PONE-D-20-20572

CHARACTERIZATION of biliary microbiota dysbiosis in extrahepatic cholangiocarcinoma

PLOS ONE

Dear Dr. Saab,

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.

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Kind regards,

Pradeep Dudeja

Academic Editor

PLOS ONE

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2. Thank you for your ethics statement : 'All patients were enrolled at Firoozgar Hospital (Teheran) for the present translational study and provided their informed consent to conduct microbial analyses on the biliary juice recovered during the ERCP (Ethics Committee approval under ID: IR.IUMS.REC.1397.115 of Iran university of medical sciences).'

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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: 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: No

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: 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: • Major comments:

i. Data is held at institute and “may” be accessible under specific request is not adhering to the journals requirements to make all data underlying findings available online, especially since this is large data that could potentially lead to incredible findings in the fields of microbiome and CCA research.

ii. He authors claim no financial support for this work, however, this manuscript demonstrates intensive work.

iii. Wald test is performed for the differential abudance analysis, but due to small sample size 30 of CCA, it is recommended that likelihood ratio test be performed.

iv. There is no information on the severity of CCA in patients (or early/late stage PSC/IBD/etc in cases of co-morbidities) and there is no information on ethnicity or race on patients studied. The authors should attempt to present this information for transparency.

v. Methods in this manuscript should be applied to established data sets, especially those cited in the discussion, to verify that similar results are found. This manuscript claims to characterize CCA bile microbiome but does not verify its findings on publicly available data, or own previously published data sets. Also, there is no comparison of microbiome from CCA-No to those of other biliary diseases or duodenal samples which may demonstrate the true novel findings of this manuscript and reduce suspicion of collection method contamination.

• Minor comments:

i. All figures say “Non” instead of “No” when indicating comorbidity existence in CCA or control patients.

ii. Differential abundance should be presented in linear modeling graph for data presentation to reduce confusion of the base mean and log fold change.

iii. The discussion does not speculate the on the role Anoxybacillus, Geobacillus, and Meiothermus play in the biliary milieu. Additionally. There is no mention of bile altering bacteria levels which would be a great addition to this manuscript since bile flow, bile composition and cholestasis are interrelated in CCA and other biliary diseases.

iv. Introduction and discussion should be edited. There is limited information on the impact of biliary microbiome dysbiosis in CCA development or how important dysbiosis of microbiome composition is in other diseases. The detection method of CCA is not of high importance for the findings in this manuscript unless it is to address any potential contamination from method of collection (which should be reserved for discussion and not introduction).

v. Figures should be combined (all figure 1 together) rather than spread out. The figure legends should be placed outside of manuscript flow since that interrupts the readers comprehension.

Reviewer #2: The current manuscript submitted by Massa Saab et al was designed to characterize biliary microbiota in consecutive patients with a histologically proven CCAe, and their results were compared to a series of patients with benign biliary affected diseases (PBBs), which were considered the control. Using 16S RNA sequencing has demonstrated significant differences in the composition of the biliary microbiota between the two populations, which could implicate CCA-associated dysbiosis in the biliary carcinogenesis. Overall, 32% of CCA and 22% of control patients displayed another associated disease, such as diabetes, pancreatitis, inflammatory bowel disease, or primary sclerosing cholangitis. Comparisons considered associated diseases. Principal coordinate analysis (PCoA) detected a significant disparity of the biliary microbiota composition between the CCAs and controls without an associated disease. Levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher in CCA patients’ biliary microbiota, without an associated disease, than in the controls. A specific CCA-related dysbiosis is identified as compared to controls independently from associated diseases, therefore a microorganism community might be involved in the CCA pathogenesis, as suggested by authors. It is an interesting paper and the experiments were properly performed. However, the mechanisms between biliary microbiota and CCA development/progression are undefined, and the data on biliary carcinogenesis are descriptive. Additionally isolated CCA cells need to be better characterized, to make sure that they display the CCA phenotypes associated with biliary microbiota as expected. Moreover, the data is too preliminary and lack of the significance and mechanistic studies to define the specific biliary microbiota associated cancer signaling pathways.

Comments:

1. The major issue of the current manuscript is the mechanisms between biliary microbiota and CCA development/progression are undefined, and the data on biliary carcinogenesis are descriptive. Actually several manuscripts have proved the associations among gut microbiota, bile acid metabolism and cytokines in cholangiocarcinoma development and progression. Therefore further functional characterizations for CCA development and progression are need for the current manuscript.

2. The isolated/primary CCA cells from human CCA tissues were not well characterized and the authors failed to show the CCA phenotypes associated with biliary microbiota as expected. Therefore their cancer associated biliary microbiota phenotypes are questionable.

3. There is some confusion in the manuscript to prove that the levels of Bacteroides, Geobacillus, Meiothermus, and Anoxybacillus genera were significantly higher in CCA patients’ biliary microbiota, without an associated disease, than in the controls. More detailed mechanistic studies should be carried out to define the specific mechanisms of malignant transformation and their down stream signaling mechanisms.

4. The rationale should be provided in introduction. Summary is missing in the discussion section and the conclusion needs to be detailed.

5. Statistic analysis should be included in Figure 1 2 with some detailed CCA cancer stage and survival information.

6. There are not enough data in the main manuscript. Therefore the supplementary information should be moved to the main text.

7. English writing skills need to be improved. The format of the fonts and some technical terms should be consistent.

8. The discussion needs to be modified and focused. Some of the descriptions are disconnected from the central focus in the discussion section.

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Reviewer #1: No

Reviewer #2: No

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CHARACTERIZATION of biliary microbiota dysbiosis in extrahepatic cholangiocarcinoma

PONE-D-20-20572R1

Dear Dr. Saab,

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,

Pradeep Dudeja

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

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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

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 all the comments and the manuscript is suitable for publication. The authors have added important data and figures

Reviewer #2: The authors have addressed almost all the concerns from the previous review cycle and the revised manuscript has been significantly improved.

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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