Association of a CHEK2 somatic variant with tumor microenvironment calprotectin expression predicts platinum resistance in a small cohort of ovarian carcinoma

Izabela Ferreira Gontijo de Amorim; Carolina Pereira de Souza Melo; Ramon de Alencar Pereira; Sidnéa Macioci Cunha; Thalía Rodrigues de Souza Zózimo; Fábio Ribeiro Queiroz; Iago de Oliveira Peixoto; Luciana Maria Silva Lopes; Laurence Rodrigues do Amaral; Matheus de Souza Gomes; Juliana Almeida Oliveira; Eduardo Batista Cândido; Paulo Guilherme de Oliveira Salles; Letícia da Conceição Braga

doi:10.1371/journal.pone.0315487

Peer Review History

Original SubmissionJune 4, 2024
4 Jun 2024 Author Response https://doi.org/10.1371/journal.pone.0315487.r001
15 Jul 2024 Decision Letter - Alvaro Galli, Editor PONE-D-24-16271Association of a CHECK2 somatic variant with tumor microenvironment calprotectin expression predicts platinum resistance in a small cohort of ovarian carcinomaPLOS ONE Dear Dr. Braga, 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. Please submit your revised manuscript by Aug 29 2024 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. 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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: Partly 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: No 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 perform a search for biomarkers of Platinum Resistance among HGSOC patients. Comments: 1) It is stated: Yes - all data are fully available without restriction. Unfortunatelly, data were not included into the submission. Please, provide the raw data, either as supplementary csv files or through a repository, such as, e.g., the Mendely data. 2) The authors did LOOCV to assess performance of the selected two predictors. However, they failed to embed into cross-validation framework the feature selection itself. As a consequence, the findings may be misleading. 3) Please complement the already performed survival analysis by the predictive approach: Use the time-dependent Brier score to quantify how well the model can predict survival. Use time-dependent ROC to quantify how well can the model discriminate. Check, whether the predictions of survivals are well-calibrated. 4) Please use the Adjusted survival plots based on the fitted CoxPH model, rather than the Kaplan-Meier plots. Reviewer #2: Major comments: 1. Lines 229-238 1.a. The authors used a decision tree classifier to identify variables and value cutoffs that discriminate between the two classes PR and PS. The classifier successfully discriminates between the two classes when using all the classes to fit the model. I believe this model is the one that produces the cutoff value of 84 for the L1 levels. I think this should be mentioned. 1.b. For the leave one out cross-validation (LOOCV). How did you choose which sample to leave out? Did you fit the 17 possible classification models? If so, is the 14/17 accuracy the best or the worse accuracy among all models? 1.c. I suggest that the authors use the term “decision tree classifier” instead of just “machine learning”. 2. Data and code availability 2.a. The authors performed analysis using RStudio. It would be beneficial if the corresponding scripts are included in the submission as supporting information, along with the necessary files for the code to run. Minor comments: 1. There is a typo in the title. The gene name should read CHEK2 instead of CHECK2. 2. Line 168. duplicated word “Table” 3. Line 191. Choose one way to write PDL1 or PD-L1 ****** 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. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0315487.r002
Revision 1
5 Sep 2024 Author Response Response to Reviewers Article title: Association of a CHEK2 Somatic Variant with Tumor Microenvironment Calprotectin Expression Predicts Platinum Resistance in a Small Cohort of Ovarian Carcinoma Authors: Izabela Ferreira Gontijo de Amorim, Carolina Pereira de Souza Melo, Ramon de Alencar Pereira, Sidnéa Macioci Cunha, Thalía Rodrigues de Souza Zózimo, Fábio Ribeiro Queiroz, Iago de Oliveira Peixoto, Luciana Maria Silva Lopes, Laurence Rodrigues do Amaral, Matheus de Souza Gomes, Juliana Almeida Oliveira, Eduardo Batista Cândido, Paulo Guilherme de Oliveira Salles, Letícia da Conceição Braga Date: September 5, 2024 Submission Number: PONE-D-24-16271 We would like to express our gratitude to the reviewers for their detailed and constructive comments on our manuscript. Your observations have significantly improved the quality of our work. We have comprehensively revised the manuscript to enhance the clarity and accuracy of our descriptions. In response to your specific comments, we have made the following revisions: ________________________________________ Journal 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 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 Answer 1: All file names have been revised to meet PLOS ONE’s requirements. 2. Please ensure that you include a title page within your main document. We do appreciate that you have a title page document uploaded as a separate file, however, as per our author guidelines (http://journals.plos.org/plosone/s/submission-guidelines#loc-title-page) we do require this to be part of the manuscript file itself and not uploaded separately. Could you therefore please include the title page into the beginning of your manuscript file itself, listing all authors and affiliations. Answer 2: We are sorry for the inconvenience. We have now included the title page in into the beginning of the manuscript file. 3. Please ensure that you include a title page within your main document. You should list all authors and all affiliations as per our author instructions and clearly indicate the corresponding author. Answer 3: We are sorry for the inconvenience. We have now included the title page in into the beginning of the manuscript file. ________________________________________ Review Comments to the Author Reviewer #1 Comment 1: It is stated: Yes - all data are fully available without restrictions. Unfortunately, the data were not included in the submission. Please provide the raw data, either as supplementary CSV files or via a repository, such as Mendeley Data. Answer 1: We apologize for this inconvenience. We have taken your comment seriously and have thoroughly revised all tables containing clinical data and other relevant datasets. These are now being submitted along with the manuscript as S2 Table, ensuring the integrity and transparency of our research. During the revision process, we found a mistake in two event/censored patients. The correction resulted in a slight change in hazard ratio values, which have been adjusted in the manuscript. The topic is Survival Analysis of the Results session. ________________________________________ Comment 2: The authors performed LOOCV to assess the performance of the two selected predictors. However, they failed to incorporate the feature selection itself into the cross-validation framework. Consequently, the findings may be misleading. Answer 2: Thank you for your insightful comment. We understand the importance of embedding feature selection within the cross-validation framework to avoid potential bias. However, we would like to clarify that the selection of the two predictors was performed independently before the application of the LOOCV, and these predictors were fixed throughout the entire validation process. This means that the feature selection was not part of the LOOCV process itself but was conducted as a preliminary step. The LOOCV was then applied to evaluate the performance of the classifiers using these pre-selected predictors. Therefore, there was no need to include the feature selection within each fold of the LOOCV, as the attributes used in the model were already determined in advance. This approach ensures that the model's performance is assessed based on the chosen predictors without any additional feature selection bias during the validation phase. We hope this clarifies our methodology and are open to further discussion if necessary. ________________________________________ Comment 3: Please complement the survival analysis already performed by the predictive approach: Use the time-dependent Brier score to quantify how well the model can predict survival. Use time-dependent ROC to quantify how well the model can discriminate. Check whether the survival predictions are well-calibrated. Answer 3: Thank you for pointing out these complementary analyses. They are now presented in Supplementary Files (S6 Table, and S5 and S6 Figures). A brief citation was also included in the Statistical Analysis topic of the Material and Methods session and in the Survival Analysis topic of the Results session. ________________________________________ Comment 4: Please use the survival plots adjusted based on the adjusted CoxPH model, instead of the Kaplan-Meier plots. Answer 4: As recommended, the Kaplan-Meier plots (Figure 5 b-d) were substituted by correspondent adjusted survival curves for the CoxPH Model. A correction was also made in the Statistical Analysis topic of the Material and Methods session. ________________________________________ Reviewer #2 Comment 1a: Lines 229-238. The authors used a decision tree classifier to identify variables and cutoffs that discriminate between the two classes PR and PS. The classifier successfully discriminates between the two classes when using all classes to fit the model. I believe this model is the one that produces the cutoff of 84 for L1 levels. I think this should be mentioned. Answer 1a: Thank you for your suggestion. This information was inserted in the Results session, line 286. ________________________________________ Comment 1b: For leave one out cross validation (LOOCV). How did you choose which sample to leave out? Did you fit all 17 possible classification models? If so, is the 14/17 accuracy the best or worst accuracy among all models? Answer 1b: We appreciate your question regarding the LOOCV process. In the LOOCV approach, each of the 17 samples was systematically left out once, and the model was trained on the remaining 16 samples. This process was repeated 17 times, leaving out a different sample each time. The accuracy of 14/17 reflects the number of correct predictions out of the 17 folds. To clarify, this accuracy is calculated by averaging the performance across all folds rather than representing the performance of a single model. Therefore, it reflects the overall generalizability of the model across the entire dataset rather than being the best or worst model accuracy. We will ensure this explanation is more clearly articulated in the revised manuscript. ________________________________________ Comment 1c: I suggest the authors use the term “decision tree classifier” instead of just “machine learning”. Answer 1c: Thank you for your suggestion. The text became clearer and more focused with this change. The revisions have been made on lines 49, 285, and 315. ________________________________________ Comment 2a: Data and code availability. The authors performed the analysis using RStudio. It would be helpful if the corresponding scripts were included in the submission as supporting information, along with the files needed to run the code. Answer 2a: All scripts used in RStudio are described in Supplementary Methods Box 2. ________________________________________ Comment 3: There is a typo in the title; the gene name should be CHEK2 instead of CHECK2. Line 168, duplicate word “Table” and line 191, and please choose a way to spell PDL1 or PD-L1. Answer 3: Thank you for your careful review of our writing. All suggestions have been accepted and successfully corrected. You can track the changes in the following lines: • a. Corrected the gene name in the title. • b. Deleted one of the duplicate words, "Table," on line 212. • c. We apologize for the inconsistency in referring to the biomarker. The correct form is PD-L1, which has been corrected throughout the text. We hope we have addressed correctly all the requirements and questions. Kind regards, Letícia Braga Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0315487.r003
14 Oct 2024 Decision Letter - Alvaro Galli, Editor PONE-D-24-16271R1Association of a CHEK2 somatic variant with tumor microenvironment calprotectin expression predicts platinum resistance in a small cohort of ovarian carcinomaPLOS ONE Dear Dr. Braga, 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. Please submit your revised manuscript by Nov 28 2024 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. 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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 #3: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: 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 #3: 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 #3: 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 #3: The manuscript is interesting and covers the contemporary problem of predicting chemo-resistance in HGSOC. The main weakness of the study is its low patients number. My main point of interest is how did Authors choose the tumors for the study. I realize that all of them were HGSOC, but there are several subtypes according to the immunoreactivity of the host against tumor. The immunological composition of all the tumors studied seems to be quite uniform in its TIL's pattern. Could Authors explain this? ****** 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 #3: 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. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0315487.r004
Revision 2
2 Nov 2024 Author Response Review Comments to the Author Reviewer #3: The manuscript is interesting and covers the contemporary problem of predicting chemo-resistance in HGSOC. The main weakness of the study is its low patients number. My main point of interest is how did Authors choose the tumors for the study. I realize that all of them were HGSOC, but there are several subtypes according to the immunoreactivity of the host against tumor. The immunological composition of all the tumors studied seems to be quite uniform in its TIL's pattern. Could Authors explain this? Thank you for your suggestion. This is a retrospective study that initially reviewed the medical records of 52 patients diagnosed with ovarian cancer treated at the Mário Penna Institute between 2015 and 2019. Of these, 24 patients were included in the study for meeting the inclusion criteria: confirmed diagnosis of high-grade serous carcinoma (HGSOC) with a pathological report, as well as complete clinical and follow-up data. The remaining 28 patients were excluded for the following reasons: 16 had histology different from HGSOC; 8 underwent surgery at another institution, resulting in loss of follow-up; and 4 had inadequate biological material (slides and paraffin blocks) for the necessary processing. To provide a clear understanding of the criteria applied, we included a flowchart in the Supplementary Files (S1_Fig), detailing the selection process for the patients with HGSOC included in this study. To clarify this modification, we added the following text in the “Patients and tissue samples” section – lines “117-120” of the Materials and Methods: “For this, the medical records of 52 patients diagnosed with ovarian cancer between 2015 and 2019 were initially reviewed. After applying the inclusion and exclusion criteria, 24 patients with high-grade serous carcinoma were selected” Despite the apparent uniformity in the pattern of TILs, patients exhibited different distribution and quantification patterns. For instance, some showed a diffuse profile with a higher infiltration rate compared to others. However, due to the small sample size, it was not possible to identify or classify a significant difference between the groups or patients. This is evident in the results presented in graph (S2_Fig). We acknowledge that the sample size is relatively small, limiting the generalizability of our findings. However, with an increased sample size, the apparent uniformity of the infiltration may not be sustained. Besides that, exploratory studies are essential for generating hypotheses. Additional studies with a larger number of patients and using other molecular biology techniques may provide more robust validation of these results. It is also important to consider the significant presence of several variants, including indels, intronic, missense and nonsense. In the context of cancer genomes, they can lead to altered or truncated proteins. When these altered proteins are processed by the proteasome, and the peptide fragments are presented by HLA molecules on the surface of tumor cells, these peptides can act as neoantigens, making them potential targets for the immune response. Although the presence of neoantigens may explain the occurrence of TILs in the majority of tissue samples, it is important to note that TIL analysis alone is insufficient to demonstrate the immunoheterogeneity of HGSOC. To address these limitations more clearly and highlight the study's significant contributions, we have revised the ‘Conclusion section’. We now explicitly state, “Despite the study's limitations, such as the small sample size, its unicentric design, and convenience sampling, its primary strength lies in its potential for improved stratification of ovarian cancer patients regarding Pt-C resistance. Additional studies with a more significant number of patients could improve and validate these results.” We hope we have addressed correctly all the requirements and questions. Kind regards, Letícia Braga Attachments Attachment Submitted filename: Response to reviewers.pdf https://doi.org/10.1371/journal.pone.0315487.r005
27 Nov 2024 Decision Letter - Avaniyapuram Kannan Murugan, Editor Association of a CHEK2 somatic variant with tumor microenvironment calprotectin expression predicts platinum resistance in a small cohort of ovarian carcinoma PONE-D-24-16271R2 Dear Dr. Braga, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Avaniyapuram Kannan Murugan, M.Phil., Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0315487.r006
Formally Accepted
Acceptance Letter - Avaniyapuram Kannan Murugan, Editor PONE-D-24-16271R2 PLOS ONE Dear Dr. Braga, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Avaniyapuram Kannan Murugan Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0315487.r007

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