PONE-D-20-37466

Meta-Analysis illustrates possible role of lipopolysaccharide

(LPS)-induced tissue injury in nasopharyngeal carcinoma (NPC) pathogenesis

PLOS ONE

Dear Dr. Allen,

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Kind regards,

Srinivas Mummidi, D.V.M., 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: Yes

Reviewer #2: Partly

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

Reviewer #1: I Don't Know

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: Allen et al. carried out a meta-analysis pointing to a possible role of LPS-induced tissue injury in NPC pathogenesis. The authors also performed sophisticated IPA analysis. The statistical analysis was very poorly described. My criticisms are intended to increase the quality of the paper by presenting more detailed information regarding the statistical methods.

1) On page 3 at line 95 the authors stated “. . . standard and random fixed effects models for meta-analysis was used to produce effect sizes and meta p-values.” Prior to this statement the authors indicated that the STARGEO platform was used to conduct the meta-analysis and they also cited a previous paper from their group by Hadley et al. (2017) for more information (their reference 27). There are several problems in this regard that should be addressed:

A) It is unclear what “standard and random fixed effects models” means. Do the authors mean both effects together as in a mixed effects model or one or the other as dictated by the data across studies?

B) Citing the STARGEO platform does not suffice as a description of the meta-analysis approach. Inspection of the STARGEO website did not reveal any mention of exactly how the meta-analysis is carried out. Moreover, citing Hadley et al. (2017) for more information also does not suffice because that paper does not adequately describe the meta-analysis approach.

C) How is heterogeneity (if a random or mixed effects model was used) modeled?

D) How are effect sizes combined? The authors mentioned that the Dersimonian-Laird inverse variance method was used to study weight percentages. Was this used to combine effect sizes? How is error in the combined effect size estimated?

2) The authors quoted a number of p-values as being obtained from IPA analysis (e.g., page 4 at lines 125 to 127). Please describe the specifics of the test used by the IPA software to compute these p-values.

3) On page 5 at line 159, the authors mention the use of z-scores to measure the degree of activation (if positive) or inhibition (if negative). Is this a meta-analysis z-score? If so, the answer to 1D is relevant because it involves the standard error of the meta-analysis combined effect. If not, then please describe how this z-score is obtained.

Reviewer #2: This meta-analysis examined lipopolysaccharides (LPS) as an effector of nasopharyngeal carcinoma (NPC). The authors contrasted 111 tumor samples with 43 samples of normal epithelium. The study is original, including a very common (almost ubiquitous) risk: the exposure to LPS as a possible etiology for the disease. Nevertheless, the researchers have weak points that must be addressed to strengthen the manuscript:

1. In the introduction section, the authors briefly mentioned LPS. The authors must clarify mechanistic LPS paths to reach the nasopharynx and plausible physiopathology. Should NPC be related to local microbiota?

2. The analysis did not give any clue regarding the heterogeneity between samples in the STARGEO dataset. Can the authors compute the variance attributable to the tissue samples? Do they have access to clinical features of the tissue samples? The differentiation of the histological type of NPC is relevant due to its relation with the Epstein Barr virus. The NPC type can be a source of heterogeneity and limitation for the analysis.

3. It is essential to have a correct interpretation of the contrast between pathological vs healthy tissue. The authors should clarify which type of log-ratio function was computed. As this function is a binary form, some researchers divide the log2(NPC/control) by two, others no. If the authors used the straight log2 ratio, what was the rationale to use a cutoff point of 0.15? The highest value was less than 1, therefore the researchers worked with tiny effect sizes, meanwhile, large values on z-score came from the Ingenuity analysis.

4. The researchers used a standard (perhaps "fixed") and randomized meta-analysis. How did they adjust by confounders? Or they did not consider these adjustments? (Perhaps due to restriction from the original dataset). Please comment on the discussion.

5. The use of Etanercept or Infliximab can induce some cancers and both have constraints as therapeutic agents for tumors. The authors should comments in deep on the pros and cons.

6. A call for prudence regarding the reference on alcohol as a risk factor for NPC. The cited study gave weak evidence to this statement: It was a retrospective design, smoking was a big confounder (except for heavy drinkers) associated with alcohol consumption, and finally, the calculated HR showed a small effect. Looks that there are few commonalities between drinking molecular pathways (large effects) and the NPC (small support from the meta-analysis).

7. Finally, the described findings are common to many other pathologies and the proposed etiology, and the suggestion that there was a common risk factor between NPC and hepatic cirrhosis is alcohol does not have strong support.

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Reviewer #1: No

Reviewer #2: No

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Meta-Analysis illustrates possible role of lipopolysaccharide

(LPS)-induced tissue injury in nasopharyngeal carcinoma (NPC) pathogenesis

PONE-D-20-37466R1

Dear Dr. Allen,

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. Before your final submission, I would recommend addressing the suggestion of the second reviewer.

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,

Srinivas Mummidi, D.V.M., Ph.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

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: The clarifications incorporated in the revised version have made the paper stronger and acceptable for publication. Congratulations on the outstanding work!

Reviewer #2: The authors accomplished the requested clarifications in concepts like LPS, EBV-related samples. The authors made a good analysis of the potential oncogenicity of the recommended drugs.

The authors dropped the concept of alcohol pathogenesis because it did not have support according to the current scientific literature.

The authors have problems dealing with the sources of heterogeneity of the sample. This is concerning the scarce information on data confounders in the specific platform they used. Like any other gene repository, it has its constraints. Strictly speaking, this paper is not like a literature Meta-Analysis. The PRISMA must be modified for making this specific genetic analysis.

The authors addressed the questions I had. The paper can be published with a minor clarification on the difficulty to analyze the source of heterogeneity in the gene effects, this difficulty is inherent to the platform limitation on including confounders, and then con not be implement the used statistical methodology.

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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