Baseline Gut Microbiome and Metabolite Profiles Associate with Treatment Response in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

1. Summary

We thank the reviewers for taking the time to review this manuscript and for their constructive comments, that helped us to improve the quality and clarity of our work. We have revised the manuscript accordingly, and all changes are highlighted using track changes in the resubmitted version. Please find below the detailed responses (line numbering was applied based on the expanded edited version of the document).

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

-

Is the research design appropriate?

Yes

-

Are the methods adequately described?

Yes

-

Are the results clearly presented?

Can be improved

We have improved the clarity of figures and reporting of effect sizes and p values.

Are the conclusions supported by the results?

Can be improved

We have clarified the exploratory nature of the conclusions.

Are all figures and tables clear and well-presented?

Can be improved

We have improved the resolution of figures.

3. Point-by-point response to Comments and Suggestions for Authors

Reviewer 1

Comment 1: The cohort size is small that could restrict statistical power, particularly for metabolomic associations that do not withstand multiple-testing correction.

Response 1: Thank you for pointing this out. We fully agree that the limited cohort size represents a major limitation of the study and restricts statistical power. We have strengthened the discussion of this limitation and clarified the exploratory nature of our findings.

Specifically, we have revised the Discussion section (Page 17, Paragraph 4, Lines 482-488) as follows:

“Although several associations exhibited large effect sizes, suggesting potential biological relevance, most did not retain significance after rigorous correction for multiple testing. Importantly, some of these signals were partially preserved when explored in a more homogeneous TNBC sub-cohort. However, given the very limited sample size, these results should be considered exploratory, that warrant strong validation in a larger cohort.”

Also, in the Results section (Page 11, Lines 367-368) the following line was added:

“However, none of these associations remained statistically significant, after multiple testing adjustment.” 

Comment 2: In addition, residual confounding factors such as diet and lifestyle are not fully addressed. These limitations, however, are appropriately acknowledged by the authors and do not detract from the overall scientific merit of the work.

Response 2: We agree with this comment and acknowledge that confounding factors such as diet and lifestyle may have influenced the observed microbiome and metabolomic profiles. While collection of such data was not feasible within the scope of this study, we have clarified this limitation and emphasized that these confounders should be addressed in further research.

We have added the following line in the Discussion section (Page 18, Lines 582-584):

“The effect of residual confounding factors such as diet and unmeasured lifestyle variables, also cannot be excluded and should be addressed in further research.”

Comments 3: Furthermore, the resolution and clarity of some figures should be improved to ensure readability, particularly for detailed labels and graphical elements.

Response 3: We thank the reviewer for this suggestion. All figures have now been improved with higher resolution to improve readability. The revised figures are included in the resubmitted manuscript and supplementary materials.

Furthermore, alterations were made according to second reviewer’s comments regarding exploratory associations, NAC regimens as confounders and repetitiveness in Discussion section, please find the tracked changes in the updated manuscript. Also, we have revised the Results section and updated Table 2, Table 3, Table 5, Table 6 to ensure consistent reporting of effect sizes, test statistics and measures of statistical significance where applicable. In addition, several reporting inconsistencies and minor errors were corrected.

1. Summary

We thank the reviewers for taking the time to review this manuscript and for their constructive comments, that helped us to improve the quality and clarity of our work. We have revised the manuscript accordingly, and all changes are highlighted using track changes in the resubmitted version. Please find below the detailed responses (line numbering was applied based on the expanded edited version of the document).

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

-

Is the research design appropriate?

Can be improved

We have clarified limitations and exploratory nature of the study.

Are the methods adequately described?

Can be improved

We have clarified statistical reporting and potential confounders.

Are the results clearly presented?

Yes

-

Are the conclusions supported by the results?

Can be improved

We have clarified the exploratory nature of the conclusions.

Are all figures and tables clear and well-presented?

Yes

-

3. Point-by-point response to Comments and Suggestions for Authors

Reviewer 2:

Comments 1: Rephrase statements implying predictive or biomarker utility to emphasize exploratory associations rather than clinical applicability.

Response 1: We agree and have revised all statements suggesting predictive or biomarker utility to clearly emphasize the exploratory nature of the findings.

The Introduction (Page 3, Lines 104-108) was revised as follows:

“By examining microbial composition, diversity, metabolic pathways, and metabolite production, our aim was to explore microbial signatures associated with chemotherapy response, thereby contributing to the research of microbiome-based biomarker and po-tential therapeutic target development for precision medicine in breast cancer.”

The Discussion section (Page 18, Lines 568-576) was revised as follows:

“Our findings identify exploratory associations between gut microbial diversity metrics, specific taxa, and metabolite signatures, and response to neoadjuvant chemo-therapy (NAC) in breast cancer. While these results may support the potential use of gut microbiome profiling as a non-invasive biomarker for predicting response to NAC, they should be regarded as hypothesis-generating. Validation in larger, independent cohort is required to determine if microbiome biomarkers have potential relevance for patient stratification and personalized treatment approaches.”

Comments 2: NAC regimens vary substantially across molecular subtypes; this should be discussed more explicitly as a potential confounder beyond HR status adjustment.

Response 2: We agree and have added an explicit discussion of treatment heterogeneity as a potential confounder:

The following text was added to the Discussion (Page 16, Lines 467-473):

“Importantly, NAC regimens varied substantially across molecular subtypes in our cohort, in accordance with current treatment standards. This treatment heterogeneity represents another potential confounder beyond HR status alone and may have contributed to the observed differences in both observed microbial composition and metabolomic profiles and actual clinical treatment outcomes. This factor should therefore be carefully considered in the interpretation of our findings and addressed in future studies with more homogeneous distribution of treatment strategies.”

Comments 3: Explicitly label TNBC results as exploratory due to the very limited sample size and avoid overinterpretation.

Response 3: We fully agree and have revised all references to the TNBC sub-cohort to explicitly describe these analyses as exploratory:

For example, the Discussion section now states (Page 17, Lines 482-488):

“Although several associations exhibited large effect sizes, suggesting potential biological relevance, most did not retain significance after rigorous correction for multiple testing. Importantly, some of these signals were partially preserved when explored in a more homogeneous TNBC sub-cohort. However, given the very limited sample size, these results should be considered exploratory, that warrant strong validation in a larger cohort.”

Comments 4: Shorten repetitive microbiome background in the Discussion.

Response 4: We thank the reviewer for this suggestion. The Discussion section has been edited to reduce repetitive microbiome background information and to focus more directly on interpretation of the study findings. Redundant descriptions of SCFA-producing bacteria and general microbiome functions were removed or condensed (Page 17, Lines 502-557).

Comments 5: Improve consistency in reporting effect sizes vs p-values.

Response 5: We have revised the Results section and updated Table 2, Table 3, Table 5, Table 6 to ensure consistent reporting of effect sizes, test statistics and measures of statistical significance where applicable. In addition, several reporting inconsistencies and minor errors were corrected.