PONE-D-20-34162

Cytomegalovirus infection in malignant pleural mesothelioma

PLOS ONE

Dear Dr. Hunter-Schlichting,

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.

Two expert reviewers have commented on your article, and their feedback is appended below.  Overall the reviewers felt that results were publication-worthy and of interest to the scientific community, but additional information is needed before it can be published.  Three major issues need to be addressed:  1. greater detail on experimental methods overall, 2. improved presentation and discussion of DNAemia results, and 3. expanded discussion with respect to HCMV biology.  Specifically, more details on the methods is required, as noted by reviewer 1, particularly clarification on the sample preparation and analysis of HCMV serostatus.  In addition, both reviewers ask for more information and clarification on the DNAemia status, both in the results (Fig 1) and discussion sections, particularly in comparison to the general population.   Finally, the reviewers include several comments about typos, awkward working, or areas where enhanced or clarified discussion of the results is necessary.  Your revised manuscript should effectively address each of the the reviewer's comments below.

Please submit your revised manuscript by Feb 06 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Juliet V Spencer, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional 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

2. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants in both cohorts. Please ensure you have provided sufficient details to replicate the analyses such as: a) the recruitment date range (month and year), b) a description of how participants were recruited.

3. Please provide a sample size and power calculation in the Methods, or discuss the reasons for not performing one before study initiation.

4. Please include additional information regarding the survey or questionnaire used in the study and ensure that you have provided sufficient details that others could replicate the analyses. For instance, if you developed a questionnaire as part of this study and it is not under a copyright more restrictive than CC-BY, please include a copy, in both the original language and English, as Supporting Information.

5. Please note that PLOS does not permit references to “data not shown.” Authors should provide the relevant data within the manuscript, the Supporting Information files, or in a public repository. If the data are not a core part of the research study being presented, we ask that authors remove any references to these data.

6.Thank you for stating the following in the Acknowledgments Section of your manuscript:

"This work was supported in part by NIH P30 CA77598 utilizing the Masonic Cancer Center,

University of Minnesota shared resources and the sponsored by the R35 grant (00052643)"

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

 "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

7. Please include a caption for figure 1.

[Note: HTML markup is below. 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: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

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: Yes

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: Hunter-Schlichting and co-authors present an interesting preliminary study assessing the incident of HCMV infection in malignant pleural mesothelioma patients. While the study is small, the authors are reasonable in their assessment of the data and their conclusions, and fully outline the limitations to the patient cohort and reasonable analysis thereof. This is an interesting preliminary study, and the authors properly address all aspects of the patient cohort, analyses, and conclusions, with an emphasis on future study. This paper provides a useful addition to the literature and is scientifically sound. To that end, I have only a few minor comments:

1) The finding that more patients have positive DNAemia than are seropositive is surprising and quite strong even using a smaller study sample size. This finding could be highlighted more as this would be a very interesting follow up.

2) On the same note as point 1 above, the conclusions/discussion regarding a latent HCMV infection contributing to disease are not supported by the data presented here. The data suggests - both by higher DNAemia patients (although any detectable DNA typically suggests a non-latent viral state in patients) trending towards increased mortality, and the by the lack of detectable IgG in DNA+ patients; that an initial or reactivated viral infection is more likely associated. The authors do address this in the Discussion, but the emphasis on latent infection is overstated.

3) Why was the DNAemia reporting level changed between patient cohorts (page 7)? Serum was analyzed for DNA copies and grouped into three categories (undetected, DNA+, active infection (greater than 1000 copies/mL) - so negative, positive, active. Yet tissue samples, although run in the same manner were categorized as only negative or positive. Active infection could be defined using similar criteria here as in serum (i.e. greater than 1000 copies per ug tissue for example)? Or discuss limitations as to why not? This is relevant to the Discussion as well (third paragraph, last sentence), which states that "viremic levels (>100 copies/mL serum) were specific to MPM" which is not supported by the data in this cohort, although I agree that it does warrant further study in a larger cohort.

4) Additional information should be included as to whether patients were assessed for treatment status / immunosuppression at the time of sampling (table 1).

5) The prevalence of HCMV in these samples is a key piece of data. The data presented on page 8 (Table 1) however suggests that prevalence of HCMV is comparable to the general population and DNAemia is similar in similar sampled tissues (metastatic lung cancer, data not shown). This is a very key point to discuss since this could suggest that the findings of HCMV prevalence are no different than background population levels. This could be clarified in the Discussion where a couple of statements (i.e. third sentence of paragraph one - "suggest HCMV infection may be an unrecognized co-morbidity") are overstated.

6) There are some minor typos/formatting/missing punctuation, including in Table 2.

Reviewer #2: In the article entitled, “Cytomegalovirus infection in malignant pleural mesothelioma”, by Hunter-Schlichting, et al, the authors describe the prevalence of CMV in patients with malignant pleural mesothelioma (MPM). To the authors’ and this reviewer’s knowledge, this is the first investigation into the potential connection between CMV and MPM. Overall, the manuscript could be clarified in some places, which should not prove overly cumbersome but will undoubtedly improve readability. Also, some of the data should be more clearly presented (e.g. Fig 1), so the reader can appropriately interpret the data herein. Suggestions are provided below for the authors’ consideration:

Major:

1. Clarity to the methods is warranted.

a. Study population: how long between the time of consent and blood sampling?

b. Serum biomarkers: Stating “…serology was performed on the Roche e411 Analyzer…”, with no additional information as to how the samples were prepared, etc. is definitely not enough information. Similarly, how were the tissue samples prepared? In the same paragraph, what does “gBa region” refer to? Is it not just “gB”? Was an uninfected control used to determine baseline for the CMV DNA qPCR? This would allow one to determine if the primers detect background (in other words, it allows one to determine the baseline). This could also be why there was no association between IgG and DNAemia (which the authors describe as ‘unexpected’ in their Discussion, p13).

c. HCMV DNA in tissue: Were the normal tissue samples also freshly frozen? What were the methods used to extract the DNA from tissue?

d. Lung Cancer BioBank samples: “Blood was collected at specified time points during a patient’s treatment course” - Did all patients receive the same treatment course, and did treatment regimens differ between the two study sites? What were these specified time points?

2. Figure 1 is hard to evaluate. The lines are not labeled, making it difficult to interpret.

3. p11 – “HCMV…is highly prevalent in the global population and nearly ubiquitous in immunocompromised populations” – I would argue this is misleading, especially since the authors use the term ‘immunocompromised’ to describe transplant patients as well (who are technically immunosuppressed). Likewise, “HCMV infection may be a biomarker that identifies patients who are the most immune-compromised…” is misleading.

4. p12 – “…those with the longest survival had low level DNAemia” – Isn’t this not completely true when adjusted for age? Similarly, p14 (Conclusion section) – “…MPM patients with a negative or low level HCMV infection were greater than those with a high level HCM infection” – isn’t this not the case when adjusted for age? (Also, minor, but this last sentence should read “…HCMV infection”).

Minor:

1. For ease in reviewing, it would help a ton to have line numbers.

2. Various points in the manuscript should be clarified:

a. What is meant by “HCMV…has been observed as viremia in various cancer patients and the tumor environment in different cancers” (p4)?

b. p4 – “>50% of the population infected by ages 75-80.” This should be double-checked. It’s more like >50% by 40 years of age.

c. What is meant by “Less is known about the experience of HCMV viremia in cancer patients…” (p4-5)?

d. The data re: 21 normal pleura samples – this is not included in the tables is it?

e. p10, p11, p12 – “HCMV biomarkers” should actually be defined.

f. p12 – what is meant by a “robust NK cell phenotype”?

3. Figures should be presented in order of appearance. As is, the supplemental figures need to be reversed. Also, I would suggest the authors put the supplemental figures in the main body of the manuscript as opposed to including them as supplemental figures.

4. The authors should include an overall “take-home” message for their findings at the end of the results section. As is, this section just drops off, leaving the reader wondering what the overall take is.

5. p11 – The authors state that HCMV infection could put MPM patients at risk for HCMV-associated pneumonia. Do any of these patients the authors evaluated present with this symptom?

6. p12 – I appreciate the authors’ idea that pleural tissue may serve as a site of latency. But it may also be worth noting that infiltrating, latently infected monocytes that are recruited to the ‘injured’ pleura could be a source of reactivating virus in this tissue. Just a thought.

**********

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.

Cytomegalovirus infection in malignant pleural mesothelioma

PONE-D-20-34162R1

Dear Dr. Hunter-Schlichting,

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Juliet V Spencer, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: