PONE-D-23-07350Diagnostic utility of quantitative analysis of microRNA in bile samples obtained during endoscopic retrograde cholangiopancreatography for malignant biliary stricturesPLOS ONE

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Additional Editor Comments:

This paper is well written and the data are well presented. I think this paper quality can be improved if authors can provide more evidence such as bioinformatics analysis from TCGA.

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Reviewers' comments:

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Reviewer #1: Partly

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: Kuniyoshi et al. evaluated the level of a large set of miRNAs in bile samples from PC (n=3), BTC (n=3) and control (n=3) patients and after qPCR validation of 6 miRNAs, they highlighted 4 significantly up-regulated. Finally, they suggest that the levels of miR-1275 and miR-6891-5p in bile may help in the diagnosis of PC and BTC after ERCP.

The manuscript is well written, with a considerable cohort of samples and the aim well defined. Moreover, although they are cautious with the conclusions, the utility is far from the clinical use.

I have some comments:

- Although significant, the differences between tumor patients and the controls is low talking about Ct. Which would be the threshold to consider that a blind bile sample analyzed has the miRNA high or low and therefore would be consider as malignant or benign?

- Although in Material&Methods it is described (page 7, lines 96-98), I am not sure I understood it correctly. Patients included in the study were diagnosed with a malignancy in the samples collected during the ERCP. According to Fig 1 there are control samples with high levels of miRNAs (comparable to tumor patients). Have you follow these patients to know whether they were later diagnosed with a tumor? This would really improve the utility you would anticipate the diagnosis.

- Authors have used miR-16 as housekeeping for the qPCR analysis and all the conclusions are based on those results. Although they justify that other authors used miR-16 as housekeeping, the other studies were done in plasma or tissue samples. Authors should include a supplementary figure reporting the miR-16 value for the 113 samples analysed to demonstrate that it is a good housekeeping.

- I found contradictory that in page 25- lines 380-383 authors said that there is one 1 paper about miRNA in bile for PC diagnosis (the ref 14), and then in page 29- lines448-451 they said that there are several reports on qPCR analysis of miRNAs in bile. The difference is the type of tumor? I mean that in these “several reports” they do not have PC patients? Or what? Moreover, references for these statement in page 29 are missing.

- I also find weird that contradictory results are found between this study and Lei et al in terms of miR-1275 levels in PC patients (REF 38). Of course as authors discussed I agree with the difference in the sample used (tissue vs bile), but normally there should not be these discrepancy. I think it would be important to analyzed some (n=10-15) pair samples (tissue-bile) in your hands.

- To perform the miRNA expression analysis 130 ng of total RNA from each sample was used. Does this mean that 45 ng of each of the 3 samples were pooled?

- Titles in Results section should be more informative than a simple “Patients” or “Quantitative PCR”.

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Reviewer #1: No

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Diagnostic utility of quantitative analysis of microRNA in bile samples obtained during endoscopic retrograde cholangiopancreatography for malignant biliary strictures

PONE-D-23-07350R1

Dear Dr. Imazu,

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,

Gopal Krishna Dhali, MBBS, MD, DM

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for the amendments.