Title: Intestinal microbiota in adults with cholangiocarcinoma identifies the dysregulated
Blautia species and bile acid metabolic pathways
Version: 2 Date: 26 May 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
1. Small Sample Size and Power
The study includes only 23 CCA patients and 29 controls, which may limit statistical power
and generalizability. PJustifythe sample size (e.g., power analysis). Acknowledge this limitation
more explicitly in the discussion, particularly regarding its impact on model performance and
biomarker discovery.
2. Lack of Validation Cohort
Findings are based on a single cohort without independent validation.
Include an external validation dataset, even if from a public database (e.g., GMrepo or HMP), or
clearly state this limitation and suggest that future studies will validate findings in independent
populations.
3. Causal Interpretation
While the study is observational, some interpretations suggest causal relationships between
dysbiosis and CCA. Rephrase these sections to avoid overinterpretation. Emphasize correlation
rather than causation unless supported by functional assays or longitudinal data.
4. Dietary and Environmental Confounders
While the authors state participants had similar diets, no detailed dietary or lifestyle data is
reported.Include more information about dietary intake, BMI, alcohol consumption, and other
potential confounders, or at least acknowledge this limitation in the discussion.
5. Lack of In-depth Functional Validation
Functional predictions are based on metagenomic annotation (e.g., KO pathways) without
metabolomic or transcriptomic validation. Discuss this as a limitation and, if available,
incorporate fecal bile acid profiling or qPCR validation of key taxa.
6. Insufficient Clinical Characterization of CCA Subtypes
CCA includes diverse subtypes (intrahepatic vs extrahepatic); these are not stratified in the
analysis. Clarify whether subtypes were included and explore whether microbial signatures
differ by subtype. If not feasible, at least report the subtype distribution.
7. Language and Clarity
While largely well-written, some sentences are overly complex or passive. Minor language
polishing for clarity and conciseness, particularly in the Discussion.
8. Data Availability Statement
The manuscript states data are available upon request but does not provide accession numbers
or repository information. Deposit raw sequencing data in a public repository (e.g., NCBI SRA)
and include accession numbers in the manuscript.
Title: Intestinal microbiota in adults with cholangiocarcinoma identifies the dysregulated
Blautia species and bile acid metabolic pathways
Version: 2 Date: 26 May 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
1. Small Sample Size and Power
The study includes only 23 CCA patients and 29 controls, which may limit statistical power
and generalizability. PJustifythe sample size (e.g., power analysis). Acknowledge this limitation
more explicitly in the discussion, particularly regarding its impact on model performance and
biomarker discovery.
2. Lack of Validation Cohort
Findings are based on a single cohort without independent validation.
Include an external validation dataset, even if from a public database (e.g., GMrepo or HMP), or
clearly state this limitation and suggest that future studies will validate findings in independent
populations.
3. Causal Interpretation
While the study is observational, some interpretations suggest causal relationships between
dysbiosis and CCA. Rephrase these sections to avoid overinterpretation. Emphasize correlation
rather than causation unless supported by functional assays or longitudinal data.
4. Dietary and Environmental Confounders
While the authors state participants had similar diets, no detailed dietary or lifestyle data is
reported.Include more information about dietary intake, BMI, alcohol consumption, and other
potential confounders, or at least acknowledge this limitation in the discussion.
5. Lack of In-depth Functional Validation
Functional predictions are based on metagenomic annotation (e.g., KO pathways) without
metabolomic or transcriptomic validation. Discuss this as a limitation and, if available,
incorporate fecal bile acid profiling or qPCR validation of key taxa.
6. Insufficient Clinical Characterization of CCA Subtypes
CCA includes diverse subtypes (intrahepatic vs extrahepatic); these are not stratified in the
analysis. Clarify whether subtypes were included and explore whether microbial signatures
differ by subtype. If not feasible, at least report the subtype distribution.
7. Language and Clarity
While largely well-written, some sentences are overly complex or passive. Minor language
polishing for clarity and conciseness, particularly in the Discussion.
8. Data Availability Statement
The manuscript states data are available upon request but does not provide accession numbers
or repository information. Deposit raw sequencing data in a public repository (e.g., NCBI SRA)
and include accession numbers in the manuscript.
Please review this paper.
user
Title: Intestinal microbiota in adults with cholangiocarcinoma identifies the dysregulated
Blautia species and bile acid metabolic pathways
Version: 2 Date: 26 May 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
1. Small Sample Size and Power
The study includes only 23 CCA patients and 29 controls, which may limit statistical power
and generalizability. PJustifythe sample size (e.g., power analysis). Acknowledge this limitation
more explicitly in the discussion, particularly regarding its impact on model performance and
biomarker discovery.
2. Lack of Validation Cohort
Findings are based on a single cohort without independent validation.
Include an external validation dataset, even if from a public database (e.g., GMrepo or HMP), or
clearly state this limitation and suggest that future studies will validate findings in independent
populations.
3. Causal Interpretation
While the study is observational, some interpretations suggest causal relationships between
dysbiosis and CCA. Rephrase these sections to avoid overinterpretation. Emphasize correlation
rather than causation unless supported by functional assays or longitudinal data.
4. Dietary and Environmental Confounders
While the authors state participants had similar diets, no detailed dietary or lifestyle data is
reported.Include more information about dietary intake, BMI, alcohol consumption, and other
potential confounders, or at least acknowledge this limitation in the discussion.
5. Lack of In-depth Functional Validation
Functional predictions are based on metagenomic annotation (e.g., KO pathways) without
metabolomic or transcriptomic validation. Discuss this as a limitation and, if available,
incorporate fecal bile acid profiling or qPCR validation of key taxa.
6. Insufficient Clinical Characterization of CCA Subtypes
CCA includes diverse subtypes (intrahepatic vs extrahepatic); these are not stratified in the
analysis. Clarify whether subtypes were included and explore whether microbial signatures
differ by subtype. If not feasible, at least report the subtype distribution.
7. Language and Clarity
While largely well-written, some sentences are overly complex or passive. Minor language
polishing for clarity and conciseness, particularly in the Discussion.
8. Data Availability Statement
The manuscript states data are available upon request but does not provide accession numbers
or repository information. Deposit raw sequencing data in a public repository (e.g., NCBI SRA)
and include accession numbers in the manuscript.
assistant