PONE-D-20-23411

Presence of myxoid stromal change and fibrotic focus in pathological examination are prognostic factors of triple-negative breast cancer: Results from a retrospective single-center study

PLOS ONE

Dear Dr. Ishida,

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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 paper the authors studied stromal reaction in TNBC tumors as prognostic factors. Specifically they assessed that the presence of myxoid change and fibrotic focus are poor prognostic factors in TNBC patients in term of overall survival and disease free survival and that the combination of both parameters was an independent poor prognostic factor.

The major limitation of this study lies on the difficulty to generalize the clinical value of the prognostic factors described due to the fact that the authors analyzed only a cohort of 62 TNBC patients without a validation cohort.

The paper is potentially interesting anyway a number of compulsory aspects need to be clarified and the following comments should be addressed before the manuscript can be considered for publication:

Major points:

• The authors analyzed only a cohort of 62 TNBC patients. Please add a validation cohort.

• As reported in Table 1, 56.5% of patients received adjuvant chemotherapy and 38.7% did not received chemotherapy. The analysis of OS and RFS for all stromal prognostic factors considered should be performed considering separately patients who received/not received treatment. Please add these analyses.

• In figures 1a and 2a the authors report only a representative image at large magnification for myxoid change and fibrotic focus respectively. Please add also images at lower magnification and images of tumor sample negative for myxoid change and fibrotic focus.

• Correlation analysis with clinicopathological factors, reported in Table 2 was done only considering patients with both mixoid change and fibrotic focus together. Please add correlation analysis considering separately the two stromal characteristics.

Minor points:

• In the introduction section the authors should better support sentences at lines 62-65 with references from literature

• Table 1: tumor size is considered as a continuous variable. Could the authors indicate tumor size as they do in Table 2 and 3 for correlation and multivariate analyzes?

• As regards the analysis of TILs, the paper the authors refer to, considers also “intermediate TILs as 10%<tils≤59%”. analysis="">

Overall the manuscript can be interesting but it cannot be accepted in the present form. A major revision is mandatory before publication.</tils≤59%”.>

Reviewer #2: Hirotsugu Yanai and collaborators examined on 62 triple negative breast cancers the clinical meaning of the histopathological assessment of myxoid change and FF fibrotic focus. Although their results seem to be novel and promising there are important issues that need to be addressing.

Major issues

Methods

1. Although there are two cited papers regarding the criteria to detect myxoid change it would be useful to clearly explain the characteristics taking into account for the analysis.

2. There is any score to classify positive and negative tissues (%, number of cells, area)?, what was to cutoff to consider a positive tissue?

Results

1. Please include at least two more representative pictures of myxoid changes and FF, and clearly indicate by arrows the area and changes of interest. Please also include images from patients in which the myxoid changes or FF were not observed.

2. On table 2 why positive patients to FF or Myxoid change were grouped with negative patients?. I would group the tumors as following to get a better idea of the independent and coordinate changes: 1) Myxoid change and fibrotic focus-positive (N=11) 2) Myxoid change positive-only (N=5) 3) fibrotic focus-positive (N=10) and 4) Myxoid change and fibrotic focus-positive negative (N=36).

3. As previously mentioned on table 3 it is necessary to show multivariate analysis of stroma changes occurring independently and in co-occurrence, to get an idea of how this phenomena impact tumor biology alone or in combination.

4. Although this is one of the first efforts to associate the presence myxoid changes and FF with clinical outcomes and patients features on TNBC, unfortunately by the limited number of included tumors there is not an optimal statistical strategy that can be robustly applied and the conclusion are not strongly supported, thus I encourage the authors to include some independent cohorts to confirm some of their findings. Since hematoxylin and eosin is the only necessary test to evaluate stroma reactions, I will suggest including some triple negative TCGA tumors that can be evaluated from https://cancer.digitalslidearchive.org/#!/CDSA/brca and clinical and TILS information is available.

5. Since most of the triple negative tumors are now being treated in neoadjuvant therapy, it is possible to examine this stroma reactions from biopsy tissues?. The authors described that they discard an important set of tumoral samples from their original cohort due to neoadjuvant treatment. So 1) It is possible to compare the histopathological assessment of the surgical analyzed tissues, included in this study, with their matched diagnosis biopsies to define how much a biopsy tissue can recapitulate what is detected in a larger tissue (surgical sample) 2) it is possible to include the analysis of this excluded patients based on their biopsy tissue? Please discuss this point.

Minor

Since a Fisher test was computed to examine the significance of the association between two kinds of classification the most accurate term for table 2 and methods section is association instead of correlation.

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Reviewer #1: No

Reviewer #2: Yes: Sandra Romero-Cordoba

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PONE-D-20-23411R1

Presence of myxoid stromal change and fibrotic focus in pathological examination are prognostic factors of triple-negative breast cancer: Results from a retrospective single-center study.

PLOS ONE

Dear Dr. Ishida,

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 January 17, 2021. 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

We look forward to receiving your revised manuscript.

Kind regards,

Elda Tagliabue

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

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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: Yes

Reviewer #2: Partly

**********

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: The authors have comprehensively addressed the queries raised in my previous review, except the request to analyze a new cohort to validate results obtained using the cohort of 62 TNBC patients. Anyway the authors have made efforts to enhance the quality of the paper, and this merits consideration.

Only four minor points need to be clarified before the manuscript can be considered for publication.

• Significant results shown in Table 2 and Table 3 need to be reported also in Result section as the authors did for Table 4.

• Figure 2 a, b and c: please use 100X and 200X magnification as done in figure 1. Please add magnification bars in figure 1 and 2.

• “Clearly” line 316 pag 24: since the authors analyzed only a cohort of patients, this adjective should be omitted.

• Sentences lines 337-342 pag 25: since the analysis of prognosis was done considering surgically resected specimens, the conclusion concerning the importance of biopsy should be revised

Reviewer #2: In this revised text Hirotsugu Yanai and collaborators improved their analysis and presented some novel results. However, I continue having the feeling that their small set of evaluated tumors prevent them to conclude some of the statements they are describing throughout the text. Thus, I think this needs to be addressed before publishing.

Point:

1. Since it is not possible to evaluate an independent validation cohort, and given the limited number of tested samples and consequently the poor number of clinical events analyzed, findings from this report should be interpreted with more caution, and thus a mention of this important limitation in the discussion is not enough. Authors should avoid to extend their finding to all TNBC patients, despite, they limited their results to their "cohort" in some sections but not through the entire manuscript as seen by the tittle, and conclusion sections, this need to be addressed.

2. An interesting data is presented by the comparison of myxoid change between pre-operative biopsy specimens and surgically-resected specimens. Can you please add a brief commentary on how this changes are established. How therapeutical interventions can cause this phenomena?

3. Since the "Presence of myxoid change and FF was significantly associated with low/intermediate TILs in the stroma (p=0.013)", what is the advantage to test FF and myxoid change over TILS, which is now a days an standardized pathologic test made on clinical samples, and more robust international guidelines had being described for its evaluation and interpretation. An although some controversial results, this immune measurement has been also related with prognosis and survival outcomes. Please comment this.

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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.

Presence of myxoid stromal change and fibrotic focus in pathological examination are prognostic factors of triple-negative breast cancer: Results from a retrospective single-center study.

PONE-D-20-23411R2

Dear Dr. Ishida,

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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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,

Elda Tagliabue

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: 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: The authors have addressed the queries raised in my previous review.

The manuscript in the present form is suitable for publication.

Reviewer #2: The authors addressed most of the points, so I consider the text is now suitable to be published in the journal

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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 #2: No