Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis

Hiroki Harada; Yusuke Nie; Ippeita Araki; Takafumi Soeno; Motohiro Chuman; Marie Washio; Mikiko Sakuraya; Hideki Ushiku; Masahiro Niihara; Kei Hosoda; Yusuke Kumamoto; Takeshi Naitoh; Takafumi Sangai; Naoki Hiki; Keishi Yamashita

doi:10.1371/journal.pone.0260303

Peer Review History

Original SubmissionMay 1, 2021
4 Jun 2021 Decision Letter - Hiromu Suzuki, Editor PONE-D-21-14491 Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis PLOS ONE Dear Dr. Yamashita, 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 manuscript was carefully reviewed by 2 experts, and both of them found several concerns which need to be addressed before acceptance. For instance, both reviewers require more careful explanation for the definition of haploinsufficiency of MLH1. Please respond to each of the reviewer comments. Please submit your revised manuscript by Jul 19 2021 11:59PM. 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Email us at plosone@plos.org if you have any questions. 3.Thank you for stating the following in the Acknowledgments Section of your manuscript: "This study was 22 also supported by the Integrative 1 Research Program grant of the Graduate School of 2 Medical Science, Kitasato University and Parents’ Association Grant of Kitasato 3 University School of Medicine." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. 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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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: To authors: In this study, the authors showed that haplo-MSI is asscoated with clinicopathological features of gastric cancer. Although the result of this study is interesting, there are some minor concerns. 1. The authors demonstrated that there is a subgroup that has a very low level of MLH1 methylation (MLH TaqMeth value range 0.23 to 2.23) and haplo-MSI including this subgroup is associated with clinicopathological features of gastric cancer. Are there any characteristics in this subgroup compared with MSS or other MSI groups? 2. The authors shoud showed which threshold of TaqMeth value was used for the assessment of MLH1 methylation in normal gastric mucosa in Fig 2g. 3. The definition of haplo-MSI is slightly difficult to understand, please clarify the range of MLH Taqmeth value in Fig 5f. 4. The labels of methylation level in the lower panel of Fig 1b should be showed. Reviewer #2: In the current study, the authors investigated promoter methylation of MutL homolog 1 (MLH1) in primary gastric cancer. They revealed MLH1 hypermethylation validated in the quantitative methylation specific PCR (qMSP) assessment, was correlated with microsatellite instability (MSI)-High/MSI-Low status in MSI-associated gastric cancer. Gastric cancer patient with MLH1 hypermethylation showed better prognosis. Based on the MLH1 methylation status, MSI could be further divided into MSI-H, MSI-L, and haploinsufficiency of MSI (MSI-haplo). This study offers a molecular classification of MSI-associated tumor may be beneficial for development of therapeutic strategy for gastric cancer. Following are some comments for this study. 1. In the introduction (page 5), the authors stated that “Most of the data on MLH1 hypermethylation have been assessed by old fashioned non-quantitated conventional methylation specific PCR (MSP), and have not been properly validated in the quantitative assessment.” However, several studies demonstrated quantitative hmlh1 methylation using qMSP in gastric cancer (Taqman or methylight, PMID: 22513802, 19957042), qMSP (sybergreen, PMID: 21599969) or bisulphite pyrosequencing (PMID:17408633). Can the authors further discuss the difference between the current studies and those studies in terms of primer location and the association to clinical parameters. And why the current is superior than the others. Otherwise, it has to be rephrased. 2. The current study found that gastric cancer patient with hMLH1 hypermethylation showed better prognosis. However, several studies showed that hMLH1 methylation associated with poor prognosis in gastric cancer, for example ( PMID:29719511) 3. It is difficult for this reviewer to understand how the author define MLH1-Taqmeth value 0.23 as haploinsufficiency of MLH1 expression. 4. Morphological types should be defined as intestinal, diffuse and mixed type. Association between hMLH1 methylation with HP status is missing. 5. Clinicopathologic characteristics of MSI-associated gastric cancer needs to be delineated more in the introduction. 6. Figure 1a, is the bold line in qMSP (original) indicates the location of Taqman probe, it has to be stated in the figure legend. 7. Figure 1b, lower panel, label of Y-axis is missing. ****** 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.] 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. 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Revision 1
9 Jul 2021 Author Response Reviewer #1: To authors. In this study, the authors showed that haplo-MSI is asscoated with clinicopathological features of gastric cancer. Although the result of this study is interesting, there are some minor concerns. 1) The authors demonstrated that there is a subgroup that has a very low level of MLH1 methylation (MLH TaqMeth value range 0.23 to 2.23) and haplo-MSI including this subgroup is associated with clinicopathological features of gastric cancer. Are there any characteristics in this subgroup compared with MSS or other MSI groups? Thank you for your pertinent comments. Following Reviewer's suggestion, we examined the clinicopathological traits of True MSS, Haplo-MSI, Low-MSI, and High-MSI and identified the factors associated with their respective characteristics. As the MLH1 methylation value increased in the order of True MSS, Haplo-MSI, Low-MSI, and High MSI, the gastric cancers with lower tumor site (P = 0.0313), the synchronous gastric cancers (P = 0.0122) and the histologically differentiated types (P = 0.0052) increased (Table S4). The above contents have been added to Table S4 and Results (P17, Line7-10). 2) The authors should show which threshold of TaqMeth value was used for the assessment of MLH1 methylation in normal gastric mucosa in Fig 2g. Thank you for your pertinent comments. From the viewpoint of field cancerization, we initially supposed that MLH1 methylation may also be observed in both the synchronous tumor and corresponding normal mucosa as well as in the main tumor. In fact, however, this was not true, and not all mucosa harbored hypermethylation of the MLH1 and methylation patterns were rather random. Thus, threshold was not set to discriminate the corresponding normal mucosa from tumor tissues. The above contents have been added to Results (P13, Line7-8). 3) The definition of haplo-MSI is slightly difficult to understand, please clarify the range of MLH Taqmeth value in Fig 5f. Thank you for your appropriate suggestions. We are considering that haplo-MSI phenotype could be microsatellite-instable despite MSS defined by the current definition. The definition of MSI-H/L is based on the specific relevant markers (Bethesda panel), and they could not be covered with minor abnormalities of microsatellites caused by haplo-MSI that is corresponding to the lowest hypermethylation of MLH1 (> 0.23) (Fig. 5f). Prognostic stratification defined by the most optimal cut-off value (0.23, but not 2.3) might propose bona-fide entity of MSI phenotypes including haplo-MSI in gastric cancer. The position of the MLH1-Taqmeth value in Figure 5f has been changed to clarify the range of each MSI phenotype. The above contents have been added to Discussion (P19, Line15-21). 4) The labels of methylation level in the lower panel of Fig 1b should be showed. Thank you for your appropriate suggestions. A label for the methylation level is displayed on the Y-axis in the lower panel of Figure 1b. Reviewer #2: In the current study, the authors investigated promoter methylation of MutL homolog 1 (MLH1) in primary gastric cancer. They revealed MLH1 hypermethylation validated in the quantitative methylation specific PCR (qMSP) assessment, was correlated with microsatellite instability (MSI)-High/MSI-Low status in MSI-associated gastric cancer. Gastric cancer patient with MLH1 hypermethylation showed better prognosis. Based on the MLH1 methylation status, MSI could be further divided into MSI-H, MSI-L, and haploinsufficiency of MSI (MSI-haplo). This study offers a molecular classification of MSI-associated tumor may be beneficial for development of therapeutic strategy for gastric cancer. Following are some comments for this study. 1) In the introduction (page 5), the authors stated that “Most of the data on MLH1 hypermethylation have been assessed by old fashioned non-quantitated conventional methylation specific PCR (MSP), and have not been properly validated in the quantitative assessment.” However, several studies demonstrated quantitative hmlh1 methylation using qMSP in gastric cancer (Taqman or methylight, PMID: 22513802, 19957042), qMSP (sybergreen, PMID: 21599969) or bisulphite pyrosequencing (PMID:17408633). Can the authors further discuss the difference between the current studies and those studies in terms of primer location and the association to clinical parameters. And why the current is superior than the others. Otherwise, it has to be rephrased. Thank you for providing these insights. Both reports using Taqman or MethyLight (PMID 22513802, 19957042) examined quantitative hMLH1 methylation by qMSP in gastric cancer, but did not clarify its clinicopathological relevance including prognostic factors such as TNM. On the other hand, our current study quantified MLH1 methylation of primary gastric tumors by qMSP using Taqman probe and evaluated its clinical pathological relations. Moreover, we for the first time proposed that quantitative values in MLH1 methylation may have a gradual effect on the MSI phenotype and prognosis in Q-MSP based on studies which originally defined positive control and negative control by cloned sequence. Therefore, our current study is considered to be unique for investigating clinicopathological factors using quantitative values of MLH1 methylation in gastric cancer. However, as the reviewers pointed out, "Most of the data on MLH1 hypermethylation have been assessed by old-fashioned non-quantitated conventional methylation specific PCR (MSP), and have not been properly validated in the quantitative assessment." The phrase was not appropriate. "Most of the data on MLH1 hypermethylation were evaluated by non-quantitative methylation-specific PCR (MSP). In previous similar reports evaluated by quantitative MSP, the quantified methylation values were not applied to investigate clinicopathological factors." We changed it to the phrase. The above contents have been added to Introduction (P5, Line3-6). 2) The current study found that gastric cancer patient with hMLH1 hypermethylation showed better prognosis. However, several studies showed that hMLH1 methylation associated with poor prognosis in gastric cancer, for example (PMID:29719511) Thank you for your pertinent comments. We have confirmed the report of the meta-analysis pointed out by the reviewers (Peng Y. Front Physiol 2018). It was reported that hMLH1 methylation in gastric cancer has a considerable association in lymph node metastasis, microsatellite status, Lauren classification, Helicobacter pylori infection, and hMLH1 protein expression. On the other hand, we could not find the results regarding the prognosis in the manuscript. We also reviewed all reports used to analyze lymph node metastases that may the most strongly affect prognosis, but none reported prognostic analysis (Ferrasi AC. World J Gastroenterol 2010, Hiraki). M. An Surg Oncol 2010, Jin J. Genet Mol Res 2014, Kolesnikova EV. Ann NY Acad Sci 2008, Moghbeli M. J Gastrointest Cancer 2014, Nakajima T. Int J Cancer 2001, Pinto M. Lab Invest 2000, Song B. Pak J Med Sci 2013, Xiong HL. Asian Pac J Cancer 2013). Smyth et al reported that surgery alone for resectable gastroesophageal cancer had the best prognosis of MSI-H or Mismatch repair (MMR) deficiency (including MLH1 methylation) groups compared to MSI-L or MSS groups (Smyth EC. JAMA Oncol 2017). On the other hand, the prognosis of MSI-H or MMR deficiency group with chemotherapy plus surgery was poor. Therefore, MSI-H or MMR deficiency groups may be resistance to chemotherapy. An et al also found that methylated MLH1 tended to have a better prognosis than unmethylated (MST: 68.4 vs 28.4 months, P = 0.49), and that CIMP-high due to multiple methylated genes including MLH1 was CIMP-low. They reported that the prognosis was significantly better than that of CIMP-negative (P = 0.04) (An C. Clin Cancer Res 2005). Based on the above reports, hypermethylation of MLH1 may be involved in chemotherapy resistance, but there are no reports that it affects the poor prognosis. Therefore, the prognostic analysis results of this study were considered to be more acceptable allowing for the previous reports. The above contents have been added to Discussion (P21, Line13-21). 3) It is difficult for this reviewer to understand how the author define MLH1-Taqmeth value 0.23 as haploinsufficiency of MLH1 expression. Thank you for your very important point. We are considering that haplo-MSI phenotype could be microsatellite-instable despite MSS defined by the current definition. The definition of MSI-H/L is based on the specific relevant markers (Bethesda panel), and they could not be covered with minor abnormalities of microsatellites caused by haplo-MSI that is corresponding to the lowest hypermethylation of MLH1 (> 0.23) (Fig. 5f). Prognostic stratification defined by the most optimal cut-off value (0.23, but not 2.3) might propose bona-fide entity of MSI phenotypes including haplo-MSI in gastric cancer. The position of the MLH1-Taqmeth value in Figure 5f has been changed to clarify the range of each MSI phenotype. The above contents have been added to Discussion (P19, Line15-21). 4) Morphological types should be defined as intestinal, diffuse and mixed type. Association between hMLH1 methylation with HP status is missing. Thank you for your suggestions. The Lauren classification is considered to be a classification method that divides the histological, but not morphological, classification in gastric cancer into intestinal, diffuse, and mixed types (Lauren P. Acta Pathol Microbiol Scand. 1965; 64: 31-49.). 5) Clinicopathologic characteristics of MSI-associated gastric cancer needs to be delineated more in the introduction. Thank you for your pertinent comments. It has been reported that the clinicopathological factors in the MSI-associated gastric cancer were elderly, female, intestinal type in Lauren classification, and tumors located in the middle to lower site of the stomach (TCGA. Nature 2014). The above contents have been added to Introduction (P4, Line13-15). 6) Figure 1a, is the bold line in qMSP (original) indicates the location of Taqman probe, it has to be stated in the figure legend. Thank you for your appropriate suggestions. As you pointed out, the bold line in qMSP (original) indicates the location of the Taqman probe. The above contents have been added to Figure Legends (P29, Line6-7). 7) Figure 1b, lower panel, label of Y-axis is missing. Thank you for your pertinent comments. A label for the methylation level is displayed on the Y-axis in the lower panel of Figure 1b. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0260303.r002
4 Aug 2021 Decision Letter - Hiromu Suzuki, Editor PONE-D-21-14491R1 Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis PLOS ONE Dear Dr. Yamashita, 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. ============================== The authors addressed most of the issues raised by the editors. However, reviewer 2 suggests an additional experiment to ensure the reliability of qMSP. Please respond to the reviewer comment. ============================== Please submit your revised manuscript by Sep 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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. 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). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Hiromu Suzuki, M.D., Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 #1: All comments have been addressed Reviewer #2: (No Response) ******** 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes 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 #1: Yes 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 #1: (No Response) Reviewer #2: The authors have addressed most of the comments. However, to avoid false positivity of qMSP, bisulphite sequencing of hMHL1 should be performed in selective samples in the haplo-MSI and low/high-MSI group ****** 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 #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.] 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. 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Revision 2
24 Oct 2021 Author Response PLoS One October 14, 2021 Dear Prof. Hiromu Suzuki, Editor Thank you for inviting us to submit a revised draft of our manuscript entitled, “Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis” to PLoS One. We also appreciate the time and effort you and each of the reviewers have dedicated to providing insightful feedback on ways to strengthen our paper. Thus, it is with great pleasure that we resubmit our article for further consideration. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We also hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you and the reviewers have noted. To facilitate your review of our revisions, the following is a point-by-point response to the questions and comments delivered in your letter. 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 #1: All comments have been addressed Reviewer #2: (No Response) 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 #1: Yes Reviewer #2: Yes 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #1: Yes 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 #1: Yes 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 #1: (No Response) Reviewer #2: The authors have addressed most of the comments. However, to avoid false positivity of qMSP, bisulphite sequencing of hMHL1 should be performed in selective samples in the haplo-MSI and low/high-MSI group Thank you for your pertinent comments. Following reviewer's suggestion, we examined eight cases using direct sequencing. As a result, methylation could be detected in only 3 cases. The remaining 5 cases were MSI-L or MSS cases. Therefore, it was considered that the sensitivity to detect methylation of direct sequencing is limited to MLH1 hypermethylation cases, and MSI-haplo can only be detected by real-time PCR. The above contents have been added to Table and Discussion (P19, Line23-P20, Line 3). 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 #1: No Reviewer #2: No In response to this revision, we asked the following doctor to give advice on the experiment, analysis and correct the rebuttal letter. Therefore, we would like to add the following one as co-author. Yusuke Nie 2, Department of General-Pediatric-Hepatobiliary Pancreatic Surgery, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0375, Japan Ippeita Araki 1, Department of Upper-gastrointestinal Surgery, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0375, Japan We have found some typographical errors in the base sequence of Table S2. Therefore, we corrected it. Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission. Sincerely, Hiroki Harada, MD, PhD Keishi Yamashita, MD, PhD, FACS Corresponding Author: Keishi Yamashita Division of Advanced Surgical Oncology, Department of Research and Development Center for New Medical Frontiers, Kitasato University School of Medicine 1-15-1, Kitasato, Minami-ku, Sagamihara, KANAGAWA, 252-0374, JAPAN E-mail: keishi23@med.kitasato-u.ac.jp Tel: +81-42-778-8111 / Fax: +81-42-778-9556 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0260303.r004
8 Nov 2021 Decision Letter - Hiromu Suzuki, Editor Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis PONE-D-21-14491R2 Dear Dr. Yamashita, 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, Hiromu Suzuki, M.D., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 #1: All comments have been addressed 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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 #1: Yes 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 #1: Yes 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 #1: The authors addressed all data adequately, and the results were well discussed. So I have no concern about publishing this paper. 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 #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0260303.r005
Formally Accepted
13 Dec 2021 Acceptance Letter - Hiromu Suzuki, Editor PONE-D-21-14491R2 Haploinsufficiency by minute MutL homolog 1 promoter DNA methylation may represent unique phenotypes of microsatellite instability-gastric carcinogenesis Dear Dr. Yamashita: 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. Hiromu Suzuki Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260303.r006

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