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3. Results | PMC10454381 | |||
3.1. Baseline Characteristics | Demographic and ANBQ chewing history characteristics are presented in The following variables were compared between randomization groups: (a) age, (b) sex, (c) race/nationality, (d) study site, (e) participant group size, (f) number of ANBQ chews per day, (g) BQDS score, and (h) education level. The results revealed several substantial differences in baseline characteristics between the intervention and control conditions. Intervention condition participants were less likely to be female compared to control condition participants (30.7% versus 46.6%). Intervention condition participants also tended to be younger than control condition participants (mean years of 26.3 and 30.6 years, respectively), and participants in Guam were more likely to be randomized to the control condition as compared to participants in Saipan (see | PMC10454381 | ||
3.2. Cessation Outcomes | REGRESSION | The current analyses included 88 participants in the intervention group and 88 participants in the control group. All participants included in these analyses provided outcome data at the 22-day follow-up. Although we aimed to schedule the initial follow-up assessments exactly 22 days following baseline, scheduling and logistical issues required that 22 days be an approximate timeframe for the follow-up assessments. The results indicated that 34 out of 88 (38.6%) participants in the intervention condition had self-reported ANBQ cessation (i.e., ex-chewers, according to the 22-day survey assessment), whereas 8 of 88 (9.1%) control condition participants self-reported cessation.The proportional hazards regression model revealed a significant effect for the treatment group, with a Wald Chi-square test using a robust variance estimator (df = 1) = 7.6159, An additional analysis of the treatment effect adjusted for the participant characteristics that were not in balance between treatment groups: sex (male or female), age (continuous), and location (Saipan or Guam). This analysis revealed a significant effect for the study condition: robust Wald Chi-square (df = 1) = 7.2017, | PMC10454381 | |
4. Discussion | EVENTS, RECRUITMENT, RECRUITMENT | This analysis of BENIT outcomes revealed strong self-reported intervention effects at the 22-day post-baseline assessment. Participant self-reports of 38.6% cessation for those in the intervention condition versus 9.1% cessation for those in the control condition met the Stage 3 criteria for early termination of the trial. This finding suggests that intensive cessation programs such as BENIT should be further developed and implemented in larger populations. Although the results of the current study are robust, several limitations should be noted. Bio-verification results for BENIT have been reported in a separate publication [The results presented are ‘per protocol’ and did not include 29 participants (14 for control and 15 for intervention) who withdrew from the study prior to the 22-day assessment. Had these 29 cases been included (and counted as failures to quit), cessation rates would have been 33.0% for intervention (34 out of 103) compared to 7.8% (8 out of 102) for control. Randomization did not result in balanced arms for all potentially relevant variables. Specifically, several substantial differences in baseline characteristics were observed between intervention and control condition participants, particularly with regard to sex and age, though controlling for these variables did not affect the significance of the results. These differences were likely due to the allowance for groups to be randomized together. Further, the study involved many logistical and scheduling challenges (e.g., scheduling conflicts, postponements, and weather events). Recruitment was on a rolling basis and involved word-of-mouth effects (i.e., non-random effects). Thus, controlling for imbalances between intervention and control conditions needed to be accomplished statistically.Recruiting participants for BENIT was challenging. There were several reasons for this. First, the populations of BQ chewers in Guam and Saipan are limited because the populations of these islands are not large. Second, the study was limited to those who wanted to quit (among other inclusion criteria). Third, participation in BENIT required a substantial commitment of time and energy on the part of participants (five in-person sessions). Fourth, our modest budget and research team limited the time we could spend on recruitment.We acknowledge that we were not able to accomplish all of our original objectives as described in Paulino et al. [Despite these limitations, it appears that the rationale for BENIT is supported and that a cessation program focused on ANBQ chewers who want to quit holds great potential for ANBQ chewers in Guam and Saipan, and possibly other countries in the western Pacific and other regions where ANBQ consumption is high. The results suggest that additional studies of intensive ANBQ cessation interventions are warranted. ANBQ cessation research should be expanded in many directions. Future developments should include: (1) continued refinement of biomarkers; (2) development of pharmaceutical adjuvants (similar to those used for tobacco cessation); and (3) additional treatment modalities such as electronic adjuvants for ANBQ cessation (for example, mobile applications).Mehrtash et al. [ | PMC10454381 | |
Author Contributions | Conceptualization; funding acquisition; supervision; writing—original draft, T.A.H. Formal analysis, L.R.W. and G.B. Data curation; resources, A.J.P.M. Project administration; investigation, A.A.F. Project administration; investigation, P.P. Data curation, J.S.N.C., L.F.T. and P.P.S. Project administration; resources, C.T.K. Conceptualization; funding acquisition; writing—review and editing, Y.C.P. All authors have read and agreed to the published version of the manuscript. | PMC10454381 | ||
Institutional Review Board Statement | MAY | The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of the University of Guam (#16-04, approved 13 May 2016). The University of Guam IRB monitored the study for both study locations (Guam and Saipan) because Saipan does not have an IRB suitable for monitoring a randomized trial. | PMC10454381 | |
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study, and all participants were compensated for their time. | PMC10454381 | ||
Data Availability Statement | Cancer | CANCER | Data will be shared pursuant to the data and specimen sharing policies and procedures of the Pacific Island Partnership for Cancer Health Equity (PIPCHE). Contact information: Hali R. Robinette at the University of Hawaii Cancer Center ( | PMC10454381 |
Conflicts of Interest | The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. | PMC10454381 | ||
References | ORAL CANCER | CONSORT flow diagram for the Betel Nut Intervention Trial.Description of the intervention sessions for the Betel Nut Intervention Trial.Introduction to the program. Obtain signed, informed consent.Health risks of areca nut and betel quid (ANBQ) chewingIntroduce self-monitoring logsIntroduce ‘trigger’ logsDiscuss how to incrementally cut back on chewing prior to cessationSaliva collection and baseline surveyDiscuss trigger logs and self-managementDiscuss lifestyle changes that encourage ANBQ cessationPreparing “excuses” for not chewing and the use of ANBQ substitutesPrepare for the upcoming Quit Day (morning of the next session)QUIT DAYEncourage participants to be helpful and supportive to other participants regardless of chewing statusEmphasize to participants that withdrawal symptoms generally last for 2 weeks before abatingReinforce coping techniques to prevent relapseUtilize social support to maintain chewing abstinenceEmphasize alternative behaviors to chewing (e.g., physical activity)Review and discuss quitting experiences, including coping strategies and different methods that participants used to manage triggers and avoid high-risk situations. Emphasize the immediate benefits of ANBQ cessation on healthReview the negative health risks of ANBQ chewing, including the increased risk of oral cancer and other oral diseasesEmphasize additional strategies for managing urges to chew ANBQContinue to support maintenance of chewing abstinence for those who quitEncourage participants who relapsed to chewing to attempt cessation againTeach approaches for managing thoughts and feelings that can result in relapseReinforce lifestyle changes that are compatible with quitting ANBQReview the use of “excuses” for not chewing and employing “fake chew” and other ANBQ substitutesPlan for the future, and discuss how to maintain ANBQ abstinence over time.Saliva collection and administer (22-day) survey assessmentBaseline participant characteristics. | PMC10454381 | |
Background | pain, OLP | CAVITY, ORAL LICHEN PLANUS, CHRONIC INFLAMMATORY DISEASE | Oral lichen planus (OLP) is a chronic inflammatory disease of the oral cavity that affects many patients’ daily living activities. Topical corticosteroids are the first-line drug for treating OLP. The Oral Impact on Daily Performances index (OIDP) is an Oral-Health-Related Quality of Life (OHRQoL) measure developed to assess the ultimate impacts. The aims of this study were to evaluate the clinical, pain and OHRQoL responses after treating OLP patients with topical corticosteroids for 1 month, and secondly to assess the relationships of changes in the clinical sign score, pain score, and OHRQoL. | PMC10662109 |
Methods | NRS, OLP | Seventy-two OLP patients were treated by topical corticosteroids based on their dentists’ clinical judgments. Clinical and patient-based outcomes were assessed at baseline and follow-up visit. The clinical outcomes were evaluated by the highest and total Thongprasom sign score. Patient-based outcomes were evaluated by numeric rating scale (NRS) and OIDP. The self-rated overall changes in quality of life during the 1-month treatment period using the Patient Global Impression of Change (PGIC) were also recorded at the follow-up visit. | PMC10662109 | |
Results | This study comprised 59 women and 13 men. All clinical and patient-based outcomes were significantly reduced after 1-month treatment with topical corticosteroids ( | PMC10662109 | ||
Conclusions | pain, OLP | Topical corticosteroids were significantly effective after 1-month treatment of OLP based on the clinical and patient-based outcomes. The OHRQoL improvement was significantly related to the reductions in pain and clinical severity. | PMC10662109 | |
Trial registration | TCTR | The trial was registered at the Thai Clinical Trials Registry (TCTR identifier: TCTR 20221110001). | PMC10662109 | |
Keywords | PMC10662109 | |||
Background | pain, OLP | CAVITY, ORAL LICHEN PLANUS, CHRONIC INFLAMMATORY DISEASE | Oral lichen planus (OLP) is a common chronic inflammatory disease of the oral cavity that affects the daily living activities in many patients. Most OLP patients are middle-aged women; the most common lesion sites are the buccal mucosa followed by the tongue, and gingiva [Currently, OLP clinical trials should use a tripartite approach comprising clinical signs, symptoms, and quality of life [Because OLP was a chronic inflammatory disease that is very difficult to completely cure, patients suffer from a burning sensation, pain, and discomfort [The Oral Impact on Daily Performances Index (OIDP) [The previous studies about oral mucosal lesions that used OIDP were cross-sectional and case-control studies, whereas most OLP longitudinal studies used OHIP and OHQoL for OHRQoL evaluation. None of the OLP longitudinal studies used OIDP to measure OHRQoL. Therefore, the aim of this study was to evaluate the clinical, pain and OHRQoL responses after a 1-month treatment with topical corticosteroid in OLP patients. Secondly, to assess the relationships of the changes in the clinical sign score, pain score, and OHRQoL. | PMC10662109 |
Methods | PMC10662109 | |||
Study subjects | OLP | The sample size was calculated using 80% power and 95% confidence level according to data from McGrath et al. [The patients were recruited from the Oral Medicine clinic, Faculty of Dentistry, Chulalongkorn University. The inclusion criteria were being over 18 years old, patients diagnosed with OLP or compatible with OLP following clinical and histopathological criteria [ | PMC10662109 | |
Data collection | PMC10662109 | |||
Demographic data | palate, OLP | Age, sex, systemic condition, medication taking and OLP duration since the first diagnosis were collected from the history taking at the first visit. In addition, the OLP locations (e.g. lips, labial mucosa, buccal mucosa, gingiva, tongue, floor of the mouth, and palate), experience with OLP treatment (i.e., new or follow-up patient) and prescribed topical corticosteroids based on their dentists’ clinical judgments were also recorded. | PMC10662109 | |
Clinical outcomes | palate, OLP lesions, OLP | ATROPHIC | Regarding the clinical data collection, the highest Thongprasom sign score was based on the original Thongprasom sign scoring system, i.e., “0” indicated no lesions; “1” indicated white striae only, “2” indicated white striae with an atrophic area of less than 1 cmAs mentioned earlier that Thongprasom sign scoring system did not take into account the number of OLP lesions, so we purposed the total Thongprasom sign score, which was the sum of all lesions’ Thongprasom sign scores. The OLP distribution was recorded as the outer and inner lips, right and left buccal mucosa, upper right, upper central, upper left, lower left, lower central, and lower right gingiva, dorsal, left and right ventrolateral tongue, floor of the mouth, hard palate, and soft palate [ | PMC10662109 |
Patient-based outcomes | pain, OLP | ULCERATION | The patients were asked about the chief symptoms of OLP that made them visit the dentist at baseline, such as a burning sensation, ulceration, and roughness. The patients could have more than 1 symptom or have no symptoms. In the aspect of pain, the OLP patients reported their NRS score for their worst pain during 1 month at baseline and the follow-up visit [The patients’ OHRQoL was assessed using the Thai version of the OIDP [At the 1-month follow-up visit, the differences in each outcome were calculated by subtracting the follow-up data from the baseline data. Negative values represented a worsened effect, whereas positive values represented an improved effect. The differences in the average values were determined. In addition, the OLP patients were asked to self-rate the overall changes in quality of life during the 1-month treatment period. The rating criteria from the Patient Global Impression of Change (PGIC) measure [
Patient flow chart (Participant flow) | PMC10662109 |
Data analysis | pain | Statistical calculations were performed with SPSS software (SPSS 22.0 for Windows; SPSS, Chicago, IL, USA). Descriptive statistics were used for the demographic and clinical data. The Kolmogorov-Smirnov normality tests were performed to determine the normal data distribution in each outcome. The pain score and total Thongprasom sign score between baseline and after the 1-month treatment were assessed using the paired t-test. The highest Thongprasom sign score, total OIDP percentage score, and performance scores of each daily activity between baseline and after the 1-month treatment were analyzed by the Wilcoxon signed-rank test. The differences in the highest Thongprasom sign score, total Thongprasom sign score, pain score and total OIDP percentage score between two groups of patients who reported no changes to moderately-improved and greatly-improved PGIC were tested using the Mann-Whitney U test. Pearson’s correlation was used to assess the relationship between the differences in the total OIDP percentage score, highest Thongprasom sign score, total Thongprasom sign score and pain score. The significance level was set at | PMC10662109 | |
Results | PMC10662109 | |||
OLP patient characteristics | Diabetes mellitus- Thyroid disease-, thyroid disease, Anti-dyslipidemia, Lips- Labial mucosa- Buccal mucosa-, palate, diabetic, hypertensive, OLP, gout, hypertension, dyslipidemia | THYROID DISEASE, DYSLIPIDEMIA, DIABETES MELLITUS, CAVITY, HYPERTENSION, FLOOR OF MOUTH, ULCERATION, GOUT, HYPERTENSION, DISEASES, DYSLIPIDEMIA | The 72 patients (100% response rate) in this study consisted of 59 women (81.9%) and 13 men (18.1%). The mean age of the patients in this study was 53.3 ± 12.4 years old (range 22–81 years old). Forty-six patients (63.9%) were between 30 and 60 years old. Twenty-three patients (31.9%) were more than 60 years old and 3 patients (4.2%) were less than 30 years old. Fifty-one patients (70.8%) had their OLP less than 1 year before seeing the dentists, however, 14 patients (19.4%) were diagnosed 1–3 years earlier. Seven patients (9.8%) had their lesions for more than 3 years. The mean duration of OLP was 21.3 ± 30.1 months (range 1–120 months).Regarding patients’ systemic conditions, 40 patients (55.6%) reported no medical problem and were not taking any medication. Twenty-two patients (30.6%) were diagnosed as dyslipidemia and took anti-dyslipidemia drugs (e.g. simvastatin, rosuvastatin, etc.) Furthermore, 20 (27.8%) of the patients had hypertension and 9 (12.5%) had diabetes mellitus. Both diseases required more than one group of drugs, namely, 27 patients (37.5%) required hypertensive drugs (e.g. amlodipine, atenolol, enalapril etc.) and 13 patients (18.1%) required diabetic drugs (e.g. metformin, glipizide, etc.). Four patients (5.6%) with thyroid disease and 2 patients (2.8%) with gout had taken levothyroxine and allopurinol, respectively.Some patients reported more than one chief complaint at the first visit. Common chief complaints were burning sensation (80.6%), ulceration (65.3%), and rough oral mucosal surface (19.4%). One patient had no symptom (1.4%). Sixty patients (83.3%) were new patients and 12 patients (16.7%) were follow-up patients.OLP occurred in various sites in the oral cavity, with the most commonly affected area being the buccal mucosa (80.6%) followed by gingiva (76.4%), tongue (18.1%), lips (16.7%), and floor of the mouth (11.1%). Less than 10% of the patients demonstrated OLP at the palate (6.9%) and labial mucosa (5.6%).The most frequently prescribed topical corticosteroids were 0.1% fluocinolone acetonide in orabase (37.5%), followed by 0.05% dexamethasone mouthwash (31.9%), 0.1% triamcinolone mouthwash (16.6%) and 0.05% fluocinolone mouthwash (15.3%). Other topical corticosteroids, 0.05% clobetasol propionate in orabase (2.8%) and 0.1% fluocinolone solution (1.4%), were infrequently prescribed. (Table
Baseline characteristics and clinical data of the OLP patients (N = 72)- Women- Men59 (81.9)13 (18.1)- None- Dyslipidemia- Hypertension- Diabetes mellitus- Thyroid disease- Gout40 (55.6)22 (30.6)20 (27.8)9 (12.5)4 (5.6)2 (2.8)- None- Anti-dyslipidemia drugs- Anti-hypertensive drugs- Anti-diabetic drugs- Levothyroxine- Allopurinol40 (55.6)22 (30.6)27 (37.5)13 (18.1)4 (5.6)2 (2.8)- Burning sensation- Ulceration- Roughness- No symptoms58 (80.6)47 (65.3)14 (19.4)1 (1.4)- New patients- Follow-up patients60 (83.3)12 (16.7)- Lips- Labial mucosa- Buccal mucosa- Gingiva- Tongue- Floor of mouth- Palate12 (16.7)4 (5.6)58 (80.6)55 (76.4)13 (18.1)8 (11.1)5 (6.9)- 0.1% Fluocinolone acetonide in orabase- 0.05% Clobetasol propionate in orabase- 0.05% Dexamethasone mouthwash- 0.1% Triamcinolone mouthwash- 0.05% Fluocinolone mouthwash- 0.1% Fluocinolone solution27 (37.5)2 (2.8)23 (31.9)12 (16.6)11 (15.3)1 (1.4) | PMC10662109 |
Discussion | gingiva, palate, pain, OLP | MINOR, CAVITY | Nowadays, the importance of utilizing patient-based outcomes alongside clinical outcomes has been recognized. The current study’s findings could support patient-centered care in evidence-based dentistry in relation to OLP treatment with topical corticosteroid. Most of the patients participating in this study benefited from using topical corticosteroids, as demonstrated by the improvement in the clinical and patient-based outcomes. We found that the highest and total Thongprasom sign scores for OLP were significantly decreased after 1-month treatment with topical corticosteroids compared with baseline. Several reviews revealed that topical corticosteroids are clinically effective and are considered the treatment of choice for OLP [OLP affects patient’s quality of life, however, effective treatment can improve their ability to perform everyday activities [The PGIC, the patients’ perception on their changing OHRQoL, results revealed that most patients reported improvement in various scales. However, the difference in the reduced clinical, pain and OIDP scores between those reporting up to moderately improved and greatly improved were not significant. Feine et al. [When considering the relationships between the changes in the clinical and patient-based outcomes after 1-month treatment with topical corticosteroids, we found significant relationships for the differences in patient-based outcomes (pain score and total OIDP percentage score) and the differences in clinical outcomes (highest Thongprasom sign score and total Thongprasom sign scores). These findings were as expected and confirmed the abovementioned discussion on the close relationship between outcomes assessed by different instruments belonging to the same concepts, either clinical or patient-based. Regarding the relationships between the difference in clinical outcomes and the difference in patient-based outcomes, the total Thongprasom sign score better correlated with patient-based outcomes, compared with the highest Thongprasom sign score. A possible explanation might relate to the different impacts from different OLP locations. A study reported that OLP occurring at different locations caused different degrees of symptoms, minor symptoms could be expected for lesions occurring at the buccal/labial mucosa, gingiva and palate, while the symptoms could be greater for tongue lesions [Our study’s weak point was the various types and forms of topical corticosteroids with different potencies used in the clinic. Most of our patients received 0.1% fluocinolone acetonide in orabase and 0.05% dexamethasone mouthwash for treating OLP. Thongprasom et al. [Another weak point was the different characteristics of the patients in this study, such as age, sex, duration of OLP, systemic conditions, medication, experience in OLP treatment and types of topical corticosteroid. Moreover, different numbers and locations of OLP in the oral cavity may have impacted the patient-based results and relationship between the clinical outcomes and patient-based outcomes as discussed above. The current study’s objectives did not include the analyses of the association between these factors and patients’ OHRQoL. Therefore, further studies using a larger sample size are recommended to explore the impacts of such factors, for example, type of topical corticosteroid, on clinical and patient-based outcomes after treatment. Such findings may be helpful for medical communication or further insurance planning. Lastly, the current study used the OIDP index, while many other studies have utilized other OHRQoL indices such as the OHIP. Thus, findings from different indices cannot be compared, which consequently, limit the building up knowledge in this field. Future research using the same index as studies in other countries is recommended in order to compare and combine the results through more impactful analyses, which would bring a more understanding on the impacts of OLP on dental patients globally and to support the pragmatic use of OHRQoL index in dental clinics. | PMC10662109 |
Conclusion | pain, OLP | After a 1-month OLP treatment with topical corticosteroids, the highest Thongprasom sign score, total Thongprasom sign score, and pain score were significantly reduced. The patients’ overall OHRQoL and daily performances were significantly improved, except for Relaxing including sleeping of which none of the patients reported a problem before treatment. The differences in the clinical and patient-based outcomes in the group reporting up to moderately improved did not significantly differ from those in the group reporting a greatly improved quality of life. There were significant relationships between the improvement in two clinical outcomes and the improvement in two patient-based outcomes. The decrease in the total Thongprasom sign score was also significantly related to the improvement in OHRQoL. | PMC10662109 | |
Acknowledgements | ORAL DISEASE | The authors thank the participants in this study. We appreciate the staff of the Department of Oral Medicine and the Research Unit in Oral Diseases, Faculty of Dentistry, Chulalongkorn University for their assistance. We also express our thanks to Dr. Kevin Tompkins for English language editing. | PMC10662109 | |
Authors’ contributions | PP, SK contributed to the study conception and design, methodology, project administration, supervision, and writing-review & re-editing. WK contributed to the data collection, formal analysis and interpretation of the data, and writing-original draft. PP contributed in the funding acquisition. All authors read and approved the final manuscript. | PMC10662109 | ||
Funding | This research was supported by the Faculty Research grant (DRF 65043), Faculty of Dentistry, Chulalongkorn University and the Royal College of Dental Surgeons of Thailand grant. | PMC10662109 | ||
Data availability | All data generated or analyzed during this study are included in this published article. | PMC10662109 | ||
Declarations | PMC10662109 | |||
Ethics approval and consent to participate | The present study was performed with informed consent following protocols approved by the Human Research Ethics Committee of the Faculty of Dentistry, Chulalongkorn University (certificate number HREC-DCU 2021-090 approved on 3 December 2021). All methods were performed in accordance with the Declaration of Helsinki. | PMC10662109 | ||
Consent for publication | Not applicable. | PMC10662109 | ||
Competing interests | The authors declare no competing interests. | PMC10662109 | ||
References | PMC10662109 | |||
Background and purpose | both-bone forearm fractures, loss of flexion and extension of the elbow | previous RCT compared short-term results of above-elbow cast (AEC) with early conversion to below-elbow cast (BEC) in children with non-reduced diaphyseal both-bone forearm fractures. After 7 months both groups had comparable function. Our primary aim was to investigate whether forearm rotation improves or worsens over time. Secondary aims were loss of flexion and extension of the elbow and wrist, patient-reported outcomes measures, grip strength ratio, and radiographic assessment. | PMC10561100 | |
Patients and methods | We performed long-term follow-up (FU) of a previous RCT. All patients were invited again for the long-term FU measurements. Primary outcome was limitation of forearm rotation. Secondary outcomes were loss of flexion and extension of the elbow and wrist compared with the contralateral forearm, the ABILHAND-Kids questionnaire and the Disabilities of the Arm, Shoulder and Hand (DASH) questionnaire, grip strength ratio, and radiographic assessment. | PMC10561100 | ||
Results | The mean FU was 7.5 (4.4–9.6) years. Of the initial 47 children, 38 (81%) participated. Rotation improved in both groups over time, with no significant difference in the final forearm rotation: 8° (SD 22) for the AEC group and 8° (SD 15) for the BEC group with a mean difference of 0° (95% confidence interval –13 to 12). Secondary outcomes showed no statistically significant differences. Finally, children < 9 years almost all have full recovery of function. | PMC10561100 | ||
Conclusion | both-bone forearm fracture, fractures | Long-term follow-up showed that loss of forearm rotation after a non-reduced diaphyseal both-bone forearm fracture improved significantly compared with that at 7 months, independent of the initial treatment and children aged < 9 will have almost full recovery of function. This substantiates that the remaining growth behaves like a “friend” at long-term follow-up.Forearm fractures of both bones account for 40% of all pediatric fractures [Previous studies showed a persisting loss of forearm rotation of up to 15% of the normal range of motion, especially in diaphyseal located fractures [ | PMC10561100 | |
Patients and methods | fracture | Our study is a long-term follow-up of a previous RCT. All 47 patients who had been previously included in the RCT by Colaris et al. [Criteria for reduction of the fracture of radius and/or ulna based on anteroposterior and/or lateral radiographs | PMC10561100 | |
Outcome measures | angulation | SECONDARY, COMPLICATION | Our primary outcome was change in loss of forearm rotation in comparison with the contralateral side and a comparison between the 2 casting methods. Secondary outcome measures were change in loss of flexion and extension of the elbow and wrist compared with the contralateral forearm, the Dutch version of the DASH and ABILHAND-Kids questionnaire, grip strength, and radiographic assessment of the radius and ulna. Cosmetics was measured as parents/child VAS 0–10 and surgeon VAS 0–10 (10 being the optimal score). An independent orthopedic surgeon (LD) measured the forearm rotation, flexion, and extension of the elbow and wrist in a standardized manner with the use of a goniometer. Before each measurement, the examiner ensured that the child was standing in an upright position. Meanwhile, the elbow was positioned firmly against the torso to eliminate compensating forearm rotation by using movements of the elbow and shoulder. The elbow was flexed at 90° with the forearm in mid-position and the wrist in neutral. Grip strength was determined using a JAMAR dynamometer (JLW Instruments, Chicago, IL, USA) on both sides and calculating a ratio of grip strength of the affected forearm/contralateral side. Furthermore, patients were asked to fill in the DASH and the ABILHAND-Kids questionnaires. The radiographic assessment included measurement of the coronal and sagittal angulation of the radius and ulna conducted by 1 of the authors (PE). Analyses were done using locally available analysis programs such as PACS and JiveX.To rule out potential effects of attrition we undertook a representability analysis. The included population was compared with the loss to follow-up population. This analysis showed no significant difference in age, sex, forearm rotation, or secondary outcomes such as ABILHAND-Kids questionnaire, VAS, and complication rate, concluding that the follow-up group was representative of the whole original study group (Representability of the lost to follow-up and included population. Values are mean (SD) unless otherwise statedCI = 95% confidence interval.ABILHAND-Kids questionnaire score 0–42. 42 is optimal score.VAS score 0–10. 10 is optimal score. | PMC10561100 |
Statistics | pronation, supination | SECONDARY | We compared the baseline characteristics and functional outcome at 7 months and at 7.5 years between the included patients and those lost to follow-up to rule out potential effects of attrition. Long-term results of primary and secondary outcomes were compared between the 2 groups (AEC vs. BEC) using independent T-testing and crosstabs. Results are presented as mean with 95% confidence interval (CI) and standard deviation (SD) because of normal distribution. Furthermore, to address the issue of missing data, linear mixed-model analyses were done for the primary outcome to compare the between-group differences over the different time points where means with 95% CI are presented.We performed a subgroups analysis for both groups on loss of forearm rotation. The pronation and supination were scaled using the grading system as described by Daruwalla with excellent, good, fair, and poor results for, respectively, 0–10°, 11–20°, 21–30°, and ≥ 31° of limitation [ | PMC10561100 |
Ethics, registration, consent, funding, and disclosures | For this post-trial follow-up study ethics approval was again obtained from the regional medical ethical committee (Dnr NL41839.098.12). The original RCT was registered in | PMC10561100 | ||
Results | angulation, loss of flexion–extension of the elbow, fractures, coronal radial angulation, mean loss of forearm rotation, ’ loss of rotation to 7 months (P = 0.02) | SECONDARY | 38 of 47 patients (17 of 23 with AEC and 21 of 24 with BEC) responded to the follow-up invitation and were clinically and radiologically assessed for long-term follow-up. The mean length of follow-up was 7.5 (4.4–9.6) years. Baseline characteristics were similar between the 2 groups (Baseline characteristics of the population with non-reduced diaphyseal forearm fractures. Values are number of patients unless otherwise specifiedAEC = above-elbow cast.BEC = AEC with early conversion to below-elbow cast.Values in years are mean and (range)Consort patient flow diagram. Intervention was above-elbow cast (AEC) or AEC with early conversion to below-elbow cast (BEC)Our primary outcome, loss of forearm rotation, showed improvement in both groups over time, with no significant difference between the 2 groups. The AEC group went from a mean loss of rotation of 30° (SD 29) at 2 months of follow-up, to 23° (SD 22) at 7.2 months to 8° (SD 22) at 7.5 years of follow-up. The BEC group went from 31° (SD 24) loss of forearm rotation at 2 months, to 18° (SD 17) at 7.2 months, to 8° (SD 15) at 7.5 years of follow-up. There was no significant difference between the two groups at 7.5 years of follow-up. Subgroup analyses based on the amount of loss of rotation showed no significant difference in mean limitation of prosupination (Loss of forearm rotation of the fractured arm with subgroup analysis. Values are number of patients unless otherwise statedAEC = above-elbow cast. BEC = AEC with early conversion to below-elbow cast. CI = 95% confidence interval.Values are mean (SD)Significant change over time compared with the previous value in time (linear mixed-models analysis).To address missing data, linear mixed-model analyses were performed, which showed a decrease in loss of forearm rotation over time in both groups. In the BEC group, a significant decrease was found when comparing 2 months’ loss of rotation to 7 months (P = 0.02), and 7 months to 7 years (P < 0.001).Secondary outcomes (loss of flexion–extension of the elbow and wrist, PROMs, and grip strength) revealed no statistically significant differences between the 2 treatment groups (Data on primary and secondary outcomes at 7.5-year long-term follow-up of non-reduced diaphyseal forearm fractures. Values are mean (SD)AEC = above-elbow cast. BEC = AEC with early conversion to below-elbow cast. CI = 95% confidence interval.ABILHAND-Kids questionnaire score 0–42. 42 is optimal score.DASH score 0–100, 100 being the worst score.JAMAR ratio = grip strength affected wrist/collateral side.At long-term follow-up we found no statistically significant differences in radiographic outcomes between the 2 groups. There was an increase in coronal radial angulation observed in both groups over time and an increase in maximal bowing (Data on radiographic outcomes, angulation at final follow-up. Values are mean (SD) unless otherwise statedAEC = above-elbow cast. BEC = AEC with early conversion to below-elbow cast. AP = anteroposterior. CI = 95% confidence interval.Significant change over time compared with the previous value in time (linear mixed-models’ analysis).We also performed a subgroup analysis comparing 2 subgroups: age < 9 years and ≥ 9 years, combining the AEC and BEC groups. Our study showed a remaining mean loss of forearm rotation of 29° in patients ≥ 9 years of age after 7.5 years, compared with 5° in children < 9 years of age (mean difference –24°, CI –38 to –9.0). There was significantly more improvement of function in patients < 9 years of age to almost no impairment. All patients, regardless of age, improved their function over time. | PMC10561100 |
Discussion | diaphyseal both-bone forearm fractures, fracture, angulation, coronal radial angulation, loss of flexion and extension of the elbow | SECONDARY | Our primary aim was to investigate whether forearm rotation improves or worsens over time. We showed that rotation improved in both groups over time, with no significant difference in the final forearm rotation: 8° (SD 22) for the AEC group and 8° (SD 15) for the BEC group with a mean difference of 0 (CI –13 to 12). Early conversion to BEC in children with a non-reduced forearm fracture of both bones is also safe at long-term follow-up of 7.5 years with both groups showing equal improvement in function with minimal persisting loss of rotation at long-term follow-up.Our secondary aim was to analyze loss of flexion and extension of the elbow and wrist, patient-reported outcomes measures, grip strength ratio, and radiographic assessment. In accordance with the short-term follow-up, long-term follow-up of 7.5 years again showed no clinically relevant differences in either clinical or radiographic aspect between treating non-reduced minimally displaced diaphyseal both-bone forearm fractures in children with conventional AEC or with an early conversion to BEC. There were no statistically significant differences between the 2 groups for PROMs or radiographic angulations. However, it did show a significant decrease over time for radial angulation in sagittal views for both groups, which is probably due to the excellent capacity to remodel over time.The remarkable increase in coronal radial angulation and maximal bowing after 7.5 years in both groups can be explained by the natural increase in angulation of the radius in children [ | PMC10561100 |
Previous research | fractures, both-bone forearm fractures, re-fracture, malunion | A systematic search of the literature on previous long-term follow-up studies for pediatric forearm fractures resulted in less than 15 studies in total, and none of them was an RCT. Most studies were published more than 5 years ago, and half of the studies were case reports. Therefore, our prospective randomized multi-center study presenting long-term results on diaphyseal pediatric forearm fractures is unique and an important base for substantiated statements on how to treat these fractures.To understand why early conversion to BEC is just as good as AEC in treating pediatric both-bone forearm fractures when looking at loss of forearm rotation, it is important to understand which factors could possibly influence forearm rotation. Previous studies addressed several factors involved: malunion of the radius and/or the ulna [Colaris et al. showed that association of a re-fracture and a diaphyseal location are both risk factors for loss of forearm rotation with, respectively, an odds ratio of 14.1 and 2.7. Furthermore, intervention of a physiotherapist resulted in a decrease in loss of forearm rotation, which suggests that reversible soft tissue contractures might be of influence in the loss of rotation [A malunion that is located near the more active distal physis has the highest capacity to remodel. The diaphyseal part has the lowest capacity for correction [ | PMC10561100 | |
Study limitations | intramedullary nailing [The primary limitation, malunion | Overall, this is a relatively favorable group, as they have little displacement with no need for reduction and, with that, they have little risk of malunion and rotation limitation. This contrasts with dislocated cases that often re-dislocate causing malunion or rotation problems, resulting in more intramedullary nailing [The primary limitation was the inevitable non-blinding of the clinical assessment. Again, at long-term follow-up the initial cast morphology revealed which group the patients were in. However, radiological assessments again were blinded.Second, this study was conducted with a small sample size of only 38 patients of the 47 primarily included patients. But with a follow-up percentage of 70%, in this young population with a relatively long follow-up period, this is a good response. Furthermore, a previous study showed that when cases are missing at random or completely at random, up to 60% loss to follow-up is acceptable, without influencing the outcome [ | PMC10561100 | |
1. Introduction | ± | Aspartate supplementation has been reported to improve endurance performance by facilitating the tricarboxylic acid cycle flux. The present study was performed to investigate the effects of aspartate supplementation on repeated-sprint performance and blood pH. Following an overnight fast, fourteen healthy males completed three sets of 10 × 6 s maximal sprints after consuming sodium L-aspartate (ASP) or placebo (PLA), in a double-blind manner. Both supplements were taken twice on each test day (2 × 4.5 g). Exercise performance (e.g., cadence and power output) and blood variables (e.g., pH and plasma amino acid levels) were measured. The ASP trial evidenced significantly higher plasma aspartate concentration during the first (ASP, 45.3 ± 9.2 μM; PLA, 6.1 ± 0.8 μM) and the second exercise sets (ASP, 24.2 ± 4.5 μM; PLA, 6.6 ± 0.9 μM) and peak cadence during the second set (ASP, 153 ± 3 rpm; PLA, 152 ± 3 rpm) compared with the PLA trial (all Aspartate is a non-essential amino acid that is a precursor of oxaloacetate, an intermediate of the tricarboxylic acid (TCA) cycle [In various team and racket sports, athletes are required to repeatedly engage in brief (≤10-s) bouts of maximal sprints with incomplete (≤60-s) recovery throughout the game. The ability to recover and reproduce power output during successive sprints is termed RSA (repeated-sprint ability) [In the present study, we determined the effects of aspartate supplementation on performance and blood pH during a repeated-sprint exercise. We hypothesized that aspartate would mitigate blood pH reduction and improve the RSA relative to the placebo (PLA). | PMC10746991 | |
2. Materials and Methods | PMC10746991 | |||
2.1. Study Design and Participants | RECRUITMENT, DISEASES | The present study was a randomized, double-blind, placebo-controlled, crossover study. A total of 15 healthy active males were recruited in the present study. Fourteen participants completed all experiments (means ± standard errors; age 22.3 ± 0.3 years, height 172.1 ± 1.7 cm, weight 65.4 ± 2.2 kg), while a participant was excluded because he could not complete all the experiments. We recruited individuals who (1) were aged between 20 and 29 years, (2) were healthy Japanese males, (3) had exercise habits, and (4) were capable of independent decision-making and the provision of written informed consent. The exclusion criteria were as follows: the participants who (1) were considered to have difficulty in exercising due to medical or surgical diseases, (2) were participating in other research or participated in a study requiring the chronic intake of medicines or specific foods within the preceding month, and (3) had undergone blood tests or given blood, with a collection of more than 200 mL of blood, within 12 weeks of this study. All participants were non-smokers and were asked to avoid intense exercise, caffeine, alcohol, and supplements for 24 h before each experiment. They were not accustomed to the repeated-sprint exercise with maximal effort on the cycle ergometer, so the familiarization was conducted before the main experimental trials. The diet pattern on the day before experimental trials was not controlled, but the participants were asked to maintain regular dietary habits throughout the study. Participant recruitment into the study began in June 2019 and ended in July 2019. The last participants completed a cross-over trial in August 2019. This study was conducted in accordance with the Declaration of Helsinki. All participants were given an overview, potential risks, and benefits of the present study and provided written informed consent. The study was approved by the Research Ethics Committees of Ritsumeikan University, Japan (BKC-IRB-2018-071), and Ajinomoto Co. Inc. (2018-013). It was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN000037306). | PMC10746991 | |
2.3. Measurements | PMC10746991 | |||
2.3.1. Repeated-Sprint Performance | MP, fatigue | During each sprint, the peak cadence and peak and mean power outputs were recorded. The fatigue index (FI) was calculated as a percentage reduction of mean power output from the best (MP | PMC10746991 | |
2.3.2. Perceptual Response | The perceived exertions for breathing (RPE | PMC10746991 | ||
2.3.3. Cardiorespiratory Data | The HR was measured and recorded every second during the repeated-sprint exercise using a HR sensor (H2; Polar Electro Oy., Kempele, Finland) and a wireless monitor (RCX5; Polar Electro Oy., Kempele, Finland), and the mean HR was calculated for each exercise set. The HR sensor was attached with an elastic band on the chest, and the wireless monitor was used to observe the HR continuously throughout the exercise. Oxygen consumption ( | PMC10746991 | ||
2.3.4. Blood Sampling and Analysis | BLOOD, SEPARATION | Blood samples were collected eight times through each trial (at baseline, immediately before and after each exercise set, and 60 min after exercise completion) into 15 mL and 2.5 mL syringes via a cannula inserted into the antecubital vein. Immediately after blood sampling, 12 mL of blood was transferred into an ethylenediaminetetraacetic acid-containing tube (6 mL) or a serum separation tube (6 mL). Glucose and lactate concentrations in the remaining blood samples were measured immediately using a glucose analyzer (FreeStyle Freedom Lite; Nipro Corp., Osaka, Japan) and a lactate analyzer (Lactate Pro 2; Arklay Inc., Kyoto, Japan), respectively. Plasma and serum samples were obtained by centrifugation for 10 min at 4 °C (3000 rpm, 1712× The blood pH, bicarbonate (HCO | PMC10746991 | |
2.3.5. Statistical Analysis | ± | All data are presented as means ± standard errors, and 95% confidence intervals (CI) are also presented. The test–retest reliability for peak cadence and peak and mean power outputs during the first 6 s maximal sprint was calculated. The intraclass correlation coefficients were 0.96, 0.99, and 0.93, respectively; the coefficients of variation were 0.66%, 0.66%, and 2.68%, respectively. The principal analysis was conducted with the generalized linear mixed model (GLMM) using the “lme4” package (ver. 1.1.27.1) of R (ver. 4.1.2). The linear predictors, as fixed effects, were the trial type (ASP or PLA), timepoint, interaction (trial × timepoint), sequence (trial order), and period (first or second trial). The participants were the random effects. The following model was used:When evaluating the blood pH, HCO | PMC10746991 | |
3. Results | PMC10746991 | |||
3.1. Cadence, Peak, and Mean Power Outputs during Repeated-Sprint Exercise | The changes in peak cadence during each exercise set of the repeated-sprint exercise are shown in | PMC10746991 | ||
3.2. FI, RPE, and Cardiorespiratory Data during Repeated-Sprint Exercise | There were no significant differences between trials in FI (ASP, 22.1% ± 1.7% [CI: 18.8–25.5%]; PLA, 20.9% ± 1.9% [CI: 17.1–24.7%]), RPE | PMC10746991 | ||
3.3. Blood pH, Bicarbonate, and Base Excess Levels during Exercise | The pH at baseline was 7.407 ± 0.006 [CI: 7.395–7.419] in the ASP and 7.406 ± 0.004 [CI: 7.398–7.414] in the PLA trials, and these levels were immediately reduced after repeated-sprint exercise. The ASP trial showed a trend for higher blood pH than the PLA trial ( | PMC10746991 | ||
3.4. Blood Parameters and Plasma Amino Acid Profile | No significant difference between the ASP and PLA trials was observed in the mean blood lactate (13.1 ± 1.1 and 13.3 ± 1.0 mmol/L) or glucose concentrations (103 ± 5 and 104 ± 5 mg/dL). Also, no significant difference was observed in the plasma adrenaline, noradrenaline, dopamine, and serum albumin concentrations (Compared with the PLA trial, the ASP trial evidenced significantly higher plasma aspartate concentrations before the first ( | PMC10746991 | ||
4. Discussion | reduction in blood pH | In the present study, we examined the effects of aspartate supplementation on performance and blood pH during repeated-sprint exercise in healthy males. Consequently, the ASP trial evidenced a smaller reduction in blood pH and increased peak cadence during the second exercise set than the PLA trial. To the best of our knowledge, this is the first study to indicate the effect of aspartate supplementation on RSA with concomitant data on acid-base balance.Aspartate supplementation has previously been shown to decrease blood lactate concentration and increase fat oxidation during submaximal cycling at 100 and 150 W [The high-intensity repeated exercise induces a fall in both blood and skeletal muscle pH [Another potential factor of the positive impact is the involvement of metabolites of aspartate. Our results showed that the plasma levels of aspartate metabolic pathway members (glutamate, alanine, citrulline, and phenylalanine) [Given that evidence, the improved peak cadence during the second exercise set in the ASP trial may be associated, at least in part, with the mitigation of blood pH reduction, probably attributable to the acceleration of the TCA cycle, as supported by a study demonstrating the facilitated TCA cycle flux and fatty acid oxidation by aspartate supplementation [At 60 min after exercise completion, the ASP trial evidenced a significantly higher blood pH, HCOThe present study had some limitations. Firstly, we did not control the physical activity level and diet pattern on the day before experimental trials, although participants were asked to avoid intense exercise, caffeine, alcohol, and supplements. In addition, we evaluated the acute effect of aspartate supplementation (2 × 4 g aspartate) or placebo supplementation on the trial days. Therefore, the impact of daily aspartate supplementation on exercise performance during consecutive days of training (e.g., multiple days of competition, training camp) remains unclear. Also, we utilized a relatively small dose of aspartate. Although there is evidence that smaller doses of aspartate show positive effects [ | PMC10746991 | |
5. Conclusions | Aspartate supplementation improved peak cadence during the second exercise set of repeated-sprint exercise compared with placebo supplementation. However, the performance-enhancing effect was limited, since no significant effect was observed for the mean power output throughout the exercise session. Aspartate is naturally found in general foods and no issues were reported in the other clinical studies. Thus, aspartate would be a safety supplement for sports athletes. | PMC10746991 | ||
Author Contributions | N.H. | K.Y., M.O., T.K., W.S., F.T., Y.A. and K.G. designed research. K.Y., N.H. and D.S. conducted experiments. W.S. and F.T. analyzed data. K.Y. drafted the manuscript. W.S., Y.A. and K.G. revised the manuscript. All authors have read and agreed to the published version of the manuscript. | PMC10746991 | |
Institutional Review Board Statement | This study was conducted in accordance with the Declaration of Helsinki. All participants were given an overview of the study and provided written informed consent. The study was approved by the Research Ethics Committees of Ritsumeikan University, Japan (BKC-IRB-2018-071, approved on 3 June 2019), and Ajinomoto Co. Inc (2018-013, approved on 1 November 2018) and adhered to the tenets of the Declaration of Helsinki. It was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN000037306) on 8 July 2019. | PMC10746991 | ||
Data Availability Statement | Data are contained within the article. | PMC10746991 | ||
Conflicts of Interest | The test foods were provided by Ajinomoto Co., Inc. M.O., T.K., W.S., F.T. and Y.A. are employed by the company Ajinomoto Co., Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | PMC10746991 | ||
References | BLOOD | Experimental overview.Changes in peak cadence during repeated-sprint exercise with aspartate (ASP) and placebo supplementations (PLA). Mean ± SE. * Blood pH (Cardiorespiratory responses during repeated-sprint exercise with aspartate (ASP) and placebo supplementations (PLA).Mean ± SE. HR: heart rate. Blood lactate, glucose, plasma catecholamine, and serum albumin concentrations immediately before and after each set of repeated-sprint exercise with aspartate (ASP) and placebo supplementations (PLA).Mean ± SE.Plasma amino acid concentrations immediately before each set of repeated-sprint exercise with aspartate (ASP) and placebo supplementations (PLA).Mean ± SE. BCAA: branched-chain amino acid. + | PMC10746991 | |
Background | METASTASIS | The detection of peritoneal metastasis (PM) is limited by current imaging tools. In this prospective study, we aimed to evaluate the sensitivity and specificity of peritoneal cell-free DNA (cfDNA) for diagnosis of PM. | PMC10114319 | |
Methods | tumor, Colorectal cancer, CRC | TUMOR, COLORECTAL CANCER | Colorectal cancer (CRC) patients with/without PM were enrolled. The cfDNA experimental personnel and statists were blinded to the diagnosis of PM. Ultradeep sequencing covering large genomic regions (35000X, Next-generation sequencing) of cfDNA in peritoneal lavage fluid (FLD) and matched tumor tissues was performed. | PMC10114319 |
Results | A total of 64 cases were recruited prospectively and 51 were enrolled into final analysis. In training cohort, 100% (17/17) PM patients obtained positive FLD cfDNA, comparing to 5/23 (21.7%) in patients without PM. Peritoneal cfDNA had a high sensitivity of 100% and specificity of 77.3% for diagnosis of PM (AUC: 0.95). In validation group of 11, 5/6 (83%) patients with PM obtained positive FLD cfDNA, comparing to 0/5 in non-PM ( | PMC10114319 | ||
Supplementary Information | The online version contains supplementary material available at 10.1186/s13148-023-01479-9. | PMC10114319 | ||
Keywords | PMC10114319 | |||
Background | peritoneal ascites, cancers, CRC, deaths, Colorectal cancer | CANCERS, COLORECTAL CANCER | Colorectal cancer (CRC) is one of the most common cancers worldwide, ranking the third position of new cases and deaths of all cancers [Preliminary study has suggested the feasibility of cfDNA detection in peritoneal ascites and peritoneal lavage fluid (FLD) [ | PMC10114319 |
Results | PMC10114319 | |||
Clinicopathological characteristics | CRC | A total of 64 consecutive patients with CRC were recruited prospectively. Thirteen cases were excluded for subsequent analysis, due to insufficient tissue or peritoneal lavage for NGS assays (The workflow of patient enrollment in this study. 40 CRC patients were including in the training cohort underwent NGS assays for mutational profiles of peritoneal cfDNA and 11 CRC patients were enrolled in the validation cohortThe clinicopathological characteristics of enrolled patients in the overall and training cohort | PMC10114319 | |
The concordance of gene mutations in peritoneal lavages and tumor tissues | tumor | MUTANT, TUMOR | The sequencing depth and DNA insert sizes fulfilled the quality of control in both FLD and TIS of PM and non-PM (Additional file The mutational profiling of cfDNA in matched FLD and TIS of PM and non-PM patients were compared by cluster plot. More mutations were shown in both FLD and TIS of PM patients, while no mutant in FLD in the major of non-PM patients were observed. The top five frequent gene mutations were KRAS (44%), TP53 (62%), APC(50%), PIK3CA(11%) and SMAD4(20%). Alleviations: TNM-TNM staging system, CN- copy number, TIS- tissue sample, FLD- peritoneal lavage fluidThe high concordance of gene mutations between peritoneal lavages and tumor tissues in PM, especially driver genes. | PMC10114319 |
The mutational profiling of cfDNA of driver gene in peritoneal and tumor tissues | tumor | TUMOR | We investigated the mutational frequencies of driver genes in both TIS and FLD of PM and non-PM. The top driver mutations in FLD-PM were suppressor gene The mutational frequencies and sites of driver genes in peritoneal cfDNA and tumor tissues of both PM and non-PM. In scatter plots of driver gene concordance in tissues, Furthermore, we investigated whether the Maximum allele frequency (MaxAF) values could be affected by single driver mutation in FLD of PM and non-PM. No significant differences of average MaxAF were shown ( | PMC10114319 |
Altered signaling pathways and key genes in PM | tumor | MUTANT, TUMOR | To clarify the underlying mechanism of PM, we conducted KEGG enrichment analysis to identify genetic mutational variations in the tumor tissues of PM and non-PM. The top signaling of mutant genes were comparable in PM and non-PM, including | PMC10114319 |
The cutoff value and diagnostic power of positive FLD cfDNA | tumor, CRC | TUMOR, RECURRENCE | To assess the clinical utility of peritoneal cfDNA in diagnosis of PM, we identified the cutoff value of FLD MaxAF at best positivity threshold of 6.29% performed by ROC analyses with area under curve (AUC) of 0.951 in the training cohort (Fig. The accuracy of peritoneal cfDNA for detection of PM and prediction of recurrence. To verify the diagnostic power and accuracy of peritoneal cfDNA in detection of PM, we conducted NGS assays in the FLD of randomized CRC patients in a validation cohort of 11 patients. In the validation cohort, 5/6 (83%) patients with PM obtained positive FLD cfDNA, comparing to 0/5 in patients without PM (We investigated the associations between peritoneal cfDNA and clinicopathological features in PM. No significant associations were observed between FLD MaxAF and these clinicopathological parameters including age, gender, alcohol & tobacco, tumor location, CEA, CA199, CA125, PCI scores, T stage, N stage, and histological type. However, CA153 levels was positively correlated with FLD MaxAF ( | PMC10114319 |
Peritoneal cfDNA and RFS | tumor, CRC | TUMOR, TUMOR RECURRENCE, POSITIVE | To clarify the prognostic value of peritoneal cfDNA in CRC patients without PM, 23 CRC patients in the non-PM were followed-up from the date of surgery until tumor recurrence or until Aug 31, 2022 for patients without tumor relapse. The FLD cfDNA was sampled at the aspiration of peritoneal fluid during the surgery. The median follow-up time was 13.8 months (range: 0.8–18.6 months). Positive peritoneal cfDNA was associated with tumor recurrence ( | PMC10114319 |
Discussion | tumor, non-tumor, cancers [Primary tumor, CRC | TUMOR, TUMOR RECURRENCE, MUTANT, PERITONEAL DISSEMINATION, SOMATIC MUTATIONS | In this prospective study, we reported that peritoneal cfDNA in lavage fluid by ultradeep NGS was a reliable tool for detection of PM in CRC with high sensitivity and specificity. High concordance of genetic mutational profiling and driver genes were observed between peritoneal cfDNA and tumor tissue of CRC patients. The clinical utility of peritoneal cfDNA was proven in the validation cohort. Therefore, the importance of peritoneal cfDNA as a liquid biopsy proves to be a surrogate or adjuvant biomarker instead of laparoscopy for earlier diagnosis of PM.Early detection of PM in CRC is a difficult clinical issue. CEA is current tumor biomarker in detection of tumor recurrence, but is limited by low sensitivity and specificity [Currently, interest has developed in the usage of liquid biopsy for cfDNA in early detection of cancers [Primary tumor shed cfDNA into the circulation, and the concentration of plasma cfDNA is relatively high for metastatic or recurrent CRC [To our knowledge, this is the first study to report peritoneal cfDNA for detection of CRC with PM. Peritoneal fluids are not common in CRC with early PM. Thus, we have conducted this cfDNA assays by peritoneal lavages. High similarity of gene mutations between peritoneal lavage and tumor tissue were shown in PM. Thus, mini-invasive peritoneal cfDNA could reflect tumor mutant burden and molecular features of PM instead of invasive test by tumor tissue. Ultradeep NGS can overcome the limitations of shallow sequencing depth of NGS profiling to achieve high sensitivity similar to ddPCR that is restricted to specific hotpot mutations [Peritoneal FLD and matched TIS are set to decrease false-positive germline variants and identified tumor-derived mutations of cfDNA to filter out background signal that comes from non-tumor-derived mutations. Matched cfDNA-WBC sequencing is usually applied in plasma cfDNA studies to exclude non-tumor derived cfDNA from clonal hematopoiesis in the blood [High concordance of driver genes in peritoneal lavage and tumor tissues are identified in this study. Despite the clinical values of our results, several limitations are still existing in this study. Firstly, this is a prospective diagnostic study with small sample size from one single institution. Our promising findings require to be verified in large cohorts of PM from multi centers. Secondly, although cfDNA experimental personals are blinded while clinicians know the diagnosis, selection of patients can still introduce bias. It is perfect if clinicians are blinded and they found peritoneal cfDNA without knowing intraoperative findings. In addition, patient population is heterogeneous as some of them received preoperative treatment which may impact some results. Thirdly, matched plasma cfDNA from WBC was used to match tumor tissue DNA to filter out somatic mutations.In conclusion, our data show that peritoneal cfDNA detected using ultradeep NGS is a potential sensitive and reliable biomarker for detection of PM in CRC patients. It offers a minimally invasive tool for assessment of driver gene mutations to guide selection of candidate patients for targeted therapies. Peritoneal cfDNA can predict the prognosis of PM and serve as a potential surrogate or adjuvant biomarker in active postoperative surveillance of peritoneal dissemination in high-risk CRC patients instead of laparoscopic explore. | PMC10114319 |
Methods | PMC10114319 | |||
Patients and designs | tumor, CRS, CRC | PRIMARY TUMOR, TUMOR, PATHOLOGY, BLIND | This was a prospectively, open-label, blind, and diagnostic study. Patients with CRC were recruited from the Sixth Affiliated Hospital of Sun Yat-sen University, China. All enrolled patients underwent surgical resection of primary tumor or conduct CRS procedure. CRC and PM were diagnosed by pathology. Peritoneal lavage fluids were collected at the start of surgery. After surgery, CRC patients with PM were enrolled in the experimental PM arm, while CRC patients without PM were included in the controlled non-PM arm. cfDNA experimental personnel and statisticians were blinded to the diagnosis of PM, while clinicians know the diagnosis. For stage IV cases, metastatic lesions such as liver or lung sites were preoperatively radically resected, or treated by radiofrequency ablation. Extensive PM with PCI > 20 was defined as late PM, which is not appropriate for CRS plus HIPEC according to previous studies [In the training cohort, the diagnostic power of peritoneal cfDNA by ultradeep NGS was investigated. A validation cohort was enrolled to verify the results. All enrolled patients were consented for the collection of peritoneal lavages, tumor tissue, blood and medical records under the protocols approved by local ethical institutional review board (IRB), which was in accordance with the Declaration of Helsinki and later amendments. Written informed consents were obtained from all enrolled patients. All authors reviewed and approved the final manuscript. The follow-up was conducted until at least one year after surgery by professional personnel. | PMC10114319 |
Inclusion and exclusion criteria | tumor, CRC | TUMOR, METASTASIS, SECONDARY, ONCOLOGY, PATHOLOGY | Key inclusion criteria were listed as follows: consecutive patients with diagnosis of CRC and peritoneal metastasis by pathology, preoperative imaging for tumor staging, and Eastern Cooperative Oncology Group (ECOG) performance status of 0–2. Exclusion criteria included insufficient tumor tissue or peritoneal lavages, failure of NGS assays, missing data or follow-up and ineligible for cfDNA analysis.The primary objective was to evaluate the sensitivity and specificity of peritoneal cfDNA in diagnosis of PM. The secondary objectives included positive/negative predictive values, accuracy, genomic mutation profiles in peritoneal cfDNA and matched tumor tissues, the underlying pathways and prognostic roles. The sample size was set as 1:1 ratio with each 20 cases in both arms. | PMC10114319 |
Sample collection, DNA isolation, and library preparation | MSI, tumor, Tumor, cancer, primary cancer | TUMOR, TUMOR, CANCER, MICROSATELLITE INSTABILITY, CAVITY, PRIMARY TUMOR, PRIMARY CANCER | Peritoneal lavage was collected after entering abdominal cavity during primary cancer surgery by laparotomy or laparoscopy in all enrolled patients. 300 ml of normal saline was infused into abdominal cavity through a small opening in the abdominal wall or laparoscopic trocar. About 100 ml of peritoneal lavage was aspirated for cfDNA sequencing. Then, resection of primary tumor was performed by surgeons. Peripheral blood was collected and saved in anticoagulant blood collection tube (EDTA tubes) in routine temperature. Tumor tissue specimens were obtained from resected tumor tissues. The percent of cancer cells by HE staining was ≥ 20%. If not, tumor mutational burden (TMB), CNV and microsatellite instability (MSI) tests may be affected.DNA isolation and targeted sequencing were performed in Burning Rock Biotech (Shanghai, China), according to optimized protocols described previously [ | PMC10114319 |
Ultradeep NGS sequencing | tumor, UMI | TUMOR | The DNA samples from tumor tissue were ligated with regular adapters. Target capture of tissue DNA was performed using a commercial panel which consists of 520 genes (OncoScreen Plus), spanning 1.64 Mb of the human genome. The detailed genes were listed in Additional file The cfDNA from peritoneal lavage were ligated with UMI-containing adapters. Oligonucleotides corresponding to the UMI adapters were designed and patented by Burning Rock Biotech (China). The oligonucleotides were commercially synthesized, annealed and purified following standard protocols in previous study [ | PMC10114319 |
Analysis of NGS sequencing data | Sequencing data were mapped to the reference of human genome (hg19) using Burrows-Wheeler Aligner version 0.7.10 [ | PMC10114319 | ||
Gene enrichment analysis | Gene enrichment analysis was performed using the GSEA software program [ | PMC10114319 | ||
Definitions | tumor | TUMOR | The cutoff value of positivity threshold in peritoneal cfDNA was calculated according to MaxAF by ROC analyses, which allowed the best discrimination between PM and non-PM [CNVs were analyzed based on the depth of coverage data of capture intervals. Coverage data were corrected against sequencing bias from GC content and probe design. The average coverage of all captured regions was used to normalize the coverage of different samples to comparable scales. Copy number was calculated based on the ratio between the depth of coverage in tumor samples and average coverage of an adequate number ( | PMC10114319 |
Statistical analysis | All analysis was conducted using the SPSS software (v.15.0; SPSS Inc. Chicago, IL) in this study. Student's | PMC10114319 | ||
Author contributions | ZXY, WLC, TH, HW, and JC concepted and designed the study.ZXY, DL, WLC, QYQ and JC analyzed and interpreted the data. WLC, DL, TH and JC prepared cfDNA and tissue samples. ZXY, WLC, TH, XZL, CHL, XXL, HW and JC conducted ultradeep NGS assays and bioinformatic analysis. ZXY, WG, AF, TH, HW, XZL, CHL and JC drafted and revised the manuscript. ZXY, CHL, XZL, and DL conducted statistical analysis. JC, ZXY and HW provided administrative, technical or material support. All authors read and approved the final manuscript. | PMC10114319 | ||
Funding | DIGESTIVE DISEASE | This study was supported by the National Natural Science Foundation of China grant (No. 82103038, 82103084) and Guangzhou Municipal Science and Technology Program (202201011360), and National Key Research and Development Program of China (2021YFC2101700) and National Key Clinical Discipline, The Sixth Affiliated Hospital of Sun Yat-sen University Clinical Research 5010 Program (grant numbers 2017008 and 2019021) and 1010 Program (1010CG 2022–08) and Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004). | PMC10114319 | |
Availability of data and materials | The data can be available if requested according to the journal instructions. | PMC10114319 | ||
Declarations | PMC10114319 | |||
Ethics approval and consent to participate | This study was approved by local ethical IRB of the Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. | PMC10114319 | ||
Competing interests | The authors declare no competing interest. | PMC10114319 | ||
References | PMC10114319 |
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