Genomic analysis for the prediction of prognosis in small-bowel cancer

Akiyoshi Tsuboi; Yuji Urabe; Shiro Oka; Akihiko Sumioka; Sumio Iio; Ryo Yuge; Ryohei Hayashi; Toshio Kuwai; Yasuhiko Kitadai; Kazuya Kuraoka; Koji Arihiro; Shinji Tanaka; Kazuaki Chayama

doi:10.1371/journal.pone.0241454

Peer Review History

Original SubmissionAugust 4, 2020
9 Sep 2020 Decision Letter - Anthony F. Shields, Editor PONE-D-20-24291 Genomic analysis for the prediction of prognosis in small-bowel cancer PLOS ONE Dear Dr. Oka, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. In particular, please address the statistical analysis is necessary to elucidate the contributions of TMB and SMAD4 over known prognostic factors such as R0 resection rate, stage, etc. using a Cox proportional hazards model or similar approach. Also, note the other issues rasied by the reviewers, such at the high rate of mismatch repair deficiency. Please submit your revised manuscript by Oct 24 2020 11:59PM. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study. Specifically, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent for the use of medical data and tissue samples. If patients provided informed written consent to have data from their medical records and tissues used in research, please include this information. Reviewers' comments: Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors present a genomic analysis of small bowel cancer in a retrospective study. The findings indicating that TMB-high patients had improved survival is notable; however, many other conclusions are limited by the small sample size of 24 patients, all of whom underwent surgical resection and did not include duodenal cancers. -If data on patient treatment (e.g. adjuvant therapy) were included, it would strengthen the novelty of the manuscript. -The genetics were similar to Western data, why do the offers suggest that small bowel cancer has a different biology in the Japanese population? -A more rigorous statistical analysis is necessary to elucidate the contributions of TMB and SMAD4 over known prognostic factors such as R0 resection rate, stage, etc. using a Cox proportional hazards model. -Breast is misspelled in Table 1. Reviewer #2: This is a well-written manuscript on a very interesting topic in a highly selected population of small-bowel cancer (duodenum not included). The main issue here is the number of patients included in the analysis, which make results quite "weak" and not robust enough to be reliable. Major: -TMB cut off: please clarify why 10 is used as cutoff. -MMR deficiency rates are higher then expected, sure this may have an impact both in TMB and in OS data. Please clarify this in the manuscript. -Au say "Although, we did not observe any correlation between DSS and OS, which may be due to the small number of cases analyzed, we believe that the SMAD4 mutations may be a prognostic factor for small-bowel cancer, similar to that of CRC. " Please provide more data and explanations for this statement. ******** While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. 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Revision 1
6 Oct 2020 Author Response Reviewer #1: The authors present a genomic analysis of small bowel cancer in a retrospective study. The findings indicating that TMB-high patients had improved survival is notable; however, many other conclusions are limited by the small sample size of 24 patients, all of whom underwent surgical resection and did not include duodenal cancers. -If data on patient treatment (e.g. adjuvant therapy) were included, it would strengthen the novelty of the manuscript. Reply: Thank you for pointing out this matter. We have included adjuvant therapy in Table 1a under ‘Diagnostic modality’ as follows: “Table 1 Baseline characteristics of small-bowel cancer 1a. Characteristics patients with small-bowel cancer in our study Variables Small-bowel cancer (n = 24) Age (years), mean ± SD 61.7 ± 11.7 Sex, male/female 16/8 BMI, mean ± SD 22.3 ± 6.3 Observation period (month), mean ± SD 50.0 ± 43.3 Chief complaint, +/− 22/2 Intestinal obstruction 9 (38) Abdominal pain 8 (33) Gastrointestinal bleeding 5 (21) History of other organ cancers 7 (29) Colorectal cancer 3(13) Breast cancer 2(8) Gastric cancer 1(4) Lung cancer 1(4) Bladder cancer 1(4) Pancreatic cancer 1(4) Endometrial cancer 1(4) Gallbladder cancer 1(4) Predisposing conditions FAP 0 (0) PJS 0 (0) LS 2 (8) Crohn’s disease 0 (0) Celiac disease 0 (0) Number of lesions, single/ multiple 21/3 CEA, median [IQR] 2.2 ng/mL [1.4–5.9] CA19-9, median [IQR] 8.5 U/mL [2–77.9] Diagnostic modality DBE 20 (83) CT 3 (13) CE 1 (4) Adjuvant therapy TS1 5 (21) FOLFOX 4 (17) CapeOX 3 (13) Capecitabine 2 (8) UFT/LV 2 (8) FOLFOX+Bmab 1 (4) TS1+Doc 1 (4) None 6 (25) Data represented as n (%) and mean ± SD. SD: standard deviation, BMI: body mass index, FAP: familial adenomatous polyposis, PJS: Peutz-Jeghers syndrome, LS: Lynch syndrome, CEA: carcinoembryonic antigen, CA19-19: carbohydrate antigen 19-9, IQR: interquartile range, DBE: double balloon endoscopy, CT: computed tomography, CE: capsule endoscopy” A total of 18 of the 24 patients received postoperative adjuvant chemotherapy and the regimens were as follows: 5 patients received TS-1, 4 patients received FOLFOX, 3 patients received CapeOX, 2 patients received Capecitabine, 2 patients received UFT/LV, 1 patient received FOLFOX+B-mab, and 1 patient received TS-1+ Docetaxel. Postoperative adjuvant chemotherapy was not correlated with OS (HR, 2.47; 95% CI, 0.64–16.16; P = 0.20) (Table 3). All patients (6/6) with TMB <10 mut/Mb received postoperative adjuvant chemotherapy, while 65% of the patients (11/17) with TMB ≥10 mut/Mb received postoperative adjuvant chemotherapy. All patients (6/6) with TMB <10 mut/Mb that received postoperative adjuvant chemotherapy had a worse prognosis than those with TMB ≥10 mut/Mb. Therefore, we believe that cancer genomic analysis of resected specimens may allow for indication of postoperative adjuvant chemotherapy depending on the value of TMB. -The genetics were similar to Western data, why do the offers suggest that small bowel cancer has a different biology in the Japanese population? Reply: We probably misled you when expressing our opinion without giving it much thought. The frequencies of redisposing factors of small-bowel cancer such as FAP, LS, Crohn’s disease, celiac disease, and BMI are different in Japanese and Caucasian populations. Thus, we suggest that these factors may have an effect on the development of small-bowel cancer. The genetics were indeed similar to that of Western data; however, some difference was found in the frequencies of APC mutations between our data and previous reports from Western countries. We have altered some sentences in the revised version of the paper as follows: (p.5 lines 81–88) From “However, significantly different factors are involved the development of small-bowel cancer exist between Caucasian and Japanese populations.” To “However, disease structure is different between Caucasian and Japanese populations. For example, background factors such as the frequency of celiac disease, Crohn's disease, and obesity are different between Japan and Western countries. The frequency of obesity varies across countries. According to World Health Organization (2016), a 20–40% frequency of body mass index (BMI) >30 was reported in Western countries, whereas a 4.4% frequency of BMI >30 was reported in Japan; this scenario is explained by Japanese people tending to consume diets low in fat, sugar and fructose.” -A more rigorous statistical analysis is necessary to elucidate the contributions of TMB and SMAD4 over known prognostic factors such as R0 resection rate, stage, etc. using a Cox proportional hazards model. Reply: Thank you for pointing this out. After re-examining the Cox proportional hazards model, we found that Stage and TMB were correlated with OS, and that Stage and SMAD4 were correlated with RFS. These results suggest that the presence of SMAD4 mutations and TMB status are strongly correlated. These results are presented in Tables 3–6) as follows: (p. 17–19) Table 3 Univariate association with overall survival Variables N OS HR (95%CI) P-value Log-rank P-value Sex Male 16 2.60 (0.68–16.94) 0.17 0.20 Female 8 Ref. Age >65 8 0.86 (0.23–2.73) 0.80 0.80 ≦65 16 Ref. pStage IV 9 12.13 (2.89–83.36) <0.01 <0.01 I–III 15 Ref. R0 resection R1/2 8 7.7 (2.06–37.25) <0.01 <0.01 R0 16 Ref. Adjuvant chemotherapy Done 18 2.47 (0.64–16.16) 0.20 0.23 None 6 Ref. IHC dMMR 11 1.25 (0.38–3.99) 0.71 0.71 pMMR 13 Ref. TMB <10 6 3.29 (0.84–11.24) 0.08 <0.05 ≧10 17 Ref. SMAD4 Mutation 6 0.52 (0.08–2.01) 0.37 0.39 Wild 17 Ref. Data represented as n. IHC: immunohistochemical, TMB: tumor mutational burden, mut/MB: mutations per megabyte of DNA, MMR: mismatch repair, dMMR: deficient mismatch repair, pMMR: proficient mismatch repair, HR: hazard ration, OS: overall survival (p. 18) Table 4 Multivariate association with overall survival Variables N OS HR (95%CI) P-value pStage IV 9 58.68 (7.89–1348.14) <0.01 I–III 15 Ref. TMB <10 6 11.33 (2.08–85.05) <0.01 ≧10 17 Ref. Data represented as n. TMB: tumor mutational burden, mut/MB: mutations per megabyte of DNA, HR: hazard ration, OS: overall survival (p. 20) Table 5 Univariate association with recurrence-free survival Variables N RFS HR (95%CI) P-value Log-rank P-value Sex Male 10 2.98 (0.50–56.65) 0.26 0.29 Female 6 Ref. Age >65 7 1.21 (0.23–5.61) 0.81 0.81 ≦65 9 Ref. pStage IV 3 11.43 (1.09–247.04) 0.04 0.01 I–III 13 Ref. Adjuvant chemotherapy Done 10 1.74 (0.38–12.22) 0.49 0.50 None 6 Ref. IHC dMMR 8 0.69 (0.14–3.13) 0.62 0.62 pMMR 8 Ref. TMB <10 3 2.97 (0.41–15.37) 0.25 0.18 ≧10 13 Ref. SMAD4 Mutation 5 4.15 (0.90–21.41) 0.07 0.04 Wild 11 Ref. Data represented as n. IHC: immunohistochemical, TMB: tumor mutational burden, mut/MB: mutations per megabyte of DNA, MMR: mismatch repair, dMMR: deficient mismatch repair, pMMR: proficient mismatch repair, HR: hazard ration, OS: overall survival (p. 20–21) Table 6 Multivariate association with recurrence-free survival Variables N OS HR (95%CI) P-value pStage IV 3 24.07 (1.84–654.62) 0.02 I–III 13 Ref. TMB <10 5 6.72 (1.28–49.54) 0.03 ≧10 11 Ref. Data represented as n. TMB: tumor mutational burden, mut/MB: mutations per megabyte of DNA, HR: hazard ration, RFS: recurrence-free survival Moreover, we included the following text in the Results section: (p. 17 lines 290–297) “The other covariates from the Cox proportional hazard analysis associated with OS are shown in Tables 3 and 4. Regarding the univariate analysis, pathological Stage IV (HR, 12.13; 95% CI, 2.89–83.36; P < 0.01) and R1/2 resection (HR, 7.7; 95% CI, 2.06–37.25; P < 0.01) were correlated with OS (Table 3), whereas TMB ≥10 mutations/Mb was not significantly correlated with OS (HR, 3.29; 95% CI, 0.84–11.24; P = 0.08). However, in the multivariate analysis, pathological Stage IV (HR, 58.68; 95% CI, 7.89–1348.14; P < 0.01) and TMB ≥10 mutations/Mb (HR, 11.33; 95% CI, 2.08–85.05; P < 0.01) were correlated with OS.” (p. 19 lines 315–321) “The other covariates from the Cox proportional hazard analysis associated with RFS are shown in Tables 5 and 6. Regarding the univariate analysis, pathological Stage IV (HR, 11.43; 95% CI, 1.09–247.04; P = 0.04) was correlated with RFS (Table 5), whereas mutated SMAD4 was not significantly correlated with RFS (HR, 4.15; 95% CI, 0.90–21.41; P = 0.07). However, pathological Stage IV (HR, 24.07; 95% CI, 1.84–654.62; P = 0.02) and mutated SMAD4 (HR, 6.72; 95% CI, 1.28–49.54; P = 0.03) were correlated with RFS in the multivariate analysis.” -Breast is misspelled in Table 1. Reply: Thank you for pointing this out. I corrected the spelling in Table 1. Reviewer #2: This is a well-written manuscript on a very interesting topic in a highly selected population of small-bowel cancer (duodenum not included). The main issue here is the number of patients included in the analysis, which make results quite "weak" and not robust enough to be reliable. Major: -TMB cut off: please clarify why 10 is used as cutoff. Reply: Thank you for pointing that out. The problem here is that the cut-off value of TMB for small-bowel cancer has not yet been determined. However, Shrock et al. (JAMA Oncol. 2017; 3: 1546-1553.) defined the TMB status of <10 as low, 10–20 as intermediate, and 20> as high. Therefore, in the present study, we defined TMB ≥10 mutations/Mb as being a high value. -MMR deficiency rates are higher then expected, sure this may have an impact both in TMB and in OS data. Please clarify this in the manuscript. Reply: Thank you for pointing this out. The Cox proportional hazards model showed that dMMR was not correlated with OS, but the stepwise method showed that Stage and TMB were correlated with OS. We describe this in the Results section: (p. 17 lines 290–297) “The other covariates from the Cox proportional hazard analysis associated with OS are shown in Tables 3 and 4. Regarding the univariate analysis, pathological Stage IV (HR, 12.13; 95% CI, 2.89–83.36; P < 0.01) and R1/2 resection (HR, 7.7; 95% CI, 2.06–37.25; P < 0.01) were correlated with OS (Table 3), whereas TMB ≥10 mutations/Mb was not significantly correlated with OS (HR, 3.29; 95% CI, 0.84–11.24; P = 0.08). However, in the multivariate analysis, pathological Stage IV (HR, 58.68; 95% CI, 7.89–1348.14; P < 0.01) and TMB ≥10 mutations/Mb (HR, 11.33; 95% CI, 2.08–85.05; P < 0.01) were correlated with OS.” (p. 17–19) Table 3 Univariate association with overall survival Variables N OS HR (95%CI) P-value Log-rank P-value Sex Male 16 2.60 (0.68–16.94) 0.17 0.20 Female 8 Ref. Age >65 8 0.86 (0.23–2.73) 0.80 0.80 ≦65 16 Ref. pStage IV 9 12.13 (2.89–83.36) <0.01 <0.01 I–III 15 Ref. R0 resection R1/2 8 7.7 (2.06–37.25) <0.01 <0.01 R0 16 Ref. Adjuvant chemotherapy Done 18 2.47 (0.64–16.16) 0.20 0.23 None 6 Ref. IHC dMMR 11 1.25 (0.38–3.99) 0.71 0.71 pMMR 13 Ref. TMB <10 6 3.29 (0.84–11.24) 0.08 <0.05 ≧10 17 Ref. SMAD4 Mutation 6 0.52 (0.08–2.01) 0.37 0.39 Wild 17 Ref. Data represented as n. IHC: immunohistochemical, TMB: tumor mutational burden, mut/MB: mutations per megabyte of DNA, MMR: mismatch repair, dMMR: deficient mismatch repair, pMMR: proficient mismatch repair, HR: hazard ration, OS: overall survival (p. 18) Table 4 Multivariate association with overall survival Variables N OS HR (95%CI) P-value pStage IV 9 58.68 (7.89–1348.14) <0.01 I–III 15 Ref. TMB <10 6 11.33 (2.08–85.05) <0.01 ≧10 17 Ref. Data represented as n. TMB: tumor mutational burden, mut/MB: mutations per megabyte of DNA, HR: hazard ration, OS: overall survival -Au say "Although, we did not observe any correlation between DSS and OS, which may be due to the small number of cases analyzed, we believe that the SMAD4 mutations may be a prognostic factor for small-bowel cancer, similar to that of CRC. " Please provide more data and explanations for this statement. Reply: Thank you for pointing this out. The case of recurrence with SMAD4 mutation is relatively recent, and the patient is still undergoing multidisciplinary treatment. However, there are no current cases of CR, which may affect OS and DSS in the future as we perform the long-term follow up. More detailed data are provided in the Results section: (p. 19 lines 324–329) “Of the four patients that had recurrence of small-bowel cancer, two patients died from primary cancer, whereas the other two patients are still alive and undergoing treatment with multidisciplinary treatment including additional surgery. Of the two patients that had recurrence at Stage II, one of them received postoperative adjuvant chemotherapy with capecitabine, and the other did not receive adjuvant chemotherapy.” and in the Discussion section: (p. 25 lines 412–419) “Two of the three patients diagnosed at Stage II with SMAD4 mutation had recurrence by peritoneal dissemination. Currently, adjuvant chemotherapy is not recommended for all patients with Stage II CRC in Japan; however, these results suggest that postoperative adjuvant chemotherapy should be considered for Stage II small-bowel cancer with SMAD4 mutations because of the high risk of recurrence. Although no correlation was observed between DSS and OS, possibly because of the small number of cases analyzed, further studies may indicate SMAD4 mutations as a prognostic factor for small-bowel cancer, as in CRC.” Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0241454.r002
15 Oct 2020 Decision Letter - Anthony F. Shields, Editor Genomic analysis for the prediction of prognosis in small-bowel cancer PONE-D-20-24291R1 Dear Dr. Oka, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Anthony F. Shields, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): In Table 1 please define the abbreviations used for the adjuvant treatment regimens. https://doi.org/10.1371/journal.pone.0241454.r003
Formally Accepted
11 May 2021 Acceptance Letter - Anthony F. Shields, Editor PONE-D-20-24291R1 Genomic analysis for the prediction of prognosis in small-bowel cancer Dear Dr. Oka: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Anthony F. Shields Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241454.r004

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