PONE-D-20-28259

The potential of tumour microenvironment markers to stratify the risk of recurrence in prostate cancer patients

PLOS ONE

Dear Dr. Gevaert,

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.

As you can realize both Reviewer ask for some further clarifications that would notably increase the strength of this paper. Furthermore, data from studies to be made freely available and this is not done in this case, even though it is said that it was.  Providing the data for this study would be as simple as a spreadsheet with each patient tumor score, stage, margin status, followup time to recurrence and stain scores  the analyzed patients

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We look forward to receiving your revised manuscript.

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Antimo Migliaccio, M.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: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: No

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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: No

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: The manuscript by Dr. Thomas Gevaert and colleagues is interesting since it is very important to have personalised medicine for every patient. In this paper, the authors consider also the tumor microenvironment (TME) of the prostate cancer, analysing different markers in samples derived form Patients affected by Prostate Cancer (PC). This is an emerging and appealing area of interest. They investigated different TME-markers that possibly could predict CLR in PCa.

The manuscript is appealing and a good starting point since the knowledge of the tumor microenvironment is still limited to few studies, but some English style corrections are needed.

Furthermore, I have some considerations.

Materials and Methods:

1. line 135. Are the methods similar or the same? Please improve this sentence and add information.

Results:

2.In table 1 is not visible the Gleason's score, but the Gleason's grade, while in the paragraph "Marker expression profiles in PCa versus PNT and in stroma versus epithelium" the authors cite the Gleason's score. Please add information to the table or make everything more homogeneous and clear.

3. Is there also an important marker for TME, in particolar for Carcinoma associated fibroblasts. It is FAP-1. Have the authors evidences about its expression?

4. The authous should add a table resuming the first paragraph of the results and that could support the figure 1, also specifying if the proteins analyzed are present in the epithelial or the stromal compartments.

5. Discussion section:

The authors talk about the importance of TME in prostate cancer. They should take also in consideration the importance of steroid hormones receptors expression in this compartment, discussing about their absence/presence.

They could consider for example: 1) https://doi.org/10.3389/fendo.2014.00225; 2) Endocrine-Related Cancer

(2018) 25, R179–R196 and therein refs.

In general, the authors should improve the manuscript, the tables, the English grammar for enlarging the audience and rendering the manuscript more fluent.

Reviewer #2: This manuscript describes a biomarker panel of immunohistochemical stains that are prognostic of clinical recurrence after prostatectomy. The study was a "case-control" of 24 each of recurrent and nonrecurrent cases matched for Gleason score; it appears that attempts were made to match to other clinical features as well, but details for such matching are vague. Findings included that vascular marker CD31 in nontumor areas, alpha-smooth muscle actin staining in peritumor stromal tissue and cancer stroma / normal tissue stroma progesterone receptor staining ratio were all significant predictors of recurrence on univariable analysis, while a CD31 and PR stain ratio panel could break the study population into high and low risk groups for recurrence on multivariable analysis.

Strengths of study include a hard endpoint of image-verified tumor progression, an objective image analysis approach for stain scoring and an appropriate group of IHC markers directed towards TME proteins. Also it appears that the TMAs may have been meticulously designed however some details in support of this are lacking, and should be added to further support this idea. Lastly, the data suggest that the markers under study do have value, make biologic sense and deserve further study.

Weaknesses of the study include that the cohort is small. Also, Gleason scoring is known as the strongest prognosticator of biochemical recurrence after prostatectomy (after margin status), and in fact literature shows a large gap between GG2 and GG3 with GG3 essentially comparable to GG4, yet the authors have a cohort where the outcomes for GG2 and GG3 are similar. This is very disturbing and might put some readers off as to the value of these findings, although it could be argued that it further supports the morphologic and clinical homogeneity of the cohort under study. Another weakness is that while it is stated that the cohort is relatively homogeneous in clinical, stage, grade features, differing only in outcomes, data supporting this claim seems vaguely presented and should be more clearly presented.

Particular issues for authors to consider to improve the manuscript:

Introduction: The biomarkers discussed in the intro deal with predictive markers for treating metastatic disease and have nothing to do with the work presented. Biomarker panels (oncotype, decipher, prolaris, etc) for prognosticating recurrence are discussed in the same referenced paper in the intro and do deal with the same types of cases presented here and this should really be the focus of the intro. In fact, the impetus for doing a study like this would be to develop a significantly cheaper panel for doing the same job as these panels do, but at a significantly cheaper price. Also, this should be extended to the discussion since all of these panels also include stromal (TME) markers within the panels, so how this IHC panel does or does not include these /compare to what is in commercial panels would be very informative.

Methods: TMA core size should be indicated (.6mm, 1mm or other?). It is unclear how many cores of tumor per case and normal per case were included in the TMAs and it is mentioned that there were 6 cores taken and 3 TMAs made, but unclear if it is 3 copies of the same TMA design or that it took 3 sperate TMAs to get all of the cores included? 6 cores with three tumor / three normal? Other combo? Unclear, please clarify. While these details may seem mundane, there are some investigators that believe that at least 3 tumor cores are needed per case to account for tumor heterogeneity (for example see Rubin, Pienta, et all from around 2000 or 2002 for details), although a sentence of why such triplicates are not needed for this study is also acceptable. Also, as the makeup of the stroma might be different depending on where in the normal it is taken from (peripheral, central, transistional, periurethral, how close or far from the tumors), was there any effort to be consistent about where the normal was sampled from??? How close or far from the tumor was the normal taken? This should be included somewhere since there is discussion of the tumor field effect in discussion section.

Table 1, you really should have p values for each to be prognostic of clinical recurrence, because that would indicate whether clinical variables alone are prognostic and then whether the cohort needs to be reconsidered/redesigned.

In relation to this would be something about the details of the CLRs, how many were distant (bone or soft tissue mets) vs. how many were local failures (prostate bed or local LN mets), the latter of which might be more a function of surgical technique rather than actual tumor biology. Including such information could further help the reader put these results into perspective.

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Reviewer #1: No

Reviewer #2: No

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The potential of tumour microenvironment markers to stratify the risk of recurrence in prostate cancer patients

PONE-D-20-28259R1

Dear Dr. Gevaert,

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,

Antimo Migliaccio, M.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

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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

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

Reviewer #1: 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

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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: 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 most of my questions and have written some concepts in a more attractive and understendable way. I think that addition of data in TME field is very important and in this form the manuscript is suitable for the publication.

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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