PONE-D-20-18560

Comprehensive Molecular Characterization of the 8q22.2 region identifies prognostic relevance of COX6C and OSR2 mRNA in muscle invasive bladder cancer

PLOS ONE

Dear Dr. Erben,

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Francisco X. Real

Academic Editor

PLOS ONE

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Reviewers' comments:

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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: No

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

Reviewer #1: I Don't Know

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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Reviewer #1: Yes

Reviewer #2: No

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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 the authors evaluated the possible role of amplification in 8q22.2 region in bladder cancer. Using TCGA data they found that this region shows CNV in around 10% of patients uin parallel with RNA amplification of the corresponding genes. among them, they can identify COX6C and OSR2 mRNA in correlation with specific clinicopthological characteristics.

In general the manuscript is well written and the conclusions are supported by the data. Nonetheless a possible concern comes from the fact that only TCGA data are used in the analyses. The clinicla annotation of this dataset can be controversial, therefore a possible validation including other cohorts is strictly recommended.

In particular the possible validation of the identified genes by RTqPCR in other cohorts is highly relevant.

Another aspect of potential controversial is the association with specific molecular subtypes. According the manuscript the authors observed a possible association with luminal tumors. However in TCGA there are many other subtypes besides luminal and basal BC. Therefore, it is difficult to ascertain how the aithors have performed this analysis. A more detailed study including specific markers is recommended.

Minor aspects,

The use of a z-score normalization value ≥2 is appropriate but a supplementary table providing values using more or less restrictive z-scores would help to realize the relevance of the amplicon

Reviewer #2: Uysal et al performed in silico analysis of data from 361 MIBC patients from TCGA to investigate the prognostic role of 8q22.2. The authors investigate amplicons using DNA and RNA based methods, and finally analyze genes at 8q22.2 that may be of clinical relevance.

1. Introduction: The authors should decide if this is a paper describing novel biological/clinical features associated the 8q22.2 region in bladder cancer – or if this is a more technical paper, describing the use of the methods used. This could be rewritten to focus on one or the other. It is not clear when reading the introduction what the main scope is. The final part of the introduction (line 99) should be rewritten.

2. The main focus of the paper- chromosomal region 8q22.2 – is mentioned in the introduction as amplified in the cbioportal.org website. However, as little is known about this in BC I suggest to move this to the result section. Overall, 8q22.2 may be interesting, but it is not clear why the authors choose to investigate this. This should be clearly written and expanded upon in the manuscript.

3. Methods: it is not clear if CNV data is derived from SNP arrays or from exome sequencing. Please specify. Also, as CNV is one of the main points in the manuscript, the authors should describe the different types og gains (high vs low gain, and imbalanced vs balanced). Also – how does the level of amplification affect the level of expression?

4. Are the gains at 8q22.2 associated with other genomic events? This may indicate that this event is an artefact, especially if gains are balanced.

5. Methods: patients receiving NAC are excluded. The reason for this is not clear – the full cohort receive various treatments, and delayed chemotherapy upon metastatic disease, and adjuvant chemotherapy. Please comment on this.

6. Page 6 – there seem to be redundancy in the text regarding the patient population exclusion criteria.

7. Methods: how was the z-score of >=2 chosen for mRNA overexpression ? Clarify the definition of “RNA_HIGH_GeneX” and “RNA_LOW_GeneX”.

8. Methods + Table 2: Why include variables with a p-value < 0.2 in the multivariable analysis?

9. Line 184: The authors call a p-value < 0.2 significant. However, they also state in line 192 that p-values < 0.05 were considered significant. Please clarify.

10. Line 138: the reason for splitting the 8q22.2 into a “core “ and “extended” region is not clear. An explanation is provided in line 146-147, but does this represent a continuous part of DNA at 8q22.2, or simply representation of genes where no correlation between DNA and RNA is observed throughout the region? Furthermore, the results related to this is presented in the method section. (Table 1).

11. Table 2: Luminal vs basal subtype is a strong dichotomization of the data, and it is not strange that this becomes significant. But it would be more interesting to see how the regions actually correlated to the consensus classification for MIBC.

12. Table 2: According to the supplementary excel file, no tumors below T2 are included. However, tumors are divided into T-stage ≤2 and T stage 3/4 in Table 2 and in line 153,201,229. The ≤2 T stage indicates that non-muscle invasive tumors are included and is therefore misleading. Rephrase this to T2 and not ≤T2.

13. N-stage is only significant for RNA and not for DNA, which indicated that the expression is not driven by amplification. In my opinion, it would be stronger to make a joint analysis of genes in the region – affected by amplification, and simultaneously overexpressed. As I read the paper this is not the case, and this is investigated separately. The authors touch upon this in the section starting line 278, but it is not clear why this is not performed in general through the manuscript – if the purpose is to identify genes of interest affected by gene amplification and with elevated expression.

14. Correlation to mutations and gene functions: the authors correlate to 18 gene mutations. Was correction for multiple testing performed here ? The significance reported is not high, so it would be important to correct for this.

15. Finally, the authors should investigate other public datasets, in order to validate the findings – e.g. only RNA data to validate specific genes.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-18560R1

A comprehensive molecular characterization of the 8q22.2 region reveals the prognostic relevance of OSR2 mRNA in muscle invasive bladder cancer

PLOS ONE

Dear Dr. Erben,

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 comments are minor and the paper might be acceptable without further external review.

Please submit your revised manuscript within one month. 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,

Francisco X. Real

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: 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: Authors have addressed my previous concerns adequately.

However, I have a few comments that may improve the manuscript further:

1. line 126: I think the use of "in silico" i headline and in line 127 is a bit strange. I would rewrite and just indicate that data has been produced earlier.

2. line 152: rewrite to just mention a validation cohort - it is not important to highlight the origin in the headline.

3. line 284: remove the "cell 2017" from the headline - and "the" before TCGA as it is already included in the acronym..

4. line 325: remove "in silico" again

5. line 382: remove "in silico"

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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: Yes: Jesús M Paramio

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.

A comprehensive molecular characterization of the 8q22.2 region reveals the prognostic relevance of OSR2 mRNA in muscle invasive bladder cancer

PONE-D-20-18560R2

Dear Dr. Erben,

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.

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Kind regards,

Francisco X. Real

Academic Editor

PLOS ONE

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