PONE-D-21-24357Immunological and prognostic significance of novel ferroptosis-related genes in soft tissue sarcomaPLOS ONE

Dear Dr. Feng,

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.

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Sandro Pasquali, M.D., Ph.D.

Academic Editor

PLOS ONE

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: In this manuscript Zhao et coworkers performed an in silico analysis of RNAseq data from a very large series of well characterized soft tissue sarcoma (STS) samples spanning different subtypes with the aim to explore the immunological and prognostic value of ferroptosis-related genes, in particular of 6 genes (HELLS, STMN1, EAPS1, CXCL2, NQO1, IL6).

The here presented data are interesting, but the manuscript is too descriptive and, in some points, confused. In particular, authors should address the following major issues:

-Authors should clearly specify which samples they compared to identify genes differentially express in STS. Did they compare tumor vs normal tissues?

-Authors should add in the main text a table reporting the name of the 40 ferroptosis-related genes along with their fold change.

-Results reported in figure 4B are not commented in the text. Authors should discuss the differential expression of the 6 hub genes between male and female, leiomyosarcoma and other STS subtypes, different disease progression.

-Authors should comment and discuss gene sets differentially enriched in STS TCGA samples divided on the basis of expression of the 6 hub genes.

-Paragraphs entitled ‘Association of hub genes expression and ICGs’ and ‘Association of hub genes expression and immune cells infiltration’ should be moved at the end of Results section.

-Authors should specify which deconvolution tool they used to estimate population of immune cells infiltrating the tumors.

-‘Immunohistochemical validation’ paragraph should be removed, as in my opinion it does not represent a validation of results obtained by in silico analysis.

-Extensive editing of English language is required.

Minor issues:

-In Figure 2, names of GO and KEGG networks are unreadable. May be authors should add them in the bar graph.

-In Figure 3, color legend is missing. Please, add it.

Reviewer #2: Thank you for putting together this comprehensive manuscript that identifies several novel targets for immunotherapy agents derived from the ferroptosis pathway in sarcoma.

It will be interesting to how you intend to translate these findings into clinical trials.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-24357R1Immunological and prognostic significance of novel ferroptosis-related genes in soft tissue sarcomaPLOS ONE

Dear Dr. Cui,

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.

Authors did a great job in addressing comments from a reviewer. The manuscript has been furtherly reviewed by a bioinformatician and comments provided. Also, some methodological comments on nomograms need to be addressed. 

Please submit your revised manuscript by Dec 30 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: https://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,

Sandro Pasquali, 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.

Additional Editor Comments:

Regarding the generation of prognostic nomograms, some clarifications and modifications are needed. The authors should consider the following publication "Kattan MW, Hess KR, Amin MB, et al;members of the AJCC Precision MedicineCore. American Joint Committee on Cancer acceptance criteria for inclusion of riskmodels for individualized prognosis in th epractice of precision medicine. CA Cancer J Clin. 2016;66:370–374." as a guidence to develop and present their nomograms. In this article there is checklist, which should be followed. For instance, it is unclear which is the patient population (e.g. primary or metastatic tumour) that was used to generate the nomograms and it is unclear if nomograms were validated. Although I understand that Authors do not aim at targeting inclusion in AJCC staging, if the authors cannot match the required quality standards for their nomograms it would be probably better to exclude them from the current article.

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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 #3: (No Response)

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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: (No Response)

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #3: Yes

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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: (No Response)

Reviewer #3: Yes

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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: (No Response)

Reviewer #3: Yes

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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 have addressed all my questions and made the necessary changes to the manuscript. I have no further comments.

Reviewer #3: Authors describe a panel of genes, related to ferroptosis able to have a significative discrimination power in prognostic and immunological model of soft tissue sarcomas.

This article has an innovative character and could contribute to knowledge of soft tissue sarcoma.

Minor reviews:

1) Authors should specify which metric they used for ranking genes in GSEA.

2) Authors correctly used ComBat algorithm to eliminate batch effects and, for this reason, they should make explicit in supplementary figure B and C which samples belonged to the same dataset (using different symbols or colors) to highlight the dataset independence (technical variability) from the biological variability.

3) Authors should maintain the same color scheme: in figure 1B the legend bar (add a title for describing) shows positive magnitudo of fold change in red color, and blue for negative (and do the same also for high and low expression in survival analysis), while in the volcano plot (figure 1A) the color scheme is inverted. To avoid any doubt change consistently also the color of the circles in the Venn diagram (figure 1C). Explicit also the type of p-value correction in y-label in volcano plot.

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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 #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.

PONE-D-21-24357R2Immunological and prognostic significance of novel ferroptosis-related genes in soft tissue sarcomaPLOS ONE

Dear Dr. Cui,

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 ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

Please submit your revised manuscript by Jan 29 2022 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: https://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,

Sandro Pasquali, 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.

Additional Editor Comments:

Thank you for addressing reviewers and editor comments in your revised manuscript. Considering that nomograms are not validated, they should not be considered conclusive and therefore the statement in the conclusion of abstract and manuscript about nomogram should be removed. Also, can you specify whether nomograms were generated on primary or metastatic or both tumours and patients? This should clearly stated in the results section and discussion.

[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.

Immunological and prognostic significance of novel ferroptosis-related genes in soft tissue sarcoma

PONE-D-21-24357R3

Dear Dr. Cui,

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.

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Kind regards,

Sandro Pasquali, M.D., Ph.D.

Academic Editor

PLOS ONE