Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma

Yuta Ibuki; Yukie Nishiyama; Yasuhiro Tsutani; Manabu Emi; Yoichi Hamai; Morihito Okada; Hidetoshi Tahara

doi:10.1371/journal.pone.0231116

Peer Review History

Original SubmissionNovember 11, 2019
30 Dec 2019 Decision Letter - Hyun-Sung Lee, Editor PONE-D-19-31398 Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma. PLOS ONE Dear Prof Tahara, 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. This is a retrospective, single-institute study that examines microRNAs (miRNAs) and isomicroRNAs (isomiRs) using the next generation sequencer (NGS) in serum samples from 66 patients with esophageal squamous cell carcinoma (ESCC) designed to identify circulating miR/isomiRs that could be used as novel biomarker for ESCC. You have demonstrated that your diagnostic panel index derived from the regression analysis with miR-30a-5p and isoforms of miR-574-3p and miR-205-5p had high accuracy in the diagnosis of ESCC. You have suggested that MiR/isomiRs detected by NGS can be novel biomarkers of ESCC. You have demonstrated that twenty-four miRNA/isomiR candidates are entered into a multiple regression model with stepwise selection to generate diagnostic panel for ESCC. Despite your appreciable efforts with NGS, major limitation of your study is the very small number of discovery cohort to perform regression analysis. In regression analysis, 10 observations per variable are recommended. So, generation of panel index with coefficients from stepwise regression analysis is not statistically appropriate. You would be better to combine your cohorts to maximize the number of samples, which approach would be more statistically reasonable. As the independent validation of your findings, you can use publicly available ESCC cohorts from GEO and TCGA database. Normally distributed continuous variables are reported as mean ± standard deviation; non–normally distributed continuous variables, as median and range (or IQR). Many grammar and typo errors are found. Thorough editing is required. We would appreciate receiving your revised manuscript by Feb 10 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Hyun-Sung Lee, M.D., Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Competing Interests section: "Prof. Hidetoshi Tahara is representative director of a university-originated venture, MiRTeL Co. Ltd., which provides commercial microRNA panel services. The remaining authors declare no potential conflict of interest." We note that one or more of the authors are employed by a commercial company: MiRTeL Co. Ltd.. 1.) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions 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: Yes 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: No 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: Some comments: 1. There are some english errors. 2. Spell out some words 3. Background in the abstract should be write in better way 4. Add the below references for the first sentence of "Introduction section" a. Int J Cancer. 2019 Mar 15;144(6):1215-1226. b. J Cell Physiol. 2018 Nov;233(11):8538-8550 5. Add the below references for this sentence: "MicroRNA(miR)s are classified as small noncoding RNAs (19 - 25 nucleotides) which regulate the expression of plural numbers of messenger RNAs" a. J Cell Biochem. 2017 Dec;118(12):4121-4128 b. Adv Clin Chem. 2017;82:47-70. c. J Cell Physiol. 2018 Feb;233(2):866-879. Reviewer #2: The study tried to explore the biomarkers of ESCC from the microRNA/isomiRs in circulating blood based on the technique of NGS. It is a very interesting and important study; the research was well conducted and the manuscript is well written and easy to follow. There are some specific suggestions for the revision of the paper according bellow that should help to improve the readability of the paper for the PLOS ONE audience. 1. In this nested case-control study, the authors select almost equal number (18 vs 12, 30 vs 30, 18 vs 18) of HC individuals as the patients after the age, sex matching. As I see, the result of the first group was the most important, why the number of HC was less than the case group? How did the authors select the HC individuals? If possible, a larger sample size may been more convinced. 2. In the 3rd group, the authors tried to test the “biomarkers” found from the two groups. Actually, to test the value of your discoveries, It seemly better to conduct a pure prospective case-control study instead of the ESCC patients only. How did the authors think? 3. The authors should check the spellings and grammar. Such as the “sere” in line 81. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231116.r001
Revision 1
12 Feb 2020 Author Response Responses to the comments of Editor We gratefully appreciate your review our manuscript and the helpful suggestions. Our point-by-point responses to your comments and suggestions are listed below. Comment 1 Despite your appreciable efforts with NGS, major limitation of your study is the very small number of discovery cohort to perform regression analysis. In regression analysis, 10 observations per variable are recommended. So, generation of panel index with coefficients from stepwise regression analysis is not statistically appropriate. You would be better to combine your cohorts to maximize the number of samples, which approach would be more statistically reasonable. As the independent validation of your findings, you can use publicly available ESCC cohorts from GEO and TCGA database. Response As suggested by the editor, limited number of samples was limitation of this study. NGS analysis is difficult to analyze many samples due to cost for now. We analyzed more than 100 samples of patients and control to discover biomarker of ESCC. This number is the largest effort ever reported included other cancer. It is well known that different RNA extraction methods strongly affected the NGS results, and there is no public database containing information on circulating isomiR in ESCC patients. Therefore, external validation is difficult, and internal validation was proceeded by dividing samples in this study. Thus, prospective study is needed before clinical application. Therefore, we added this sentence in the documents (‘‘We tested the application for our diagnostic panel using another cohort, but it was using retrospective single institution samples after all. Therefore, prospective confirmation study is needed before clinical application.’’; line279-281). The editor also points out about stepwise procedure. We understand there is a recommendation the number of covariates should be "10-15 observations per variable" as a "guide" for multiple regression. And we also understand there is criticism against stepwise method because there is unreproducible of variable selection and the methods select covariates without considering the clinical evaluation. However, the "guide" is just reference criteria, and the suitable number of covariates will change depend on the variance and covariance of variables. Stepwise procedure is effective method to decide the suitable combination of covariates with considering the possible number of covariates and correlation between covariates, especially multicollinearity. Moreover, stepwise procedure has potential to find undiscovered confounding factors. Therefore, we applied the covariates both of clinical important covariate and variables selected by stepwise procedure We proceeded with this analysis in consultation with a clinical statistician (Prof. Junko Tanaka; Professor of Department of Epidemiology, Infectious Disease Control and Prevention, Hiroshima university). Comment 2 Normally distributed continuous variables are reported as mean ± standard deviation; non–normally distributed continuous variables, as median and range (or IQR). Response As mentioned by the editor, we changed representation of normally distributed continuous variables as mean ± standard deviation. Specifically, we changed the representation of mean read number of patients and healthy control in Table 2. Responses to the comments of Reviewer 1 We gratefully appreciate your review our manuscript and the helpful suggestions. Our point-by-point responses to your comments and suggestions are listed below. Comment 1, 2 1. There are some english errors. 2. Spell out some words Response As suggested by the reviewer, we reviewed the text and corrected some errors (ex. ‘‘sere →were’’ in line 79). Abbreviations of micro RNA were not unified, so they were unified as ‘‘miR’’. We have also unified the description of the supporting information in the text (ex. S1 Fig. and S1 File). Comment 3 Background in the abstract should be write in better way Response As suggested by reviewer, we changed background in the abstract as following “ MicroRNA (miR)s are promising diagnostic biomarkers of cancer. Recent next generation sequencer (NGS) studies have found that isoforms of micro RNA (isomiR) circulate in the bloodstream similarly to mature miRs. We hypothesized that combination of circulating miR and isomiRs detected by NGS are potentially powerful cancer biomarker. The present study aimed to investigate their application in esophageal cancer. ” Comment 4, 5 4. Add the below references for the first sentence of "Introduction section" a. Int J Cancer. 2019 Mar 15;144(6):1215-1226. b. J Cell Physiol. 2018 Nov;233(11):8538-8550 5. Add the below references for this sentence: "MicroRNA(miR)s are classified as small noncoding RNAs (19 - 25 nucleotides) which regulate the expression of plural numbers of messenger RNAs" a. J Cell Biochem. 2017 Dec;118(12):4121-4128 b. Adv Clin Chem. 2017;82:47-70. c. J Cell Physiol. 2018 Feb;233(2):866-879. Response As suggested by the reviewer, we added these literatures in References part. Responses to the comments of Reviewer 2 We gratefully appreciate your review our manuscript and the helpful suggestions. Our point-by-point responses to your comments and suggestions are listed below. Comment 1 In this nested case-control study, the authors select almost equal number (18 vs 12, 30 vs 30, 18 vs 18) of HC individuals as the patients after the age, sex matching. As I see, the result of the first group was the most important, why the number of HC was less than the case group? How did the authors select the HC individuals? If possible, a larger sample size may been more convinced. Response One of the main reasons is that time duration of blood collection was the same between patients and controls. As the reviewers mentioned, the first group result was important. We tried to get many candidates and validate them with many patients and controls to get precise diagnostic biomarkers. In our comparative analysis method, a smaller number of samples would yield more candidates. For this reason, more numbers of samples were allocated to the second group than to the first group. We proceeded with this analysis in consultation with a clinical statistician (Junko Tanaka; Professor of Department of Epidemiology, Infectious Disease Control and Prevention, Hiroshima university). Comment 2 In the 3rd group, the authors tried to test the “biomarkers” found from the two groups. Actually, to test the value of your discoveries, It seemly better to conduct a pure prospective case-control study instead of the ESCC patients only. How did the authors think?. Response We think that prospective case-control testing is necessary for clinical use as pointed out by the reviewers. We did internal validation instead of prospective study. It is the limitations of this study. We will be conducting a prospective study based on results of the current study. We added following the following statements to line 270-272 ‘‘We tested the application for our diagnostic panel using another cohort, but it was using retrospective single institution samples after all. Therefore, prospective confirmation study is needed before clinical application.’’ Comment 3 The authors should check the spellings and grammar. Such as the “sere” in line 81. Response As suggested by the reviewer, we reviewed the text and corrected some errors including in line 79. Abbreviations of micro RNA were not unified, so they were unified as ‘‘miR’’. We have also unified the description of the supporting information in the text (ex. S1 Fig. and S1 File). https://doi.org/10.1371/journal.pone.0231116.r002
3 Mar 2020 Decision Letter - Hyun-Sung Lee, Editor PONE-D-19-31398R1 Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma. PLOS ONE Dear Prof Tahara, 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. You addressed statistical issues well after discussing your biostatistician. However, we are still worrying about overfitting issue from multivariable analysis with the small number of cohort. Our biostatistician recommends to build the model with n=30+30 as a discovery and then to validate with the smaller dataset. As for me, identification itself of circulating microRNA/isomiRs from enough number of samples can be acceptable without validation. Since you have performed pricey experiments with valuable human samples, more reasonable analysis would make your findings more convincing. However, I know it would be very challenging to reanalyze all your data. Therefore, I recommend that the statistical issue should be described as the limitation of your study. Further, you would be better to provide a table to clarify the process of multivariable stepwise selection of your candidates. We would appreciate receiving your revised manuscript by Apr 17 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Hyun-Sung Lee, M.D., Ph.D. Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. 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 #2: Yes ****** 5. 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 #2: Yes ****** 6. 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 #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes: Tao-tao, Liu [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231116.r003
Revision 2
14 Mar 2020 Author Response Responses to the comments of Editor. We gratefully appreciate your review our manuscript and the helpful suggestions. Our point-by-point responses to your comments and suggestions are listed below. Comment 1 I recommend that the statistical issue should be described as the limitation of your study. As suggested by the editor, we also think that the method of statistics needs discussion. We appreciate you for accepting our method. Our laboratory is currently preparing for a prospective validation. We hope to report this result in the future. The following description has been added to the Discussion part. (‘‘Our results were also influenced by the number of samples assigned to each group and the method of statistical analysis.’’; line270-272 and ‘‘External validation is preferred to confirm accuracy of our results, but it is difficult because there is no public database containing information on circulating isomiR in ESCC patients. Therefore we tested the application for our diagnostic panel using another cohort, but it was using retrospective single institution samples after all. Prospective confirmation study is needed before clinical application.’’). Comment 2 Further, you would be better to provide a table to clarify the process of multivariable stepwise selection of your candidates. As suggested by the editor, we described selection method of variables for the Stepwise model in Material and methods part, and shown their results in Results part. These results are also shown in S1 Fig. and S3 File. https://doi.org/10.1371/journal.pone.0231116.r004
17 Mar 2020 Decision Letter - Hyun-Sung Lee, Editor Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma. PONE-D-19-31398R2 Dear Dr. Tahara, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Hyun-Sung Lee, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0231116.r005
Formally Accepted
23 Mar 2020 Acceptance Letter - Hyun-Sung Lee, Editor PONE-D-19-31398R2 Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma. Dear Dr. Tahara: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Hyun-Sung Lee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231116.r006

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