-->PONE-D-26-08926-->-->CREBBP and KRAS codon 13 polymorphisms define the genetic landscape of intrahepatic cholangiocarcinoma in a Thai population: A comparative cohort study-->-->PLOS One

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Additional Editor Comments:

The background on the RAS oncogenes in the introduction to be improved better and the potential articles are as follows: PMID: 31255772; PMID: 27102293; PMID: 22240207.

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: Partly

Reviewer #2: Yes

Reviewer #3: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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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

Reviewer #3: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: In this manuscript, the authors investigate genetic polymorphisms associated with intrahepatic cholangiocarcinoma (iCCA) in a Thai cohort. The study includes three groups: 112 iCCA patients, 60 individuals infected with Opisthorchis viverrini (OV), and 156 healthy controls. Using PCR-RFLP and sequencing approaches, the authors examine variants in several cancer-related genes (including TP53, KRAS, CREBBP, CDKN2A, IDH1, and GZMB) as well as oxidative stress pathway genes (KEAP1 and NFE2L2). The authors report that CREBBP polymorphisms are significantly enriched in iCCA and associated with increased cancer risk, and that KRAS codon 13 variants are observed only in iCCA patients. The topic is relevant. Further improvements are suggested as follow:

1. It is unclear whether they are germline polymophisms or tumor specific mutations. The study appears to rely on DNA extracted from blood samples, suggesting the variants are germline polymorphisms. However, the manuscript frequently interprets these variants as tumor drivers and discusses them in the context of tumor biology. The authors need to clearly distinguish between germline polymorphisms and somatic mutations throughout the manuscript.

2. Claiming the variants as drivers without experimental validation is another major concern. This study, as it is, is observational and demonstrates association, rather than causation.

3. The statistical analysis relies primarily on chi-square tests and odds ratios. More rigorous statistical tests and multiple testing correlation should be included.

Reviewer #2: This article looks good to me overall. In this study, the author focuses on iCCA, a certain liver cancer type endangering Thai population, performed retrospective molecular analysis, analyzed status of TP53, KRAS, CREBBP, CDKN2A, IDH1, GZMB among three groups: control/OV infected/iCCA, drew some useful conclusions, and provided solid data to help doctors to have a fuller image of genetic landscape of Thai iCCA population.

The author has already listed limitations of this study, which covers most of the problems I discovered from this article. Apart from that, from my point of view, I found that some improvements are still required to make this research more complete and meaningful.

1.An important fact is that, given all the detection were conducted with ctDNA from serum, there’s a chance that diseases from other organs(if exists) might disrupt the results of analysis. It seems unavoidable at this moment, but could you please share your opinions on how to overcome this problem with potential solutions in the discussion section?

2.The genes selected as candidates are not well-organized. For example, G6PD was introduced in the beginning as a regulator for metabolism; however, no G6PD was detected in this article. As for other genes selected, some are unable to represent the whole story, like merely GZMB is not enough to reflect tumor immune microenvironment changes. Try to hire more factors to support the theory if allowed.

3.In the discussion section, I figured that more talk should be focused on data obtained from this current study, instead of concluding existing information on the genes tested in this study. For example, what can be derived from this study and be applied in clinical is the most important. It’d be better to put more words into illustrating how this study can benefit iCCA prevention, diagnosis, treatment and prognosis.

4.Since OV infected patient is in the transition phase, how to prevent OV infected patients from developing into iCCA is as crucial. With present data, could you please try to analyze the genetic pattern between OV and iCCA groups to see if you can discover any indicator for prediction? For example, I noticed that KRAS mutation is absent in OV group, any speculation on this phenomenon?

5.Given CREBBP combined with KRAS codon 13 polymorphisms has been identified as the most significantly enriched alteration among iCCA patients, It would be better to further verify this idea. For example, can you maybe collect CREBBP and KRAS mutant patients from online database(like TCGA), and to see whether this mutation predicts bad prognosis?

6.As described in this article, CREBBP mutation might lead to dysregulated epigenetic alterations, therefore causing silence of some tumor suppressing genes. Could you maybe identify potential CREBBP affected tumor suppressing genes and test the expression levels with sample at hands to further confirm this idea?

Despite the limitations listed above, this study is still providing us useful information on genetic landscape of iCCA among Thai population, making it a critical reference for further biomarker identification.

Reviewer #3: The authors use a targeted mutation panel to investigate the determinants of intra-hepatic cholangiocarcinoma in the presence of infection. The work represents a novel contribution to the field since the reported associations between CREBBP and KRAS genetics in Thai populations, which have a disproportionately large incidence rate of the disease, have not been previously reported on (although mutations in KRAS have been identified as a driver gene in Chinese - https://www.nature.com/articles/ncomms6696). The authors stress the difficulties of obtaining sufficient numbers of subjects to participate in the study resulting in small sample sizes for case-control and case-case comparisons. The overall conclusion is that 13 variants in two genes describe the mutational landscape of the disease / normal population. However, given the aforementioned small sample sizes, and demographic inequalities between test subjects assigned to study groups, I am unconvinced that these data support the conclusions.

In particular, their may exist many other mutations on a genome wide scale that stratify these groups in a similar, if not better manner. Considering this fact, the authors might consider ammending their grand title to more closely reflect the findings from the study. Given the sample size issues, I am not convinced the statistical tests performed here are sufficient to test the hypothesis, that the variant frequencies significantly differ between groups. For example, in the demographics section, and text, the bias in age and sexes between test subjects in the study groups is emphasized, however, statistical methods that allow modelling and testing under these conditions have not been adopted (see multivariate logistic regression, Mantel-Haenszel tests , Stratified Fisher's Exact Test or conditional logistic regression as alternatives). Since the control population is limited to females only, it would seem only appropriate to report the trend in females, or to acquire approximated estimates of these frequencies in males from one of the South East Asian BioBanks.

Given the importance of studying these mutation profiles in under-represented or difficult to obtain population cohorts, I'd consider accepting the paper after making some MAJOR changes. First, some attempt to compare / contextualise the results/mutation frequencies among other South East Asian populations (chinese/japanese for example) and published studies on iCC. I'd also like the statistical tests to be re-evaluated considering age and where possible, sex as factors, and a power calculation to exemplify why these studies can only be used as guidelines or as hypothesis generators rather than considered hard evidence. [ The raw frequency differences are large, hence I anticipate the existing trends would be preserved under different test methodologies.] The methods section simply states frequencies were generated as input to chi-square or Fishers Exact test. Again, given the sample sizes, chi-square test results would seem inappropriate in this case.

The results and discussion sections should then include information around the biology eluded to from the mutation profiling results, and the observed population differences. For example., are the rates of OV infection similar across the asian populations ... and are the particular KRAS mutations preserved. Can the results be used to make a population specific signature ?

I feel with a few additions, the work would be significantly elevated to accepted status.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Targeted Analysis of KRAS and CREBBP Mutations Uncovers a Potential Population-Specific Signature in Thai Patients with Liver Fluke-Associated Cholangiocarcinoma

PONE-D-26-08926R1

Dear Dr. Na-Bangchang,

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,

Avaniyapuram Kannan Murugan, M.Phil., 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

Reviewer #3: 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: (No Response)

Reviewer #3: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: (No Response)

Reviewer #3: Yes

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-->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: (No Response)

Reviewer #3: 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: (No Response)

Reviewer #3: Yes

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-->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 revised manuscript adequently addressed the concerns raised from the previous version of submission, with additional analysis, discussions, and justification. The reviewer believes the data in the current format largely support the conclusions, delivering genuinely interesting findings to the field of cholangiocarcinoma. Acceptance is suggested.

Reviewer #2: (No Response)

Reviewer #3: I find the revised submission a substantially improved work and recommend accepting the manuscript for publication.

I do not have any further improvements/clarification requests to add at this time.

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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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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