science activities Wiggins andCrownston classiï¬ed Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientiï¬c questions and education providingknowledge to citizens Haklay classiï¬ed CitizenScience into four levels based on participants engagement level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in deï¬ning researchstrategies data collection data interpretation and performing scientiï¬c analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is popularepidemiology in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or street science as a process in which general public communities actively engagedin deï¬ning problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is ï¬rst coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using ï¬ve senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undeï¬ned crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term crowdsourcing geographic information Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuites performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe ï¬rst approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents signiï¬cant concerns regarding privacy The digital contact tracing works on the principle ofcrowdsource data by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom TrackerThis mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and Kings College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare workersexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya SetuThis mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using apps crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identiï¬ed potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of worldTrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apples recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as SARSCoV Boulos and the MERSCoVGikonyo and other seasonal ï¬us online realtime or nearrealtime mapping of diseases occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identiï¬cationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identiï¬cation of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versionsprospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHOs ofï¬cial dataDashboard Dong httpscovid19whointGlobal Visualization of ofï¬cial daily counts ofconï¬rmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation conï¬rmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconï¬rmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISROs BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of conï¬rmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric compositionPM10 and NO2Source CAMS EAC4Meteorological datahumidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and speciï¬c humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to peoplesdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically signiï¬cant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare ofï¬cials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The signiï¬cant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is deï¬ned by Hawkins as an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism In other words when data generation processstarts behaving abnormal and reï¬ects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to ï¬nd clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity scienceecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Theï¬rst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically deï¬ned as follows¼ l N þ Sðà 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Ãð2Ãð3à 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem | Thyroid_Cancer |
Deacetylases inSkeletal Muscle Physiology andSystemic Energy HomeostasisImplications for Metabolic Diseasesand TherapyHaili Tian1 Sujuan Liu2 Jun Ren3 Jason Kai Wei Lee456 Ru Wang1 and Peijie Chen1 School of Kinesiology Shanghai University of Sport Shanghai China Department of Anatomy and Histology Schoolof Basic Medical Sciences Tianjin Medical University Tianjin China Department of Cardiology Shanghai Instituteof Cardiovascular Diseases Zhongshan Hospital Fudan University Shanghai China Department of Physiology Yong LooLin School of Medicine National University of Singapore Singapore Singapore Global Asia Institute National Universityof Singapore Singapore Singapore N1 Institute for Health National University of Singapore Singapore SingaporeSkeletal muscle is the largest metabolic an in the human body and is able torapidly adapt to drastic changes during exercise Histone acetyltransferases HATs andhistone deacetylases HDACs which target histone and nonhistone proteins are twomajor enzyme families that control the biological process of histone acetylation anddeacetylation Balance between these two enzymes serves as an essential elementfor gene expression and metabolic and physiological function Genetic KOTG murinemodels reveal that HDACs possess pivotal roles in maintaining skeletal musclesmetabolic homeostasis regulating skeletal muscles motor adaptation and exercisecapacity HDACs may be involved in mitochondrial remodelinginsulin sensitivityregulationturn onoff of metabolic fuel switching and orchestrating physiologicalhomeostasis of skeletal muscles from the process of myogenesis Moreover manymyogenic factors and metabolic factors are modulated by HDACs HDACs areconsidered as therapeutic targets in clinicaltreatment of cancerammation and neurological and metabolicrelated diseases This review will focuson physiological function of HDACs in skeletal muscles and provide new ideas for thetreatment of metabolic diseasesresearch forKeywords histone deacetylases exercise capacity skeletal muscle metabolism muscle physiologyINTRODUCTIONSkeletal muscle is the largest metabolic an in the human body consuming about of theentire body daily expenditure of energy Durnin It produces various secrete factors andparticipates in the interplay among multiple tissues and ans Pedersen and Febbraio Mizgier Muscular contraction is one of the main physiological functions of skeletalmuscles and plays a role in maintaining an and systemic metabolic homeostasis Guo Proper exercises help build up body defense to combat various diseases including obesitytype diabetes Alzheimer disease osteoarthritis and so on Luan These importantEdited byBrian James MorrisThe University of Sydney AustraliaReviewed bySean L McGeeDeakin University AustraliaViviana MoresiSapienza University of Rome ItalyCorrespondenceRu WangwangrutysuseducnPeijie Chenchenpeijiesuseducn These authors have contributedequally to this workSpecialty sectionThis was submitted toIntegrative Physiologya section of the journalFrontiers in PhysiologyReceived May Accepted July Published August CitationTian H Liu S Ren J Lee JKWWang R and Chen P Roleof Histone Deacetylases in SkeletalMuscle Physiology and SystemicEnergy Homeostasis Implicationsfor Metabolic Diseases and TherapyFront Physiol 103389fphys202000949Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise Capacityfunctions require delicate regulations in muscle from thelevel of genome to signal transduction establishing muscleplasticity and responses to environmental stress AcetylCoAas a central metabolite of amino acidfatty acidglucose notonly serves as fuel for energy expenditure but also bridgesthe gap between environmental stress and anismal responseLempradl Liu This control occursthrough posttranslational modiï¬cation on nucleosomes histonelysine residues and other signal transducing proteins whichin turn controls accessibility of DNA to regulatory factorsand protein activity Acetylation one of the most prevalentmodiï¬cations of proteinis believed to function as a keymodulator of chromatin structure and signal transduction andprovides an avenue to couple extracellular stimuli with genomeduring muscle metabolism process by regulating acetylation anddeacetylation Haberland histone deacetylasesHDACs as a family of proteindeacetylases have been demonstrated to moderate physiologicalhomeostasis and development by deacetylation Haberland Table In humans there are types ofHDACs which are classiï¬ed into four categories based onhomologous proteins in yeast namely Class I Rpd3like proteinHDAC1 Class II Hda1like proteins are classiï¬edas Class IIa HDAC4 and Class IIb HDAC6 Class III Sir2plike nicotinamide adenine dinucleotideNADdependent SIRT1 and ClassIV HDAC11 containing homologous domain with bothRpd3 and Hda1 Seto and Yoshida HDACs modulategene expression and protein activity through deacetylatingproteins When histone lysine εamino acid in the nucleosomeis acetylated it can neutralize positive charge before looseningchromatin structuretranscriptionfactors to DNA and expression of downstream target genesBy contrast histone deacetylation compresseschromatinstructure thereby inhibiting transcriptional gene expressionShahbazian and Grunstein to promote binding ofBiochemical properties of HDACs have been comprehensivelyreviewed Shahbazian and Grunstein Howeverthephysiological functions of HDACs have yet to be well examined inskeletal muscles A series of researches shed light on the potentialof drugs targeting HDACs to improve muscle ï¬tness and cardiacmuscle disease which needs further clariï¬cation of HDACsphysiological function to understand the mechanisms Bai Galmozzi Xie Zhang and Ren Gaur Ample work has revealed the role of HDACin muscle physiology such as skeletal muscles me olism and thusexercise capacity McGee and Hargreaves Table Class IHDACs can interact with myocyte enhancer factor MEF2MyoD regulating myogenesis and exercise capacity Mal Puri Ohkawa Gregoire HDAC3 participates in myocytes diï¬erentiation and is linked toskeletal muscles metabolic fuel switching Gregoire Hong Class II HDACs can be phosphorylated byHDAC kinases and are transduced from nucleus to cytoplasmin response to cellular stress mediating muscle ï¬ber switch andaï¬ecting the pathway of insulin sensitivity such as Glut4 andAKT Haberland McGee and Hargreaves Class III HDACs target a crucial mitochondrial biogenesis factorperoxisome proliferatoractivated receptor gamma coactivator alpha PGC1α in skeletal muscles liver and fat tissue Whiteand Schenk Therefore we will further discuss howthese HDACs uence respective downstream targets exercisecapacity and therapeutic eï¬ects in human diseasesCLASS I HDAC HDAC123Regulation of Myogenesis by Class IHDACsIn previous studies HDAC123 was shown to be associated withthe process of myogenesis or myocyte diï¬erentiation Mal Puri Ohkawa Gregoire HDAC1 tightly binds to MyoD and deacetylates speciï¬c sites toinhibit the expression of musclespeciï¬c genes such as MHC andMCK in myoblasts Mimicking muscle diï¬erentiation conditionby serum deprivation HDAC1 protein gradually decreasesduring myocyte diï¬erentiation and transfers to bind to the tumorsuppressor pRb accompanied by isolation of HDAC1MyoD andtranscriptional activation of musclespeciï¬c genes Puri A HDAC1 H141A mutant incapable of binding withMyoD loses its inhibitory property on musclespeciï¬c genes inmyoblast state Mal In the late stage of myocytediï¬erentiation activating factor gradually switches from MyoDto myogenin occurring with a decrease in the MEF2D inhibitoryregulator HDAC2 Simultaneous overexpression of myogeninand MEF2D can enhance the expression of the musclespeciï¬cgene MHC in the absence of MyoD Ohkawa MEF2D a key factor that controls myocyte diï¬erentiationcan only be eï¬ectively deacetylated by HDAC3 rather thanHDAC128 Gregoire In addition HDAC3 caninhibit autoacetylation of acetyltransferases p300 and p300CBPassociated factor PCAF Thus HDAC3 impedes MyoDMEF2PCAF to form a multicomplex disturbing MEF2dependentmyogenic transcription Gregoire Figure Redundant Roles of HDAC12 inMaintaining Sarcomere Homeostasis inMuscleLoss of function of HDAC12 inhibits autophagy ï¬ux in skeletalmuscles tissue accompanied by decreased LC3 III ratio andp62 accumulation under fasting condition Moresi Toxic autophagy intermediates accumulate in muscle ï¬bersin which class I HDACs deï¬ciency led to impaired exercisecapacity Moresi This oï¬ers convincing evidenceon the role of HDAC12 in stabilizing basic structure andfacilitating development in muscles Genetic models suggestthat Class I HDACs control the physiological homeostasis ofskeletal muscles A germline deletion shows that wholebodyknockout of either HDAC1 or HDAC2 leads to mortalityin mice prior to the perinatal period Montgomery However a tissuespeciï¬c single deletion of HDAC1or HDAC2 in myocardium does not cause any phenotypeMontgomery Double knockout HDAC12 in theFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityTABLE The targets of HADCs in skeletal muscles and their physiological functionsClassMembersDown targetsGenetic KOTG phenotypeReferencesIHDAC1MyoD PTEN FoxOHDAC2HDAC3HDAC8HDAC4HDAC5HDAC9HDAC7HDAC6MyoD MEF2D NFκBp65MEF2D PCAF Ampd3RCAN1 MEF2ACpt1bMEF2 Glut4 Myosin HeavyChain PGC1α and Hsc70Pax7MEF2 Glut4 Baf60cAtg7 Beclin1 LC3Dach2Myog Gdf5MEF2Pax7 MFN1 Fam65bdysferlin and MAFbxHDAC10SIRT1PGC1α STAT3IIAIIBIIIMnSOD Hexokinase II PDHsubunit E1αMalonyl CoA decarboxylaseNFκBMstnSIRT2SIRT3SIRT4SIRT5SIRT6SIRT7Functionally redundant HDAC12 DKO mice causesmitochondrial abnormalities and sarcomeredegenerationPuri Ohkawa Beharry Cho Zhu Bin mKO mice Enhanced amino acid\\lipid metabolism andendurance exercise Causes IR under basal conditionGregoire Han Yuan Hong Functionally redundant HDAC45 DKO HDAC59 DKOHDAC459 TKO increases type I ï¬ber percentage andMcKinsey 2000a Choi Niu Luo oxidation metabolismYuan Meng Niu Macpherson Zhang Dressel KO mice Causes mitochondrial oxidative damagemitofusion defect Protect against muscle wastingBalasubramanian Lee Ratti mKO mice Mediates CR induced insulin sensitivity hasno effect on glucose homeostasis or exercise capacityTG mice no effect on glucose homeostasis or exercisecapacityKO mice Exacerbates obesity and IR in HFDKO mice Exacerbates obesity and IR in HFDKO mice Increased exercise tolerance and protectagainst HFDinduced obesityRodgers Schenk Boyle Jing Lantier Laurent KO mice Abnormal hypoglycemia TG mice Protectagainst HFD mKO mice Impaired insulin sensitivity lossKanï¬ Xiao Cui Samant of muscle massHDAC11IVActivation inhibition unknown KO wholebody deletion TG wholebody overexpression DKO double knockout TKO triple knockout mKO musclespeciï¬cknockout CR caloric restriction HFD high fat diet IR insulin resistanceheart leads to severe cardiomyopathy in mice indicating theobligatory role for HDAC12 in speciï¬c tissues Montgomery In skeletal muscle double knockout of HDAC12by myogeninCre causes mitochondrial abnormalities andsarcomere degeneration disrupting fundamental structural unitsof myoï¬bers Moresi Therefore these ï¬ndingsdemonstrate that HDAC12 redundantly maintains sarcomerehomeostasis in skeletal muscleHDAC3 Functions as a Fuel Switch inMuscle Energy Metabolismthe enzymatic core of nuclearHistone deacetylases isreceptor corepressorNCoR and silencing mediator ofretinoic acid and thyroid hormone receptors SMRT corepressor correspondingly NCoR and SMRT corepressoractivate HDAC3 through its SANT domain enzyme activityKaragianni and Wong Mice suï¬er from insulin resistanceafter speciï¬cally knocking out HDAC3 in skeletal musclesmKO while their endurance exercise abilities are enhancedHong It seems a selfcontradictory phenomenonbecause former studies pointed out that increased enduranceexercise capacity enhances insulin sensitivity The study showsthat insulin signaling cascades including pAKTAKT pIRS1S1101 and pGSKGSK have no change in HDAC3 mKO musclewhile glucose uptake and insulin sensitivity are impaired inglucose tolerance test GTT and insulin tolerance test ITTHong which is called the dissociation eï¬ect byresearchers Lipid tends to be used as energy fuel in HDAC mKOmice which inhibits glucose absorption but does not uenceinsulin signaling sensitivity Hong Further workfound that the HDAC3 knockout upregulates the expressionof AMP deaminase AMPD3 the ï¬rst ratelimiting enzymein purine metabolism in skeletal muscles Fortuin Hong AMPD3 can deaminate AMP to form IMPfacilitating aspartic acid to transmit into fumarate and malate theintermediate metabolites of tricarboxylic acid cycle TCA cycleHong Research studies found that exercise inducedglucose labeled 13C6 expressed a lower glycolysis ï¬ux rate inmuscles of HDAC3 mKO but a higher expression in the TCAFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Controls of myogenesis by Class I HDACs The inhibitory effects of HDACs on myogenic factors MEF2 and MyoD maintain a primary myoblast stateDuring myocyte differentiation and myogenic process the inhibition is lifted by other factors facilitating MEF2MyoD complex formation to promote myogenesisAc deacetylationcycle intermediates Hong Gong Thus ageneral increase in TCA cycle metabolites activates the oxidationin mitochondria In vitro radioactive aspartic acid isotope tracertest showed that inhibiting HDAC3 or overexpressing AMPD3can increase amino acid metabolism rate and enhance fatty acidmetabolism thereby downregulating glucose metabolism Hong Figure In liver knockout HDAC3 causes severeliver steatosis and can be rescued by wildtype or catalyticallyinactive mutants of HDAC3 indicating an enzymatic activityindependent function Sun However the musclefuel switching in HDAC3 mKO mice cannot be rescued by anenzymatic inactivity mutant HDAC3 Song Thisï¬nding demonstrates that HDAC3 controls the fuel utilizationin skeletal muscles and is dependent on its enzymatic activityThe relationship between exercise and glucose uptake is far morecomplex than one could expectCLASS II HDACs HDAC45796Phosphorylation and NucleusCytoplasmShuttle of Class II HDACsThe MEF2 family is a class of transcription factors that areclosely associated with myogenic basic helixloophelix domainsincluding MyoD Taylor and Hughes MEF2 may interactwith Class IIa histone deacetylase HDAC45 to inhibit thetranscription of MEF2dependent genes thereby impeding thediï¬erentiation from myoblast to myotube McKinsey et al2000a During exercise Ca2 ï¬ows out from sarcoplasmicreticulum and activate calciumcalmodulindependent proteinkinase CaMK which phosphorylates HDAC45 and mediatesHDAC45 shuttle from the nucleus to the cytoplasm thusreleasing the repressive eï¬ect of HDAC45 on MEF2dependentgenes McKinsey 2000b Yuan Further researchdemonstrates that the nucleoplasmic shuttle of HDAC4 relies on binding while the shuttle of HDAC5 depends on CaMKphosphorylation at serine and sites ï¬rst and thenexporting from the nucleus by binding with McKinsey 2000b As expected HDAC7 in Class IIa also possessesthe ability to be exported from nucleus to cytoplasm Gao suggesting that there may be redundant functions of ClassIIa HDACs Except for Class IIa knocking out Class IIb HDAC6in embryonic stem cells ESCs can promote ESCs to diï¬erentiateinto myoblast cells and transplantation of ESCs with HDAC6knockdown can also help the regeneration of wound skeletalmuscles Lee type I ï¬berthe proportion ofRegulation of Class II HDACs onFiberType Switch MitochondriaRemodeling and Energy Stress inSkeletal MuscleEvidence showsinthatskeletal muscles has no change in animal knocked out aloneany member of Class IIa Potthoï¬ Howeverthe proportion of type I ï¬ber is signiï¬cantly increased inHDAC45 DKO HDAC59 DKO HDAC459 TKO miceas well as exercise capacity of skeletal muscles Potthoï¬ Altogether the ï¬nding indicated a functionalredundant mechanism in Class II HDACs family Previousstudy found that Class IIa HDACs are closely associated withthe MEF2 family and can repress their downstream targetgenes McKinsey 2000a Therefore similar to Class IIaHDACs DKO the proportion of type I ï¬ber is decreased in theMEF2C and MEF2D knockout mouse models overexpressingMEF2C results in increasing type I ï¬ber and exercise capacityHDAC45 not only regulates exercise capacity but also governsmotor adaptation Several studies had shown that exerciseFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class I HDACs regulates sarcomere structure and exercise capacity in mature muscle A schematic diagram shows Class I HDACs regulate exercisecapacity on different levels HDAC12 have redundant roles in modulating autophagy ï¬ux in muscle eliminating toxic factors in muscle HDAC3 serves as a fuelswitch and promotes muscle to use fatty acid to adapt endurance exercisepromotes phosphorylation of HDAC45 mediated by CaMK andadenosine monophosphateactivated protein kinase AMPKthen increasing a MEF2dependent transcription of Glut4 andmusclespeciï¬c genes which participates in the adaptationand plasticity of skeletal muscles McGee McGee and Hargreaves Besides exercise glucose can alsoactivate KATP channeldependent calcium signaling and CaMKwhich induce phosphorylationdependent nuclearcytoplasmtransportation of HDAC5 Meng The leavingHDAC5 releases its repressive eï¬ect on Baf60c and enhancinginsulinindependent AKT activation Meng Usingspeciï¬c inhibitor of class IIa HDAC activity myosin heavy chainPGC1α and heatshock cognate HSC70 are conï¬rmed to beone of the substrates of HDAC4 in denervationinduced atrophymuscle in comparison with acetylated protein enrichment withuntreated group Luo Taken together Class IIaHDACs play critical roles in exercise capacity remodeling andnutrient sensing of skeletal muscles Figure For Class IIbit was revealed that glucose deprivationinduced mitochondrialfusion mediated by mitofusion1MFN1 is signiï¬cantly disrupted in HDAC6 KO mice causingmitochondria degeneration which isreversed followingapplication of MFN1 acetylationresistant mutant Lee This study suggested that MFN1 deacetylation by HDAC6plays an important role in mitochondrial adaptive energyproduction and remodeling of skeletal musclesCLASS III HDACsSir2HOMOLOGSIRT1634NuclearLocated SIRT16Numerous studies have found that caloric restriction CRextends lifespan of mammals Caenorhabditis elegans and fruitï¬ies while such eï¬ect is lost in SIR2 or NPT1 mutant C elegansstrains Imai Lin SIR2 encodes thesilencing protein Sir2p and NPT1 participates in the synthesis ofNAD NAD is an essential cofactor for Sir2pClass III HDACsto exert enzyme activity rather than Class I II and IV relayingon the zinc Imai Lin In mammalsSir2 has homologous proteins and these proteins are essentialto mitochondrial energy homeostasis antioxidant defense cellproliferation and DNA repair VargasOrtiz SIRT1 Mediates Energy Stress Adaptation in SkeletalMuscles reported that SIRT1 promotes theRodgers et altranscriptional activity of PGC1α by deacetylating the PGC1αat K13 site and mediates CRinduced gluconeogenesisrelatedgenes G6P PEPCK expression in liver Further research showedthat the NADNADH ratio and the enzymatic activity of Sir2 asensor of redox state were decreased in diï¬erentiating myocyteswhich relieved the inhibitory eï¬ect on MyoD and then promotedcell diï¬erentiation Resveratrol RSV a compound that maytarget SIRT1PGC1α can improve mitochondrial function andmetabolic homeostasis owning a potential to prolong lifespanRSV loses its activating eï¬ect on PGC1α in SIRT1 MEFsLagouge Moreover RSVtreated mice at a dose of gkg increased their exercise capacity oxygen consumptionand improved insulin sensitivity against highfat induced obesityLagouge In addition RSV could enhance thedeacetylation of PGC1α in brown fat tissue BAT and skeletalmuscles which promotes mitochondrial production oxygenconsumption and thus improves metabolic syndrome Lagouge The function of SIRT1 in regulating skeletal musclesmetabolic homeostasis and exercise capacity has attracted greatattention from researchers Researchers further generated skeletalmusclespeciï¬c SIRT1mKO mice and found that mKO miceFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Control of muscle exercise adaption and mitochondrial function by Class II HDACs A Schematic of the Class IIa HDACs nucleoplasm shuttle Understress condition CaMK and AMPK could be activated and phosphorylate Class IIa HDACs Phosphorylated HDACs then bind with and shuttle to cytoplasmrelieving inhibitory effects on MEF2 and Baf60c B Class IIb HDAC6 can deacetylate MFN1 and facilitates its mitofusion function P phosphorylationlost the eï¬ect of CRinduced increasing insulin sensitivity andwere unable to deacetylate and inactivate STAT3 resulting inupregulating the phosphatidylinositol3kinase PI3K inhibitoryregulator p55αp50α expression Schenk Althoughknockout or overexpression of SIRT1 in skeletal muscles hasno eï¬ect on endurance capacity or glucose homeostasis in miceGurd Philp White Svensson some studies have shown that AMPK regulatesenergy metabolism of skeletal muscles partially mediated bySIRT1 and SIRT1 is signiï¬cantly increased after enduranceexercise Suwa Canto Overexpressionof Sirt1 in skeletal muscles by adenoassociated virus AAV1promotes the expression of oxidationrelated genes includingPpargc1a Tfam Cpt1b and Pdk4 while it has no eï¬ect on insulinsensitivity of body Vila But overexpressing Sirt1in liver by AAV8 can protect from fatty liver induced by highcarbohydrate food HCD Vila Taken togetherthe aforementioned studies suggest that SIRT1 possesses limitedregulation capacity of skeletal muscles motility and SIRT1may modulate mitochondrial homeostasis and mediate skeletalmuscles adaptation under certain physiological conditions suchas CR aging and regeneration Gomes Ryall Figure In addition there may be more beneï¬cial eï¬ectsof SIRT1 on metabolism in the liver or fat tissue Lagouge Vila Stefanowicz SIRT6 Improves Muscle Fitness andExercise CapacitySIRT6 is another Sir2like deacetylase located in the nucleusWhole body deletion of SIRT6 causes mice death around weeks owning to hypoglycemia Xiao Neverthelessthe skeletal musclespeciï¬c knockout of SIRT6 causes insulinresistance and impairs glucose homeostasis of mKO mice Cui Because the activity of AMPK is reduced inSIRT6mKO mice the glycolipids absorption and utilizationof skeletal muscles are impaired and the exercise capacity ofthe mice was also attenuated Cui Furthermoreresearchers constructed the overexpressing SIRT6 Sirt6BACmice and found that their body weight and fat content werenormal but the Sirt6BAC mice could protect from HCDinducedhyperglycemia via increasing pAKTAKT induced by insulinstimulation and glucose absorption Anderson Vitroexperiments further demonstrated that the ability of glucoseuptake was enhanced mainly in skeletal muscles not in brainiWAT eWAT tissues of Sirt6BAC mice Anderson These results shows the potential eï¬ects of SIRT6 targeted inskeletal muscles on improving exercise performance and treatingmetabolic diseases Figure MitochondriaLocated SIRT34SIRT34 Maintains Mitochondria Function in SkeletalMusclesSIRT34 are mitochondrialocalized deacetylases and responsiblefor regulating the deacetylation of proteins in mitochondria andmaintaining mitochondrial homeostasis After feeding highfatdiet HFD accelerated obesity attenuated insulin sensitivityworsened fatty liver and increased acetylation of proteins inmitochondria were observed in SIRT3 KO mice Hirschey Multitissue proteomics and physiological examinationreveals that SIRT3 is responsible for mitochondrial acetylatedFrontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityFIGURE Class III HDACs promote a stressinduced adaptation in muscle and regulate mitochondrial function Class III HDACs are NADdependentdeacetylases and may function as a redox sensor in muscle cell SIRT1 could activate PGC1alpha by directly deacetylasing K13 site and regulate insulin sensitivityby targeting STAT3 Mitochondrialocated Class III HDACs control mitochondrial function by deacetylating certain mitochondrial proteins modulating muscle fuelutilization and exercise capacityproteome regulation and metabolic fuel switching in brainheart kidneyliver and skeletal muscles DittenhaferReed Protein enrichment analysis discovers that itsregulatory proteins were mainly concentrated in lipid metabolismDittenhaferReed Knocking out SIRT3 leadsto reduction in the levels of deacetylation of manganesesuperoxide dismutase MnSOD mitochondrial complex II andpyruvate dehydrogenase PDH subunit E1α a downregulationof hexokinase II HK II binding with mitochondria resultingin impaired glucose and lipid metabolism of skeletal musclesLantier Interestingly SIRT3 liverspeciï¬c knockouthep and skeletal musclespeciï¬c knockoutskmmice did not aï¬ect glucose homeostasis under chow or HFDconditions FernandezMarcos The above resultsindicate that SIRT3 has an important role in metabolic regulationbut in speciï¬c physiological processes such as redox stateexercise and aging and its regulatory mechanism is yet deï¬nedin skeletal muscles Kong Robin Williams A recent study found that SIRT4 knockoutcan resist HFDinduced obesity and increase endurance exercisein mice by repressing malonyl CoA decarboxylase which wasa key enzyme controlling fatty acid betaoxidation and wasreported to regulate muscle fuel switching between carbohydratesand fatty acids Koves Laurent Moreover knockdown of SIRT4 by adenoviral shRNA canincrease the mRNA and protein content of SIRT1 therebyenhancing the expression of fatty acid oxidation genes andmitochondrial oxidation capacity in hepatocytes and myotubesNasrin Table THERAPEUTIC TARGETS OF HDACsAND THEIR POTENTIAL FORMETABOLIC DISEASESA comparative analysis used HDAC paninhibitor SAHA aclass I HDAC selective inhibitor MS275 and a class IIHDAC selective inhibitor MC1568 to treat C2C12 separatelyIt was found that only MS275 could signiï¬cantly stimulatemitochondria biogenesis and oxygen consumption Galmozzi In obese diabetic mice it was found that speciï¬callyinhibiting Class I rather than Class II HDACs improvedGTT and insulin sensitivityincreased oxidative metabolismof skeletal muscles and adipose tissue and reduced bodyweight Galmozzi Similar to the eï¬ect of MS275knockdown HDAC3 in C2C12 could also increase Pgc1αGlut4 Tfam Idh3α transcription suggesting that HDAC3 andits target genes played an important role in the above eventsGalmozzi For Class II HDACs studies have found that scriptaid a ClassIIa HDAC inhibitor has similar eï¬ects to exercise Six weeksof scriptaid administration signiï¬cantly improved enduranceperformance and signiï¬cantly increased the wholebody energyexpenditure and the expression of lipid oxidation related genesPdk4 Cpt1b Pgc1α Pparδ etc of C57BL6 mice Gaur One possible explanation of above observation is thepotential target of scriptaid inhibition of titin a structure proteinof sarcomere deacetylation and downstream genes of Class IIaHDACs Gaur Huang Frontiers in Physiology wwwfrontiersinAugust Volume 0cTian et alRole of HDACs in Metabolism and Exercise CapacityIn mice treated with SPT1720 another activator of SIRT1overtly enhanced endurance exercise ability was noted Moreoverthese mice were protected from HFDinduced obesity and insulinresistance owing to an upregulation of the oxidative metabolismin skeletal muscles liver and BAT tissues Pacholec In order to further explore the principle of SIRT1 activation theresearchers puriï¬ed SIRT1 in vitro and added SRT1720 and itsanalogs SRT2183 SRT1460 and RSV and they found that theenzymatic activity of SIRT1 was not enhanced suggesting thatthese drugs may indirectly activate SIRT1 Pacholec The enzymatic activity of SIRT1 largely depends on the contentof cofactor NAD This implied that the enhancement of SIRT1activity may be achieved through indirect upregulation of NADStudy has discovered that knocking out poly ADPribosepolymerase1 PARP1 which is a NAD consuming enzymecould increase NAD content and enhance the activity of SIRT1in BAT and skeletal muscles Bai PARP1 inhibitorscan upregulate the proteins of mitochondrial respiratory chaincomplexes in mice enhance the aerobic oxidation capacity ofmitochondria and improve mitochondrial defects in the primarymyotubes of obese humans Bai Pirinen et alResveratrol was initially reported as an SIRT1 activatorthat improves mitochondrial function and exercise capacity inmice and resists HFDinduced obesity Lagouge However subsequent studies have discovered that administeringthe same dose of RSV to rats or mice does not increasemitochondrial protein content Higashida Bycontrast overexpression of SIRT1 in the triceps muscle of ratsdecreases the mitochondrial protein content Higashida In a doubleblind human trial healthy and obesemen were supplied for 30day RSV in which the data showedthat RSV can improve systolic blood pressure and homeostasismodel assessment HOMA indexindicating glucose metabolismability simulating the eï¬ect of CR Timmers However some researchers found that the overexpression ofSIRT1 alone cannot mimic the CR eï¬ect in transgenic mice andthe transcriptomic changes in various tissues were quite diï¬erentor even opposite Boutant Such an opposite situationmay explain that the genetic model and compound stimulationare not completely consistent RSV as a potential metabolicsyndrome treatment drug still needs largescale populationsample veriï¬cationCONCLUSIONThe very ï¬rst mammalian histone deacetylase HDAC1 wascloned and isolated by Taunton and sabout HDACs have been published in the last years CurrentlyREFERENCESAnderson J G Ramadori G Ioris R M Galie M Berglund E D CoateK C Enhanced insulin sensitivity in skeletal muscle and liver byphysiological overexpression of SIRT6 Mol Metab 101016jmolmet201509003we know at least HDAC proteins They are responsible foreradicating epigenetic modiï¬cations establishing an epigeneticoï¬ chromatin state and regulating heritable gene expressionYang and Seto Haberland In these processeseach HDACs may play a diï¬erent role Table What kind ofgeneprotein is the speciï¬c downstream target of these HDACsIs its enzyme activity related to intracellular localization andthe formation of multiprotein complex It is still one of thekey and diï¬cult issues in this ï¬eld Based on previous researchexperience some techniques may be used to further experimentsas follows HDACs interacting proteinscomplex coIP Proteinacetylation western Histone acetylation target geneChIP High through put proteomicsacetylome with speciï¬cHDACs inhibitor etctherapeuticare potentialHistone deacetylasestargetsand clinical drugs such as SAHA Vorinostat and FK228romidepsin have been used for antitumor treatment Bolden However they have two disadvantages greattoxic and side eï¬ects and diï¬culty in speciï¬c inhibition ofHDACs activity With the development of computer simulationtechnology and structural biology it is believed that more speciï¬cHDACs inhibitorsactivators can be constructed And researchesshould attach importance to HDACs regulatory factors likePARP1 when direct targeting on HDACs fails to show its eï¬ectsFurther development of their inhibitors with more speciï¬cityand trials for the treatment of metabolic diseases may have greatpotential as well | 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 |
"researchWhat are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records dataHelen Strongman Rachael Williams2 Krishnan Bhaskaran1To cite Strongman a0H Williams a0R Bhaskaran a0K What are the implications of using individual and combined sources of routinely collected data to identify and characterise incident site specific cancers a concordance and validation study using linked English electronic health records data BMJ 202010e037719 101136bmj 2020037719 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 February Revised April Accepted July Authors or their employers Re use permitted under CC BY Published by BMJFor numbered affiliations see end of Correspondence toDr Helen Strongman helen strongman lshtm ac ukObjectives To describe the benefits and limitations of using individual and combinations of linked English electronic health data to identify incident cancersDesign and setting Our descriptive study uses linked English Clinical Practice Research Datalink primary care cancer registration hospitalisation and death registration dataParticipants and measures We implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and We calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers We described completeness of grade and stage information in the cancer registration data setResults gold standard cancers were identified Positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate Sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone For case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer When primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed Completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancersConclusions Ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority Strengths and limitations of this study º This is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in England to identify cases of the most common incident cancers º Using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º We described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º A key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataINTRODUCTIONThe Clinical Practice Research Datalink CPRD provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 Use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 In the field of cancer epidemiology CPRD primary care data linked to Hospital Episode Statistics Admitted Patient Care Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access data HES APC Office of National Statistics ONS mortality and National Cancer Registration and Analysis Service NCRAS cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment Use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for NCRAS data Research teams therefore commonly choose not to use all available linked data3 Cancer epidemiology studies can also be conducted using NCRAS and HES APC data provided by National Health Service NHS Digital and Public Health England PHE without linkage to CPRD primary care data4 This provides national coverage at the expense of the detailed health data that are available in primary care recordsValidation studies assessing concordance between CPRD GOLD HES APC and NCRAS data have estimated high positive predictive values PPVs for CPRD GOLD data and varying proportions of registered cancers that are not captured in CPRD GOLD and HES APC5 The most up to date analysis by Arhi et al included the five most common cancers and all papers focussed on concordance between CPRD GOLD only and NCRAS existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs National data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and Clinical Commissioning Groups10 We aim to describe and compare the benefits and limitations of using different combinations of linked CPRD primary care data HES APC ONS mortality and NCRAS cancer registration data for conducting cancer epidemiology studies Our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets We have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets We also describe the availability of stage grade and treatment variables over time in the cancer registration data for the CPRD linked cohort This reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesMETHODSStudy design and settingWe completed a concordance study using linked2 English CPRD GOLD HES APC ONS mortality and NCRAS data CPRD GOLD data were extracted from the January monthly release and the 13th update to CPRDs linked data The study period was from January to December with December matching the end of the NCRAS coverage periodThe CPRD GOLD database includes de identified records from participating general practices in the UK who use Vision software1 General practice staff can record cancer diagnoses using Read codes or in free text comments boxes though the latter are not collected by CPRD Diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care CPRD GOLD data are linked to HES APC ONS mortality and NCRAS through a trusted third party for English practices that have agreed to participate in the linkage programme2 HES APC data are collected by NHS Digital to co ordinate clinical care in England and calculate hospital payments12 Admissions for and related to cancer diagnoses are recorded using International Classification of Diseases version ICD10 codes National cancer registration data are collected by NCRAS which is part of PHE in accordance with the Cancer Outcomes and Services Data set13 which has been the national standard for reporting of cancer in England since January Data include ICD10 codes to identify the cancer site and more detailed information such as stage and grade ONS mortality data includes dates and causes of deaths registered in England recorded using ICD10 codesParticipants exposures and outcomesOur underlying study population included male and female patients registered in CPRD GOLD practices who were eligible for linkage to HES APC NCRAS and ONS mortality data and had at least days of follow up between January and December Start of follow up was defined as the latest of the current registration date within the practice and the CPRD estimated start of continuous data collection for the practice up to standard date End of follow up was determined as the date the patient left the practice ONS mortality date of death or practice last collection dateIdentification and classification of cancer codesWe used code lists to classify cancer records in each of CPRD GOLD HES APC and ONS mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https data Incompletely specified cancer codes could be mapped to cancer site eg ICD10 code C689 Malignant neoplasms of urinary an unspecified was considered consistent with both bladder and kidney cancer For NCRAS we accessed coded records for the most common cancers We included cancers recorded in the clinical or referral file for CPRD GOLD cancers recorded in any diagnosis field for HES APC and the underlying or Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Gold standard algorithm to identify incident site specific cancers using all data sources HES Hospital Episode Statistics NCRAS National Cancer Registration and Analysis Service ONS Office of National Statisticsmost immediate cancer cause of death in ONS mortality dataCancer case definitions based on individual sources and combinations of sourcesWe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources NCRAS alone NCRAS and HES APC all sources CPRD GOLD HES APC and ONS mortality CPRD GOLD alone HES APC alone Multiple malignant cancers recorded on the index date in CPRD GOLD or HES APC were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the NCRAS data if available and as multiple site cancer if not For each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upGold standard cancer case definitionWe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure Cancers recorded in NCRAS alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in HES APC alone or GOLD alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period Where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in NCRAS and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in NCRAS if there were more data sources in total with records for cancer at that site than data sources with contradictory recordsWe used NCRAS data to identify stage grade and treatment where available in the cancer registry only cohort Binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisStatistical analysisFor each cancer site and each individual or combined data source we combined our applied study definitions Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordWe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex We calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourcesWe used Kaplan Meier methods to describe mortality over time for cancers identified using each definition The ONS mortality death date was used for these analysesWe used the NCRAS only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timePatient public involvementPatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsRESULTSOf research quality patients in the CPRD GOLD January build were eligible for linkage to HES ONS mortality and NCRAS data in set were excluded due to unknown sex Of the remainder and had at least year of follow up between January and December and were included in the study population Using the gold standard algorithm incident cases of cancer were identified The number of patients identified with each cancer is presented in online supplementary appendix table Half n82 of these patients were male aged to aged to and aged or olderFigure presents PPVs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm When using NCRAS data alone to of cancers were confirmed by the algorithm for out of cancer sites the NCRAS only case definition gave the highest PPV Case definitions using data sources not including NCRAS generally had lower PPVs ranging from to for individual cancer sites For the four most common cancers breast lung colorectal and prostate PPVs were at least for all case definitions Minimal differences in PPVs were observed between age groups years and sexes online supplementary appendix figures Figure presents sensitivity values for each case definition Sensitivity was generally higher for the case definitions that included NCRAS data ranging from to for individual cancer sites except bladder cancer identified using NCRAS data alone and ¥ for the four most common cancers breast lung colorectal and prostate Sensitivity was also generally high for definitions using a combination of CPRD GOLD HES APC and ONS mortality data ranging from to ¥ for the four most common cancers Sensitivity was lower for case definitions that used CPRD GOLD alone range to for individual cancer sites or HES APC alone range to Sensitivity values for CPRD GOLD alone and HES APC alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures Post hoc analysis suggested that the low sensitivity of CPRD GOLD only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary an cancer codes n1108 in CPRD GOLD rather than contradictory information about the first cancer record n625 These incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 Bladder cancers that were not recorded in NCRAS data n3445 were commonly recorded in both HES APC and CPRD GOLD n2228 or in HES APC only with a subsequent unspecified or bladder cancer record in HES APC within months n995 Table describes the number of days median IQR and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used Case definitions using NCRAS alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using CPRD GOLD alone and HES APC aloneFigure describes mortality over time following incident cancer diagnoses ascertained from each case definition Minimal differences in mortality were observed between cancers identified from different case definitions Where variability was observed cancers identified using CPRD GOLD only had the lowest mortality rates eg kidney cancer and cancers identified using HES APC only or NCRAS only had higher mortality rates eg prostate cancer and bladder cancer respectivelyFigure describes completeness of grade and stage for cancers identified using NCRAS only Recording of grade was highly variable between cancers with gradual increases in completeness over time Completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in Post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure Online supplementary appendix figure describes recording of treatment modalities identified using NCRAS Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Positive Predictive Value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm Percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system NHL Non Hodgkin's lymphoma CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service ONSOffice of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0celitnecrep hthtRQI inadeMelitnecrepRQI inadeM hthtelitnecrep htht inadeMRQIelitnecrep hthtCPASEH DLOGDRPC ytil atromSNOdna CPASEH DLOGDRPC CPASEHdnaSARCN secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emTi l ebaT access recnaC lanoitaN SARCN atad eraC tneitaPdetti mdA scitsitatS edospEi latipsoH CPASEH knil ataDhcraeseR ecitcarP lacniilC DRPC metsys suovren lartnec SNCl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iiA setis recnac nommoc tsom ruoFDCiI nosrev sesaesDi fonoitacfissaCliscitsitatS lanoitaN rof ecfifO SNOi atadnoitartsgerrecnac ecvreS ssyanAdna liinoitartsgeRi lanoitanretnI eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaCi snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmuN ot ot ot ot ot Cl amoeym epitluMl ot Ci ameakueL ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtSARCN inadeMRQI ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot C ytivac larOC laegahposeOC hcamotS CC latcerooClrecnaC amonaeml tnangilaMC saercnaPC gnuLC suretUC etatsorPC seiravOC yendKiC tsaerBCi xvreCCC SNCnarBiC reddaBlC amohpmyl s'inkgdo HnoNC idoryhTC revLiStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access Figure Mortality following first ever record of cancer in each combination of sources Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites CNS central nervous system CPRD Clinical Practice Research Datalink HES APC Hospital Episode Statistics Admitted Patient Care data NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphoma ONS Office of National StatisticsStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c accessFigure Completeness of grade and stage for cancers identified using NCRAS data only Cancer sites are ordered according to corresponding codes from the International Classification of Diseases version ICD10 Four most common cancer sites Grading information is not applicable to brainCNS sarcoma or haematological cancers and not required by in the national data standard COSD for prostate cancer Core staging is not applicable to haematological and gynaecological cancers Other types of staging are recommended by COSD CNS central nervous system COSD Cancer Outcomes and Services Data set NCRAS National Cancer Registration and Analysis Service NHL Non Hodgkin's lymphomaStrongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0c access only Missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordedDISCUSSIONStatement of principal findingsWe investigated the use of different sources of electronic health record data to identify incident cancers For all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers Use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers Combining CPRD GOLD HES APC and ONS mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers Sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone Use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date Finally while we observed minimal changes in PPVs and sensitivities between and completeness of NCRAS cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 Completeness of cancer treatment recording was difficult to assess due to the absence of a missing categoryStrengths and weaknesses of the studyThe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from CPRD to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies We have also assessed the value of using NCRAS cancer registration data to measure stage grade and cancer treatment modalitiesA limitation of the study is that our gold standard algorithm is not validated We feel that we were justified in pre weighting NCRAS data as more reliable that other data sources as NCRAS is a highly validated data set that matches merges and quality checks data from multiple sources4 We did not consider NCRAS to be the outright gold standard as it is plausible that NCRAS does not identify all tumours diagnosed and treated in primary and secondary care For most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in HES APC CPRD GOLD or ONS mortality data but not in NCRAS These tumours may have been diagnosed and coded as invasive in primary or secondary care but not by NCRAS been incorrectly coded in HES APC CPRD GOLD or ONS mortality data not have been notified to NCRAS eg tumours treated in private hospitals or be the result of linkage errors between the data sets The proportion of cancers identified in HES APC but not in NCRAS is particularly high for bladder cancer This is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in NCRAS which are greatest for bladder cancer4 This explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in NCRAS compared with other data sources To identify incident cancers we required months of research quality follow up in CPRD GOLD prior to inclusion in the study Previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 The identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as NCRAS data collection started in HES APC in and ONS mortality data in and by the inclusion of all diagnostic codes in HES APC assuming that the first ever primary or secondary record identified incident cancer Reassuringly PPVs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases Requiring internal confirmation within months for cancers recorded in CPRD GOLD alone in our GOLD standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up Our data cut only included NCRAS data for the top cancers earlier cancers at other sites will have been missed in this studyIt is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesStrengths and weaknesses in relation to other studies discussing important differences in resultsThe most up to date study describing concordance between linked CPRD GOLD HES APC and NCRAS data sets demonstrated that to of the five most common cancers recorded in CPRD GOLD are not confirmed in either HES APC or cancer registration data and to of registered cancers are not recorded in CPRD GOLD8 For cancers recorded in both sources the diagnosis date was a median of to days later in CPRD GOLD than in the cancer registration data Using CPRD GOLD alone to identify these Strongman a0H et a0al BMJ 202010e037719 101136bmj 2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis Use of HES APC only identified a higher proportion of patients with the correct diagnosis date than CPRD GOLD but over represented older patients and those diagnosed through the emergency route The majority of registered cancers were picked up using both CPRD GOLD and HES APC ranging from for lung cancer to for breast cancer Previous research demonstrated similar results with substantial differences between cancer types5 Additionally a study using data from to found that using HES data in addition to NCRAS data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16Our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancersWe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards Levels of data completeness of staging information in the CPRD extract in were similar to those reported by the United Kingdom and Ireland Association of Cancer Registries UKAICR9Meaning of the study possible explanations and implications for clinicians and policymakersUse of NCRAS cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately Case definitions based on a combination of CPRD GOLD HES APC and ONS mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersThese findings | Thyroid_Cancer |
"Sleep disturbance is an issue reported by caregivers Waking at night is a feature of dementia and byproxy sleep disturbance among caregivers is reported to be high Little is known about the characteristics ofdementia caregivers sleep and the factors that may influence sleep disruptionThe purpose of this study was to investigate the sleep characteristics and disturbances of Australian caregivers of aperson living with dementia In addition it evaluated the psychological wellbeing of caregivers by evaluatingassociations between mood and sleep in this populationMethods This study used a crosssectional descriptive correlation design Participants were recruited with theassistance of Alzheimers Australia Dementia Australia and targeted social media advertising In total adultprimary informal caregivers of people with dementia participated completing a questionnaire on demographiccharacteristics the Depression Anxiety and Stress Scale DASS21 and the Pittsburgh Sleep Quality Index PSQIResults In this study of caregivers were female who had been caring for someone living with dementia onaverage for years of participants had two or more comorbidities namely cardiovascular disease osteoarthritisand diabetes of participants were poor sleepers with with difficulty initiating sleep and reporting havingdifficulty maintaining sleep Overall psychological distress was common with high levels of moderate to severedepression anxiety and stress Global PSQI scores were significantly positively associated with depression and anxietywith the strongest correlation seen with stress scores Depression scores were also moderately associated with daytimedysfunction Stress was identified as a significant predictor of overall sleep qualityConclusions Sleep problems are common within the population of dementia caregivers Due to the nature andduration of caregiving and the progression of dementia of the care recipient there is the potential for a decline in thecaregivers mental and physical health Caregivers of those living with dementia are more likely to have comorbiditiesdepression anxiety and stress Sleep quality is correlated with emotional distress in dementia caregivers although thedirection of this association is unclear Therefore sleep and psychological wellbeing may be intertwined withimprovements in one aspect resulting in a positive impact in the otherKeywords Carers Caregivers Sleep Mood Psychological wellbeing Dementia Correspondence aislingsmythecueduau1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA AustraliaFull 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 0cSmyth BMC Geriatrics Page of BackgroundDementia is an inclusive term used to describe a numberof neurological conditions resulting in cognitive impairment and can include Alzheimers Disease senile dementia frontotemporal dementia vascular dementia LewyBody dementia Korsakoff syndrome alcohol related braininjury and younger onset dementia [] Globally thenumber of individuals with a formal dementia diagnosisrose from million in to million in []A further million new cases of dementia worldwideare predicted each year [] as populations around theglobe continue to live longer [] These figures could bevery much under estimated as it is widely accepted thatbetween and of people affected have no formaldementia diagnosis [] This growing epidemic effects notonly the individuals living with dementia but also theirfamilies caring for them the communities they live in andthe health care systems they rely uponFamily or friends are often key informal caregivers forpeople living with dementia [ ] Globally over billion hours ofinformal care is provided to peopleliving with dementia [ ] Whilst providing this carecan be highly rewarding it has also been described as achronic stressor with caregivers reporting low qualityand quantity of sleep [] and high levels of stress anddepression [] In fact sleep disruption is prevalentamongst dementia caregivers with over of caregiversexperiencing sleep disturbances []The National Sleep Foundation recommend that olderadults ¥ years require seven to h sleep for optimalphysical and psychological wellbeing [] Yet numerousstudies have illustrated that dementia caregivers sleepsignificantly less than that with estimates reporting mostcaregivers sleep less than h per night [] Not only arecaregivers sleeping less than they should the sleep theydo get is of significantly lower quality than their noncaregiver counterparts []Poor sleep is associated with a myriad of negative physical and psychological outcomes including hypertensionobesity mood disorders and dementia [] A disruptedsleep pattern is also recognised as a significant factor inpredicting caregiver strain [ ] and perhaps more importantly in predicting placing an individual into long termcare [] Enabling people living with dementia to stay athome rather than transfer to longterm care is the optimaloutcome for many families However this cannot be to thedetriment of the caregivers own physical andor psychological wellbeing Therefore in order to support the personliving with dementia PLWD to remain in the communitymaintaining caregiver health is vital Given the pivotal rolesleep has in a myriad of physiological processes it is essential to optimise and preserve caregivers sleepDespite the important role sleep plays in dementiacaregiver health it remains an understudied populationparticularly within the Australian context In fact arecent report by Carers Australia identified noAustralian studies on sleep disruption in dementia caregivers Only sixteen international studies were identifiedwhich subjectively measured sleep in the dementiacaregivers population with the majority of studies notreporting on the causes or consequences of disturbedsleep []In order to address this paucity of Australian data thepurpose of this study was to elucidate the sleep characteristics and disturbances of Australian caregivers ofPLWD Furthermore this study will determine whetherthere is a relationship between sleep and psychologicalwellbeing among caregivers of communitydwellingpeople living with dementia Lastly we will aim to identify significant predictors of poor sleep which in turncould offer a therapeutic target of poor sleep in dementia caregiversMethodsRecruitmentThis study used a crosssectional descriptive correlationdesign One hundred and four informal caregiversof people with dementia living in the community wereenrolled in the study Participants were invited via anumber of anisations including Alzheimers AustraliaDementia Australia and targeted social media advertising Mail outs were conducted and online questionnairesdistributed The inclusion criterion required the participant be an adult years or older primary informalcaregiver of a communitydwelling person living withdementia Power analysis was undertaken to computeminimum number of sample size required A samplesize of at least participants is necessary to detect amedium to large effect with power assuming atwotailed ttestEthicsThis study was granted ethical approval by Edith CowanUniversity Human Research Ethics Committee No Informed written consent was obtained from allparticipants prior to participation Participants receivedno incentive for taking partMaterialsThe survey collected demographic characteristics including gender age body mass index BMI as well as information about preexisting medical history caregivinghistory and use of respite The survey also included the questions from the Depression Anxiety and StressScale DASS21 [] and the Pittsburgh Sleep QualityIndex PSQI []The DASS21 is a selfreport questionnaire assessinglevels of caregiver stress anxiety and depression over the 0cSmyth BMC Geriatrics Page of previous seven [] day period The results provide anindication of the individuals perception of their experience of depression anxiety and stress The DASS21 isoften used as a screen for high levels of distress whenthe depression andor anxiety scores are high and as ascreen for the presence of a significant life event orproblem if the stress score is elevated DASS scorescorrespond to ranges of severity normal mild moderate severe extremely severe for depression anxiety andstress individually []The PSQI [] is a selfreport questionnaire assessinglevels of perceived quality and patterns of sleep over theprevious month across seven components subjectivesleep quality sleep latency sleep duration habitual sleepefficiency sleep disturbances use of sleep medicationand daytime dysfunction Each component has a potential score of three with a higher score indicating poorersleep related performance The total score for the sevencomponents creates a global score range to wherea score greater or equal to five ¥ indicates that theperson is a poor sleeper with severe difficulties in atleast two of the seven components or moderate difficulties in three or more components []Presence of comorbidities was determined by participantshaving one or more chronic conditions As per WorldHealth anisation WHO chronic conditions wereidentified as those requiring ongoing management over aperiod of years or decades covering a wide range of healthproblems such as heart disease diabetes asthma immunodeficiency disorder depression and schizophreniaStatistical analysesData analysis was undertaken using IBM SPSS Version and GraphPad Prism Descriptive statistics weregenerated for demographic characteristics DASS21 andPSQI scales Spearmans rho r correlation analysis wasused to assess the relationship between categorical components of DASS21 depression anxiety and stress andboth PSQI global scores and individual componentscores subjective sleep quality sleep latency sleep duration sleep efficiency sleep disturbances use of sleepingmedication and daytime dysfunction For logistic regression all variables were categorical age in years genderBMI length of care in months comorbidities DASS21component scores An r of was considered a mediumcorrelation and an r of was considered a large correlation [] Nonpaired ttests were used to determine ifthese values were statistically significant Results wereconsidered statistically significant if p Multivariatestepwise regression analysis was undertaken to determinethe predictive factors of global PSQI scores and identify variables significantly associated with sleep quality Independentvariables included in multivariate stepwise regression analyses were age gender BMI length of care comorbiditiesand DASS21 subscale scores Subjects with missing data oneither PSQI or DASS21 were excluded from inclusion incorrelation and regression analysesResultsCharacteristics of the caregiversOne hundred and four n surveys were completedin either hardcopy n or online n Table As the surveys were widely distributed by service providers and links made available online the response ratecannot be determinedParticipating caregivers were predominantly femalen with a mean age of years Range years The average BMI for respondents was kgm2range kgm2 Table which lies within theoverweight category While half of the participants n had a BMI within the healthy weight range twenty two participants were classified as underweight BMI less than and participants were classified as overweight The averagelength of time in the caregiving role in this study was months years ranging from months to yearsOnly n of participants reported using formalrespite services and all were female n of participants reported no comorbidities of participants n reported one comorbidity and n reported two or more comorbidities Themost commonly reported comorbidities were cardiovascular disorders n mainly hypertension and hypercholesterolemia bone and joint disorders n mainlyTable Demographic of participantsCharacteristicGendern or mean SDMaleFemaleAllAgeBMI kgm2Caregiver role MonthsComorbiditiesNoneOneTwo TotalUse of RespiteYesNoTotal99a94b96c ± ± ± a n missingb n missingc n missing 0cSmyth BMC Geriatrics Page of osteoporosis and arthritis and endocrine disorders n mainly prediabetes diabetes and thyroid dysfunctionTable Characteristics of Caregivers sleepMeasuren Subjective Sleep qualityThe majority of participants n reported aglobal sleep score equal or greater than which indicated they had clinically significant sleep issues in thepreceding month with a mean global score of ± Table The highest scoring individual subcomponentsincluded sleep latency ± subjective sleep quality ± and sleep disturbances ± n of participants had a sleep latency period greater than min indicative of issues initiating sleep Table ofparticipants had sleep efficiencies less than suggestingissues with maintaining sleep while in bed of participants had taken sleep medication at least once a weekover the previous month Participants were invited to addany additional relevant comments to their PSQI sleep assessment Forty participants provided additional information regarding their sleeping issues and identified issueswhich fell broadly into three themes Sleep disruption dueto caregivers physical needs such as pain and restless legs caregivers emotional distress such as stress anxietyand worrying and responding to care recipientneeds Depression anxiety and stress in caregivers of participants reported mild depression scores of participants reported mild anxiety scores and of participants reported mild stress scores Over athird of respondents reported moderate to extremelysevere stress levels n and depression n and more than a quarter n of respondents reported moderate to extremely severe anxietylevels Table In the bivariate analyses numerous subcomponents ofthe PSQI scale were significantly associated with measures of depression anxiety and stress Table Theglobal PSQI score is significantly positively associatedwith depression r anxiety r and stressscores r Stress scores also significantly correlatedwith other PSQI subcomponents including subjectivesleep quality r sleep latency r and daytimedysfunction r Depression scores were moderatelyassociated with daytime dysfunction r Global ScoreSubjective Sleep QualityVery GoodFairly GoodFairly BadVery BadSleep Latency¤ mins min min minsSleep duration h h h hSleep Efficiency¥ Sleep DisturbancesDaytime DysfunctionFrequency of SleepingMedicationNeverOnce per weekTwice per weekThree per week PSQI ComponentMean SD ± ± ± ± ± ± ± ± Predictors of Sleep quality in caregiversTo understand the relationship between the predictorsand global PSQI scores multivariate stepwise regressionanalyses were conducted to assess variables significantlyassociated with sleep quality The dependent variablewas the global PSQI score and the independent variableswere age gender BMI use of respite length of carecomorbidities and scores on the subscales of DASS21scale Depression Anxiety and Stress Stress was theonly significant covariate of global PSQI scores Stressscores could statistically significantly predict PSQIGlobal scores accounting for of variance Thestandardised coefficient was which was statisticallysignificant p as was the overall model F p All other variables were excluded from themodel due to nonsignificance Forward stepwise regression 0cSmyth BMC Geriatrics Page of Table Depression anxiety and stress in caregivers DASS21Clinical classificationTotalDepression n Anxiety n Stress n NormalMildModerateSevereExtremely Severe analyses were then undertaken with global PSQI scores asthe dependent variable and stress scores as a predictorwhile adjusting for the following confounders gender agecomorbidities of the caregiver and length of care Whilethese confounders were not statistically significant theywere retained within the model to adjust the effects ofstress on PSQI After adjusting for these factors that modelremains statistically significant F p andaccounts for of the adjusted variance in sleep scoresFor every one unit increase in stress scores PSQI Globalscores increases by a score of t p Thisregression model confirms that stress is a key significantcovariate of selfreported sleep issuesDiscussionA recent Australian report highlighted a significant gapin the literature around Australian caregivers of a PLWD[] Our study provides a comprehensive overview ofAustralian dementia caregiverssleep characteristicsassociations between psychological wellbeing and sleepand highlights a predictive role for stress in sleep qualityAll previous international studies identified an averagePSQI in dementia caregiver studies [] highlightingthe prevalence of the issue As expected and consistentwith previous studies [] Australian caregivers ofPLWD have a high prevalence of poor sleep with ofparticipants classified as poor sleepers The PSQI globalscore ± was higher than those found in somedementia caregiver studies [ ] but comparable withTable Correlation between PSQI subcomponent scores andDASS21 subcomponent score in dementia caregiversPSQI componentDASS21DepressionDASS21AnxietyDASS21StressPSQI Global ScoreSubjective sleep qualitySleep latencySleep durationSleep efficiencySleep disturbancesUse of sleeping medicationDaytime dysfunctionp p others [] The PSQI subcomponents which greatestcontributed to the overall score were sleep latency timetaken to fall asleep sleep quality overall subjectivequality and sleep disturbances Sleep latency and sleepdisturbances have previously been identified as the mostcommon contributors to sleep quality in caregivers ofPLWD in a recent systematic review [] Given thedearth of Australian specific data this study identifiedimportant depth of detail around caregiverssleepcharacteristics of caregivers took longer than therecommended min to fall asleep only of caregivers slept more than h of caregivers had sleepefficiency lower that the recommended and ofcaregivers used sleep aiding medication in the previousmonth Taken togetherfindingspresent a novel and ominous overview of the poor sleephealth conditions that Australian dementia caregiversexperiencethese descriptiveCaregivers of community dwelling people living withdementia reported poor sleep quality and high levels ofdepression anxiety and stress This study found that poorsleep was correlated with subjective feelings of depressionanxiety and stress which were in keeping with previousliterature around caregiving and psychological distressMore than half of the caregivers surveyed reported symptoms of depression and stress and respectivelyand reported anxiety These findings are in keepingwith that of a recent study of dementia caregivers in ruralVictoria where of caregivers reported depression orstress and reported anxiety []Previous work has identified an association betweendepression and poor sleep among dementia caregivers[ ] Reduced quality of sleep and depression arehigher among caregivers of people living with dementia[] Furthermore caregivers of people living withdementia who were also depressed experience a greatervariation in sleep patterns []This study reveals sleep scores were significantly associated with measures of depression anxiety and stressThese findings illustrate the interplay between sleepquality and quantity and psychological wellbeing incaregivers providing care for an individual living withdementia Further exploration of predictive factors incaregiverssleep quality identified stress as a keypredictor of poor overall sleep quality whilst adjustingfor age gender comorbidities and length of care This isa novel finding and represents a potential therapeutictarget to improve sleep quality in dementia caregiversDespite the high prevalence of selfidentified poorsleep overnight respite was only used by of participants which is in keeping with previous research thatcites of Australian caregivers have never usedrespite services [] Access to respite care remains oneof the major means of easing caregiver burden and is 0cSmyth BMC Geriatrics Page of frequently identified as needed by caregivers yet remainsunderutilised Whilst a high proportion of dementiacaregivers report a need for respite services there isinsufficient awareness of and access to respite servicesfor caregivers [] Respite service availability can beinvaluable with caregivers reporting lower stress levelsand improved health after use [] Sleep disturbanceshave also been shown to be partially reversed for caregiversduring periods of respite care [] perhaps due to reversalof hyper stressed state Respite services have been cited asan important measure to allow caregivers time to attend totheir own health imperative to supporting the caregiver tocontinue in providing care for their loved one []A recent Australian parliamentary inquiry into sleephealth identified sleep as a foundation of positive healthand wellbeing alongside diet and exercise [] Furthermore the report urged government to prioritise sleephealth as a national priority given its pivotal role inmaintaining health It is clear that caregivers of PLWDhave suboptimal sleep which is associated with poorerpsychological wellbeing and potential increased risk ofdeveloping chronic health conditions such as diabetesand cardiovascular disease [] Interestingly cardiovascular disease and endocrine disease such as diabeteswere the most prevalent comorbidities in our studypopulation Previous studies have also identified highblood pressure diabetes and arthritis as the most prevalent chronic disease in the dementia caregiver groupwhich is in keeping with our findings []Managing sleep and its associated mediators will havedirect impact on both the caregivers own health as wellas the caregiver care recipient relationship It is imperative to provide educational support around sleep considerpractical interventions such as overnight respite and toaddress stress management interventions for caregivers inturn reinstating and preserving sleep of this critical population of informal caregiversLimitationsA limitation of this study was the small sample size despitenumerous attempts to recruit participants and involvementof national anisations Although over participantsprovided demographic details only participants completed all questionnaires and were included in statisticalanalyses Challenges related to recruiting caregivers andparticularly caregivers of people living with dementia hasbeen described in numerous publications [ ]Caregivers of people with dementia living in the community are more likely to be female and this was representedin our population Furthermore caregivers who are underthe greatest stress may also be those least likely to participate However this remains one of the larger studies ofAustralian dementia caregivers sleep Another limitationwithin this study was that no data was collected regardingthe severity of dementia or behavioural disturbances whichmay impact sleep of the caregiverConclusionSleep problems are widespread within the population ofdementia caregivers Given that the majority of peopleliving with dementia are reliant on family caregiversminimising health and psychologicalimpact on caregivers should be of major concern Furthermore sleepquality is correlated with emotional distress in dementiacaregivers so improving the sleep of caregivers may inturn improve their psychological wellbeing Converselystress is a significant predictor of poor sleep so managing stress may have a positive impact on sleepDementia caregivers are often older with coexistingmorbidities and high levels of psychological distress Withprolonged caregiving and the progression of dementia ofthe care recipient there is the potential for a concurrent decline in the caregivers mental and physical health In orderto support the caregiver in their role it is of the upmost importance that we promote and maximises their health andwellbeing By managing and minimising the negative factorsassociated with caregiving we can enhance caregiving satisfaction and gratification In turn this can ensure optimalcaregivercare recipient relationship supporting the personliving with dementia to remain at home as long as possibleAbbreviationsDASS21 Depression Anxiety and Stress Scale21 PLWD PersonPeopleliving with dementia PSQI Pittsburgh Sleep Quality Index WHO WorldHealth anisationAcknowledgementsThe authors acknowledge the statistical advice of Dr Mark Jenkins and DrPeter PalamaraAuthors contributionsAS was involved in study design data collection data interpretation andmanuscript preparation LW supported study design data interpretation andediting of the manuscript CV supported study design and manuscriptpreparation EQ advised on use of psychological tools interpretation of theirdata and manuscript preparation LE was involved in data collection andmanuscript preparation All authors have read and approved the manuscriptFundingThis research was supported by an Edith Cowan University ECU Early CareerResearcher Grant awarded to Dr Aisling Smyth ECU had no role in thedesign data collection and data interpretation of the studyAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was granted ethical approval by Edith Cowan University HumanResearch Ethics Committee No Informed written consent wasobtained and participants received no incentive for taking partConsent for publicationNot applicableCompeting interestsThe authors state that they have no competing interests 0cSmyth BMC Geriatrics Page of Cohen J Statistical power analysis for the behavioral sciences 2nd edHillsdale NJ Erlbaum Wilcox S King AC Sleep complaints in older women who are familycaregivers J Gerontol B Psychol Sci Soc Sci 1999543P189Simpson C Carter P Dementia behavioural and psychiatric symptomseffect on caregiver's sleep J Clin Nurs Kochar J Fredman L Stone KL Cauley JA Sleep problems in elderlywomen caregivers depend on the level of depressive symptoms resultsof the caregiverstudy of osteoporotic fractures J Am Geriatr Soc Lee D Heo S Yoon S Chang D Lee S Rhee H Sleep disturbances andpredictive factors in caregivers of patients with mild cognitive impairmentand dementia J Clin Neurol Johnson E Respite reconsidered a discussion of key issues and futuredirections for carer respite Sydney Carers Australia Phillipson L Johnson K Cridland E Hall D Neville C Fielding E et alKnowledge helpseeking and efficacy to find respite services an exploratorystudy in helpseeking carers of people with dementia in the context ofaged care reforms BMC Geriatr Oconnell B Hawkins M Ostaszkiewicz J Millar L Carers perspectives ofrespite care in Australia an evaluative study Contemp Nurse Lee D Man K Lindesay J Effect of institutional respite care on the Sleepof people with dementia and their primary caregivers J Am Geriatr Soc Parliament of the Commonwealth of Australia Bedtime Reading inquiry intoSleep health awareness in Australia Canberra Commonwealth of Australia [Available from httpsparlinfoaphgovauparlInfodownloadcommitteesreportrep024220toc_pdfBedtimeReadingpdffileTypeapplication2Fpdf] Accessed Mar Moon H DilworthAnderson P Baby boomer caregiver and dementiacaregiving findings from the National Study of caregiving Age Ageing Bristow M Cook R Erzinclioglu S Hodges J Stress distress and mucosalimmunity in carers of a partner with frontotemporal dementia Aging MentHealth McConaghy R Caltabiano ML Caring for a person with dementia exploringrelationships between perceived burden depression coping and wellbeing Nurs Health Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsAuthor details1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA Australia 2School of Arts and HumanitiesPsychological Services Centre Edith Cowan University Joondalup AustraliaReceived April Accepted August ReferencesAlzheimer's Association Types of dementia [Available from httpswwwalzalzheimersdementiawhatisdementiatypesofdementia]Ozcare Types of Dementia The six most common forms of dementia [Available from httpswwwozcareaudementiacareunderstandingdementiatypesofdementia]Dementia Australia Types of dementia [Available from httpswwwdementiaauinformationaboutdementiatypesofdementia]Global Burden of Disease Dementia Collaborators Global regional andnational burden of Alzheimer's disease and other dementias asystematic analysis for the Global Burden of Disease Study LancetNeurol Alzheimer's Disease International The Global Voice on Dementia2020 Availablefrom httpswwwalzcoukresearchstatistics] Accessed Mar James BD Bennett DA Causes and patterns of dementia an update inthe era of redefining Alzheimer's disease Annu Rev Public Health Ahern S Cronin J Woods N Brady NM O'Regan NA Trawley S et alDementia in older people admitted to hospital an analysis of length of stayand associated costs Int J Geriatr Psychiatr Allen AP Buckley MM Cryan JF Nà Chorcoráin A Dinan TG KearneyPM et alInformal caregiving for dementia patients the contribution ofpatient characteristics and behaviours to caregiver burden Age AgeingBevans M Sternberg EM Caregiving burden stress and health effectsamong family caregivers of adult Cancer patients JAMA Alzheimer's Disease International Global estimates of informal care LondonUK 2018Available from httpswwwalzcoukadipdfglobalestimatesofinformalcarepdf Accessed Mar Gao C Chapagain NY Scullin MK Sleep Duration and Sleep Quality inCaregivers of Patients With Dementia A Systematic Review and Metaanalysis JAMA Netw 201928e199891Ervin K Pallant J R C Caregiver distress in dementia in rural VictoriaAustralasian J Ageing Peng HL Lorenz RA Chang YP Factors associated with sleep in familycaregivers of individuals with dementia Perspect Psychiatric Care Hirshkowitz M Whiton K Albert SM Alessi C Bruni O DonCarlos L et alNational Sleep Foundation's updated sleep duration recommendations finalreport Sleep Health Jay S Vincent G Kovac K Dorrian J Thomas M Reynolds A Ferguson SReport Reducing Sleep Disruption in Carers Australia Carers Australia httpswwwsleephealthfoundationaufilesResearch_GrantsSpecial_reportsReport_Carers_Australia_FINALv2pdf Accessed Mar2020 Worley SL The extraordinary importance of Sleep the detrimental effects ofinadequate Sleep on health and public safety drive an explosion of Sleepresearch P T Rowe MA McCrae CS Campbell JM Benito AP Cheng J Sleep patterndifferences between older adult dementia caregivers and older adultnoncaregivers using objective and subjective measures J Clin Sleep MedSchulz R Belle SH Czaja SJ McGinnis KA Stevens A Zhang S Longtermcare placement of dementia patients and caregiver health and wellbeingJAMA Gaugler JE Yu F Krichbaum K Wyman JF Predictors of nursing homeadmission for persons with dementia Med Care Lovibond SH Lovibond PF Manual for the Depression Anxiety Stress ScalesSydney Smyth C The Pittsburgh Sleep quality index PSQI Insight Buysee DJ Reynolds CF Monk TH Berman SR Kupfer DJ The PittsburghSleep Qulaity index a new instrument for psychiatric practice and researchPsychiatry Res | 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 |
Mukonal is an active member of carbazole alkaloids isolated from Murraya koenigii It has been shown to possess remarkable biological and pharmacological activities including anticancer activity Therefore the aim of current investigation was to explore antibreast cancer activity of mukonal and to explore the underlying mechanism Results indicate that mukonal has potential to induce antiproliferative effects against MDAMB231 and SKBR3 cells with an IC50 of µM No significant toxicity of mukonal was observed in case of normal breast cells The antiproliferative effects of mukonal were found to proceed via apoptosis which was further supported by increased cleavage of PARP and caspase3 and reduced expression of Bcl2 Mukonal induced autophagic cells death in breast cancer cells as was evidenced by formation of autophagosomes and enhanced expressions of Beclin1 LC3BI and LC3BII proteins In vivo examination of antibreast cancer property of mukonal indicated that it could potentially reduce tumor weight and volume in xenografted mice models In mukonal has a remarkable potential of inhibiting breast cancer via induction of apoptosis and autophagy Mukonal also inhibited xenografted tumors models in a dosedependent manner Therefore mukonal may prove lead molecule in breast cancer drug discovery and treatment provided further investigations are recommendedKeywords Breast cancer Alkaloids Carbazole alkaloids Mukonal Autophagy ApoptosisIntroductionBreast cancer is the most frequent and devastating disease prevailing in females worldwide Alone in the year of more than a0million deaths were recorded globally due to this lethal malignancy in women Sun et a0 al Every year approximately of all cancers over a0million diagnosed in women are breast cancer United States registered over of all cancers in women were due breast cancer in the year of Siegel et a0al Metastatic nature of breast cancer leads to easy transfer of disease to distant places in the body wherein it develops Correspondence wangcqy2020163comDepartment of Thyroid Breast Surgery Gong An County Peoples Hospital Gong An Jingzhou Hubei Chinaindividually like brain lung liver and bones Early detection of the disease may lead with better overall survival chances and prognosis Screening of breast cancer is widely performed by mammography which has been proved fruitful in lowering the mortality rate effectually Several risk factors have been linked to enhance the possibility of breast cancer development like that of genetic mutations family history estrogen levels aging sex and poor lifestyle Majeed et a0al Primary development of breast cancer takes place from ductal hyperproliferation and then maturing into metastatic or benign tumors Thus far significant advances have been achieved in theoretical and clinical investigations of breast cancer The current strategies for effective management of breast cancer incorporate biologicalprevention chemoprevention 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 0cWang a0et a0al AMB Expr Page of and screening Smith and Henderson Besides recent advances and effective management breast cancer remains major cause of cancer related deaths in females of age a0 years Therefore there is a pressing need for novel and capable chemopreventive drugs that can assists us with better results in overcoming this disastrous malignancy Since time immemorial natural products have revealed health promoting effects in human beings Natural products are mostly the secondary metabolites synthesized by the plants to survive and adapt to harsh environmental conditions Williams et a0 al Alkaloids are a major class of naturally occurring secondary metabolites in plants with huge medicinal and biological properties including antidiabetic analgesic antiinflammatory antihypertensive antimalarial and anticancer Mukonal molecule is an active member of carbazole alkaloids found in Murraya koenigii Bhattacharyya and Chakraborty This molecule has shown significant antioxidant antimicrobial and anticancer activity Samanta et a0al Mukonal has been reported to show anticancer activities against different human cancer cell lines in a0vitro including laryngeal AMCHN8 cancer cells and nasopharyngeal CNE1 carcinoma cells It has shown a remarkable potential of autophagy initiation apoptosis induction modification of mitochondrial membrane potential cell cycle arrest blocking of MEKERK and PI3KAKT signalling pathways Li et a0al Guo et a0al Therefore current investigation was designed to unveil the antibreast cancer potential of mukonal along with its effects of autophagy and apoptosis inductionMaterials and a0methodsReagents chemicals and a0cell culturesMukonal with of purity by HPLC and other chemicals were bought from SigmaAldrich Darmstadt Germany unless otherwise mentioned Breast cancer cell lines MDAMB231 CAMA1 MDAMB436 and SKBR3 and normal breast cell line MB157 were procured from Type Culture Collection of Chinese Academy of Sciences Shanghai China All cell cultures were grown in RPMI1640 media maintaining fetal bovine serum Thermo Fisher Scientific Inc Waltham United States and penicillin G a0Uml and streptomycin a0µgml as suitable antibiotics Allinclusive cell cultures were placed and maintained within a humid environment of CO2 concentration and a temperature of a0°CDetermination of a0cellular proliferationThe cellular proliferation of MDAMB231 CAMA MDAMB436 and SKBR3 and a normal breast MB157 cell line were estimated after mukonal exposure by 345dimethylthiazol2yl25diphenyl tetrazolium bromide MTT assay In brief both cancer and noncancer cells were placed with a concentration of cells per well of 96well plates and precultured for a0h in a humid environment of CO2 concentration and a temperature of a0 °C Thereafter each well plate was supplemented with different mukonal doses viz and a0µM and left untouched on incubation for a0h Mukonal treated cancer and noncancer breast cells were washed twice with phosphate buffered saline PBS prior to staining with a0µl of MTT stock solution of concentration a0 mgml for a0 h The formazan crystals then evolved were dissolved with dimethyl sulphoxide followed by colorimetric analysis Finally absorbance was taken at a0nm to determine the optical density using microplate spectrophotometer BioRad Laboratories Inc Hercules United States Experiments for individual mukonal concentrations were repeated thriceColony formation assayMDAMB231 and SKBR3 were plated onto 6well plates with cells per well After a0h of preculturing the cells treatment with variant mukonal doses of and a0µM was instigated Thereafter cancer breast cells were left on incubation for days devoid of any physical and chemical disturbance After days of incubation cell colonies were stained with crystal violet Finally MDAMB231 and SKBR3 cell colonies were totaled under a light microscope OLYMPUS Japan and only colonies with cells were considered for countingAnnexin VPI staining assayAnnexin VPI dual staining assay was performed to monitor and quantify apoptosis in mukonal treated cancer MDAMB231 and SKBR3 breast cells These cancer cells were placed onto 6well plates and subjected to variant mukonal doses viz and a0µM for a0h After that cells were washed in PBS fixed in of formaldehyde and yet again washed in PBS Finally these cells were stained with annexin VPI dual staining solution and eventually analyzed through flow cytometryTransmission electron microscopyMukonal treated MDAMB231 and SKBR3 cells at variant doses and a0µM were subjected to electron microscopy for autophagy assessment Mukonal treated cancer cells were fixed in the solution of glutaraldehyde bearing a0m of sodium cacodylate Afterwards post fixation of treated cells was carried out by of osmium tetroxide followed by moisture removal using alcohol Then cells were prepared for implantation over Epon for sectioning and finally investigated under Zeiss CEM electron microscope Oberkochen Germany 0cWang a0et a0al AMB Expr Page of Assessment of a0protein expressions by a0western blottingThe expressions levels of apoptosis and autophagy allied proteins were evaluated by western blotting Mukonal treated and a0 µM and untreated controls tumerous breast cells MDAMB231 and SKBR3 were lysed with RIPA buffer for protein collection Each lysate was subjected to bicinchoninic acid assay for quantification of proteins and a0µg of proteins from each sample were run on SDSPAGE Thereafter proteins were transferred to PVDF membranes and these membranes were blocked with nonfat milk at room temperature for a0h blocked membranes were subjected to suitable primary antibodies of dilution anticaspase3 antiPARP antiBAX antiBcl2 antiLC3BI antiLC3BII and antiBeclin1 Santa Cruz Biotechnology Inc Dallas United States overnight at a0°C Afterwards secondary antibody treatment was instigated with dilutions of horse radish peroxidaseconjugated antirabbit secondary antibody Santa Cruz Biotechnology Inc Dallas United States for a0h at room temperature Finally the protein bands were visualized using ECL Advanced Western Blot Detection kit GE Healthcare Life Sciences SwedenXenograft studyXenograft studies were carried out by strictly obeying the ethical guidelines permitted by animal ethics committee Gong An County Peoples Hospital Gong An Jingzhou Hubei China Immunodeficient nude mice of 6weeks weighing a0 g were placed in sterile steel cages These cages were placed in an environment of a0 h cycle of lightdark relative humidity of and a moderate temperature of °C These mice were given subcutaneous injections of SKBR3 cells on their left flank As the tumor became superficially apparent treatment with mukonal was instigated by intraperitoneal insertion with of DMS dissolved mukonal and diluted normal saline at doses of and a0mgkg body weight This treatment procedure was repeated each second of a week and only of DMSO was injected to the control mice The tumor volume and weight was monitored after every week and the procedure lasted for weeks The mice were then sacrificed for this noble cause by inhaling of deep anesthesia with isoflurane The tumor volume was determined using the following formula V W W L2 where V is the volume W is the width and L is the length of the tumor The study was approved by the animal ethics committee of Gong An County Peoples Hospital Gong An Jingzhou Hubei China under approval number GACPH022019Statistical analysisStatistical analysis of data collected by execution of each individual experiment in triplicates was performed via analysis of variance ANOVA and followed by Tukeys posthoc test Entire data were represented as mean ± SE standard error considering p as statistically significantResultsMukonal inhibited proliferation of a0breast cancer cellsThe proliferation rate of four different cancer breast cell lines MDAMB231 CAMA1 MDAMB436 and SKBR3 and a normal breast MB157 cell line was determined using MTT assay after treatment with variant Mukonal a0µM Results revealed that mukonal significantly retarded the proliferation of these cancer breast cells with an IC50 value ranging from a0 µM to a0µM Table a0 Higher efficiency with lower IC50 value was recorded in case of MDAMB231 and SKBR3 cells Fig a0 1a Therefore rest of the studies was carried on these two cell lines The viability in case of MDAMB231 cells reduced from to almost Fig a01b and that of SKBR3 cells from to nearly with enhanced doses of mukonal from to a0µM Fig a01c The mukonal induced no significant cytotoxicity against normal MB157 breast cells and a high IC50 of a0µM was obtained Fig a0 1d Enhanced cytotoxicity was observed in case of normal MB157 cells at higher mukonal doses Clonogenic assay was used to monitor the impact of mukonal treatment over colony generation propensity of MDAMB231 and SKBR3 cells On treatment of MDAMB231 and SKBR3 cells with mukonal remarkable suppression of their colonies was observed in comparison to controls The numbers of MDAMB231 colonies left over were nearly and those of SKBR3 cells were nearly after days long treatment at a0µM of mukonal concentration Fig a0 Therefore mukonal induced remarkable toxicity against MDAMB231 and SKBR3 cells in comparison to MB157 cells which indicates specificity in anticancer activity of mukonalTable Effects of a0 Mukonal on a0 the a0 viability of a0 breast cancer cells as a0 depicted by a0 MTT assay and a0 presented as a0 IC50S noBreast cancer cell linesIC50 µMMDAMB231CAMA1MDAMB436SKBR3MB157The experiments were performed in triplicate 0cWang a0et a0al AMB Expr Page of Fig Mukonal inhibits the growth of breast cancer cells a Chemical structure of mukonal molecule b The viability of SKBR3 breast tumor cells after being subjected varying mukonal concentration as indicated SKBR3 cells were exposed to mukonal for h and then subsequently stained with MTT solution for calorimetric analysis c The viability of MDAMB231 breast tumor cells after being subjected varying mukonal concentration as indicated d The viability of normal MB157 breast cells after being subjected varying mukonal concentration as indicated All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figureFig Clonogenic analysis of MDAMB231 and SKBR3 cells after being exposed to indicated mukonal doses MDAMB231 and SKBR3 breast tumor cell lines were treated with mukonal left untouched for days and stained with crystal violet to calculate the number of colonies generated provided considering colonies with number of cells for calculation All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figure 0cWang a0et a0al AMB Expr Page of Mukonal induced apoptosis in a0breast cancer cellsThe annexin VPI assay was employed to determine the percentage of apoptosis in MDAMB231 and SKBR3 cells Results indicated that mukonal could potentially exhibit proapoptotic effects against both MDAMB231 as well as SKBR3 cells in a dosereliant fashion In comparison to control group the number of early apoptotic late apoptotic and necrotic cell percentage enhanced significantly in treated groups The percentage of apoptotic cells was raised to about in case of treated SKBR3 cells Fig a03a The apoptotic cell percentage of MDAMB231 cells at controls was but it enhanced to nearly at a0 µM of mukonal concentration Fig a03b Further western blotting analysis indicated that Mukonal enhanced the cleavage of caspase3 and PARP levels Moreover the expression of Bcl2 decreased and Bax increased with increase in the dosage of mukonal in case of SKBR3 cells Fig a03c The expressions of cleaved caspase3 and cleaved PARP enhanced significantly and Bcl2 PARP and Bax decreased with enhancing doses of mukonal in case of MDAMB231 cells Fig a03d Therefore the results from annexin VPI staining and western blotting analysis indicated that antiproliferative effects of mukonal could be mediated via its apoptosis inducing propensityMukonal induced autophagy in a0breast cancer cellsThe impact of mukonal exposure on cellular autophagy in MDAMB231 and SKBR3 cells was analyzed through transmission electron microscopy and western blotting assay Results indicated that mukonal potentially induce proautophagic effects in both of these cancer breast cell lines In comparison to control groups autophagosomes are clearly visible in both mukonal treated SKBR3 Fig a04a and MDAMB231 Fig a04b cells After exposure to variant doses a0µM of mukonal MDAMB231 and SKBR3 cell lines showed enhanced expression levels of Beclin1 LC3BI and LC3BII proteins Which indicated molecular features of autophagic cell death in mukonal treated MDAMB231 and SKBR3 cancer cells Fig a04c d Therefore it may be concluded that mukonal exhibits antiproliferative effects mediated via its autophagy inducing potentialIn vivo inhibition of a0tumor growth by a0mukonalNude mice xenografts were used to determine in a0 vivo anticancer effects of mukonal The results showed that SKBR3 tumor growth was remarkably retarded by mukonal administration in comparison to control group At the end of 6weeks the average tumor volume and weight in untreated control group was substantially advanced than mukonal treated groups Moreover the in a0 vivo anticancer effects of mukonal were dose and timedependent manner Fig a05a bDiscussionDespite significant advancements in cancer research management it still remains the most prevalently diagnosed cancer and cause of death in women globally Better approaches are needed to understand and recognize this disease at molecular levels Due to timely diagnoses of the disease North America has registered over 5year survival rate among the breast cancer patients DeSantis et a0al High frequency of occurrence late diagnosis and disastrous side effects of present day chemopreventives creates an emergency for novel therapeutic drugs that can deliver better results with higher efficacy Waks and Winer Collignon et a0 al Mukonal is a carbazole alkaloid and has revealed to possess remarkable pharmacological and biological propensities It has been reported to induce anticancer effects against different human cancer cell lines Li et a0al Guo et a0al Therefore the current study was undertaken due to accumulative evidences suggesting that mukonal has a great propensity to act as an anticancer agent The results revealed that mukonal decreased the proliferation rate of five variant breast cancer cell lines but remarkable results with IC50 of a0µM was recorded against breast cancer MDAMB231 and SKBR3 cancer cell lines Mukonal showed a minuscule toxicity against normal breast MB157 cell line indicating some specificity of inducing toxicity against cancerous breast cell lines Mukonal induced potential inhibition of colony generation by MDAMB231 and SKBR3 cells Further chemopreventive drugs target several cellular processes to induce cytotoxicity against cancer cells Apoptosis has been a focal target of chemopreventives and is often termed as typeI PCD programmed cell death Khursheed et a0 al Apoptosis remains dormant in normal cells but in case of injury malfunction aging and macromolecule accumulation in cells it plays a vital role of elimination Bonofiglio et a0al Herein mukonal induced apoptotic cell death in MDAMB231 and SKBR3 cell lines in a dosereliant fashion as suggested by annexin VFITC assay Similar results have been reported previously wherein mukonal induced apoptosis in laryngeal cancer cells Mukonal stimulated apoptosis was further supported by enhanced expression levels of cleaved PARP cleaved caspase3 and Bax and retarded expression levels of caspase3 PARP and Bcl2 proteins in both the cancerous cell lines after mukonal exposure Autophagy is another process that remains conserved in multicellular anisms activated under stressful conditions like starvation Levy et a0 al Autophagy is often termed as typeII PCD and 0cWang a0et a0al AMB Expr Page of Fig a Flow cytometric analysis of mukonal treated SKBR3 cells SKBR3 cells were cultured in 6well plates and then subjected to indicated mukonal doses Afterwards staining with annexin VPI was performed to analyze apoptosis flow cytometrically b Flow cytometric analysis of mukonal treated MDAMB231 cells MDAMB231 cells were cultured in 6well plates and then subjected to indicated mukonal doses Afterwards staining with annexin VPI was performed to analyze apoptosis flow cytometrically c Western blotting analysis was performed to assess the activity of apoptosis allied proteins in SKBR3 cells Results indicated that mukonal enhanced activity of proapoptosis proteins wherein blocking of antiapoptotic protein expression d Western blotting analysis was performed to assess the activity of apoptosis allied proteins in MDAMB231 cells Results indicating that mukonal enhanced activity of proapoptosis proteins in MDAMB231 cells wherein blocking of antiapoptotic protein expression All the experiments were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figure 0cWang a0et a0al AMB Expr Page of Fig a TEM analysis of SKBR3 cells after being exposed to indicated doses of mukonal Results revealed formation of autophagic vesiclesautophagosomes in treated groups as compared to control group Autophagosomes have been shown by arrows in the picture b TEM analysis of MDAMB231 cells after being exposed to indicated doses of mukonal Results revealed formation of autophagic vesiclesautophagosomes in treated groups as compared to control group Autophagosomes have been shown by arrows in the picture c Western blotting results of SKBR3 cells after being exposed to mukonal at indicated doses In treated groups enhanced activity of Beclin1 LC3BI and LC3BII was observed with increasing concentrations of mukonal d Western blotting results of MDAMB231 cells after being exposed to mukonal at indicated doses In treated groups enhanced activity of Beclin1 LC3BI and LC3BII was observed with increasing concentrations of mukonalalso remains as focal target of chemopreventives PoilletPerez et a0al Autophagy plays a key role in the degradation of marcomolecules and damaged anelle This process is completely hallmarked by the formation of autophagosomes which on maturation turns into autolysosomes GarcaPrat et a0 al Mukonal was observed to induce autophagic cell death in both MDAMB231 and SKBR3 cell lines Similar results have been reported previous wherein mukonal induced autophagic cell death in human nasopharyngeal cancer cells Both of these cancerous cell lines showed enhanced expressions levels of LC3BI LC3BII and Beclin1 proteins indicating autophagy initiation at molecular levelsThe in a0 vivo investigation of mukonal revealed that it noticeably retarded growth of MDAMB231 and SKBR3 breast tumor growth in comparison to untreated control group and no apparent toxicity was detected The tumor weight and volume in mice models were observed to decline comparative to increased doses of mukonalIn the results of this investigation revealed that mukonal could potentially induce antibreast cancer effects both in a0 vitro and in a0 vivo against MDAMB231 and SKBR3 cell lines The antibreast cancer effects of mukonal were observed to mediate through induction of autophagy and apoptosis These studies point towards the potential of Mukonal in the treatment of breast cancer However further in a0vitro and in a0vivo studies are required to fully establish it as a lead molecule in the development of breast cancer chemotherapy 0cWang a0et a0al AMB Expr Page of Received June Accepted July ReferencesBhattacharyya P Chakraborty A Mukonal a probable biogenetic intermediate of pyranocarbazole alkaloids from Murraya koenigii Phytochemistry Bonofiglio D Giordano C De Amicis F Lanzino M Ando S Natural products as promising antitumoral agents in breast cancer mechanisms of action and molecular targets MiniRev Med Chem Collignon J Lousberg L Schroeder H Jerusalem G Triplenegative breast cancer treatment challenges and solutions Breast Cancer Targets Therapy DeSantis CE Fedewa SA Goding Sauer A Kramer JL Smith RA Jemal A Breast cancer statistics convergence of incidence rates between black and white women CA Cancer J Clin GarcaPrat L MartnezVicente M Perdiguero E Ortet L RodrguezUbreva J Rebollo E RuizBonilla V Gutarra S Ballestar E Serrano AL Sandri M Autophagy maintains stemness by preventing senescence Nature Guo Y Hao Y Guan G Ma S Zhu Z Guo F Bai J Mukonal inhibits cell proliferation alters mitochondrial membrane potential and induces apoptosis and autophagy in human CNE1 nasopharyngeal carcinoma cells Med Sci Mon Int Med J Exp Clin Res Khursheed A Rather MA Rashid R Plantbased natural compounds and herbal extracts as promising apoptotic agents their implications for cancer prevention and treatment Adv Biomed Pharma Levy JM Towers CG Thorburn A Targeting autophagy in cancer Nat Rev Cancer Li L Huizhi L Binu W Xinxin D Longjun W Liping Y Yingying Z Anticancer activity of mukonal against human laryngeal cancer cells involves apoptosis cell cycle arrest and inhibition of PI3KAKT and MEKERK signalling pathways Med Sci Mon Int Med J Exp Clin Res Majeed W Aslam B Javed I Khaliq T Muhammad F Ali A Raza A Breast cancer major risk factors and recent developments in treatment Asian Pac J Cancer Prev PoilletPerez L Xie X Zhan L Yang Y Sharp DW Hu ZS Su X Maganti A Jiang C Lu W Zheng H Autophagy maintains tumour growth through circulating arginine Nature Samanta SK Kandimalla R Gogoi B Dutta KN Choudhury P Deb PK Devi R Pal BC Talukdar NC Phytochemical portfolio and anticancer activity of Murraya koenigii and its primary active component mahanine Pharmacol Res Siegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin Smith G Henderson IC New treatments for breast cancer Semin Oncol Sun YS Zhao Z Yang ZN Xu F Lu HJ Zhu ZY Shi W Jiang J Yao PP Zhu HP Risk factors and preventions of breast cancer Int J Biol Sci Waks AG Winer EP Breast cancer treatment a review JAMA Williams DH Stone MJ Hauck PR Rahman SK Why are secondary metabolites natural products biosynthesized J Nat Prod Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsFig Mukonal inhibits MDAMB231 and SKBR3 tumor growth in vivo a Tumor volume and b tumor weight were measured at indicated time intervals and doses of mukonal molecule All the experiments for separate drug concentration were executed three times and data was shown as mean ± SE standard error The p value of was taken as a statistical significant figureAcknowledgementsAll the author of this manuscript is thankful to Gong An County Peoples Hospital Gong An Jingzhou Hubei China to conduct the presented protocolAuthors contributionsWW and ZJ designed the protocol of the study WW ZZ XZ LC and SB performed the experimental work and collect the data for presented study WW and ZJ involve in the statistical analysis ZJ supervised the work and drafted the manuscript although all author contributed for the preparation of manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsNot applicableEthics approval and consent to participateThe study was approved by the animal ethics committee by Gong An County Peoples Hospital Gong An Jingzhou Hubei China under approval number 721GACPH022019Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0c' | 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 |
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