Microbiome Sex-Related Diversity in Non-Muscle-Invasive Urothelial Bladder Cancer

Round 1

Reviewer 1 Report

This study reveals sex-specific differences in the bladder microbiome of patients diagnosed with non-muscle invasive bladder cancer (NMIBC), specifically highlighting a decrease in bacterial diversity and richness in high-grade tumors. Furthermore, distinct bacterial communities in high-grade tumor tissue were identified between males and females. Despite limitations such as sample size and control for patient-specific factors, this research offers potential insights into the role of microbiota in the diagnosis and treatment of bladder cancer. The methods used to analyze clinical specimens in this study are considered adequate. This reviewer requests additions and corrections on the following points.

(1)  Additional data presentation regarding the specific assignment of OTUs to genera or species is requested. The description in the text is limited and a description of comprehensive results on the composition of the bacterial communities in the samples is needed.

(2)  For NGS analysis, the number of reads, the percentage of sequence data lost during quality control and assembly, and the percentage of unattributed sequences during sequence identification should be explicitly reported. If the proportion of unattributed sequences is too high, it could affect the reliability of the identified bacterial communities. This detail is critical for assessing the robustness and reliability of the microbiome analysis results obtained from the study.

Author Response

Dear Reviewer,

Thank you for Your precise and comprehensive review,

1) We understand the importance of providing detailed information regarding the assignment of Operational Taxonomic Units (OTUs) to genera. Results on the composition of the bacterial communities in the samples has been described in text lines: 137 - 143. Additionally, the data has been presented graphically (Supplementary Figure 1).

2) Key metrics of NGS analysis such as the number of reads, and the percentage of unattributed sequences during sequence identification have been provided lines: 125 - 129

Reviewer 2 Report

The research manuscript investigates sex-related diversity in the bladder microbiome of non-muscle invasive bladder cancer patients, employing 16S NGS and shotgun metagenomics on urine, tumor, and healthy bladder mucosa samples. Results reveal differences in taxa richness between sexes and varying microbial compositions associated with tumor grade, providing insights into potential implications for diagnosis and personalized treatment strategies in bladder cancer management. The study effectively addresses important point it has in bladder cancer, making it a significant topic for consideration. However, there are notable knowledge gaps and structural changes in the paper that require attention. Comments to authors are given below-

1. Clarify Sample Collection Methodology:

• Provide detailed information on the criteria used for selecting participants and collecting samples. Were there any specific inclusion or exclusion criteria?
• Describe the methods used for obtaining midstream urine, bladder tumor samples, and healthy appearing bladder mucosa. Were any precautions taken to minimize contamination during sample collection? Also add all catalogue numbers of materials, regaents and assay kit to make it repetabel fro the researchers.

2. Elaborate on DNA Isolation and Sequencing Protocols:

• Provide a step-by-step description of the DNA isolation process using the QIAamp Viral RNA Mini Kit. Were any modifications made to the manufacturer's protocol?
• Detail the sequencing methods used for 16S NGS and shotgun metagenomics, including any quality control measures implemented to ensure accurate and reliable results.

3. Specify Bioinformatics Analysis Pipeline:

• Describe the bioinformatics pipeline used for metagenomic analysis, including software tools and parameters employed for taxonomic assignment and diversity estimation.
• Clarify how hypervariable fragments of the 16S rRNA gene were selected for analysis and why the Ion Torrent Personal Genome Machine platform was chosen for sequencing. Cite a report https://0-doi-org.brum.beds.ac.uk/10.1016/j.biopha.2023.114784 with the sentence ´ Recent advancements in molecular engineering have allowed researchers to explore the intricate connections between the host and the microbiome, which has been found to exist within variety of its vital systems´

4. Provide Detailed Results Summary:

• Present a comprehensive summary of the taxa discovered in each sample type (tumor, healthy bladder mucosa, midstream urine), including their relative abundance and distribution across different taxonomic levels.
• Highlight any notable trends or patterns observed in the microbial composition between sample types and across different cancer grades.

5. Discuss Statistical Analyses:

• Provide details on the statistical tests used to compare taxa richness and diversity between sexes and cancer grades. Specify the significance threshold used and any adjustments made for multiple comparisons.
• Consider discussing the statistical power of the analyses and whether any confounding variables were accounted for in the analysis.

6. Clarify Findings Regarding Sex-Specific Discrepancies:

• Elaborate on the observed differences in taxa richness between men and women, particularly regarding the implications of smaller taxa richness in women based on the Chao1 index.
• Discuss potential factors contributing to these sex-specific differences and their relevance to bladder cancer progression and treatment.

7. Interpret Results on Bacterial Diversity in Urine:

• Provide a detailed interpretation of the findings regarding bacterial diversity and richness in urine samples, particularly in relation to tumor grade. What implications do these findings have for the diagnosis and prognosis of non-muscle invasive bladder cancer?

8. Discuss Clinical Relevance of Sex-Specific Differences:

• Explore the clinical implications of the observed differences in relative abundance of microbial communities at the family level between sexes, specifically in high-grade tumors. How might these findings inform personalized treatment strategies for bladder cancer patients?

9. Address Potential Biases and Limitations:

·         Discuss potential biases in sample selection, DNA isolation, and sequencing methodology that may have influenced the results. Cite https://0-doi-org.brum.beds.ac.uk/10.1002/cmdc.202300328 with the sentence ´ Specific microbial signatures can be used for various purposes, such as predicting the response to therapeutics´

• Address any limitations of the study, such as sample size, patient heterogeneity, or the inability to establish causality due to the observational nature of the study.

10. Add Impact of Microbiome Composition on Bladder Cancer Progression:

• Discuss the potential mechanistic links between microbial composition and bladder cancer progression, considering existing literature on the role of the microbiome in cancer development and immune modulation.

11. Highlight Novel Insights and Contributions:

• Emphasize the novel insights provided by the study regarding sex-related diversity in the bladder microbiome of non-muscle invasive bladder cancer patients. How do these findings advance our understanding of the interplay between microbial communities and cancer biology?

12. Provide Reproducibility Information:

• Include detailed information on sample metadata, sequencing data, and bioinformatics workflows to facilitate reproducibility and further exploration of the findings by other researchers.

13. Consider Future Directions:

• Discuss potential avenues for future research based on the findings of the study, such as exploring the functional implications of microbial community differences or investigating the utility of microbiome-based biomarkers for bladder cancer diagnosis and prognosis.

14. Improve Clarity and Readability:

• Ensure that the abstract and manuscript are written in clear, concise language with well-structured sections. Most of figures are low resolution and it is hard to comprehend the labels, authors must add high resolution figures. Define any specialized terminology and abbreviations to enhance understanding for readers outside the field of microbiology and oncology.

Minor editing of English language required

Author Response

Dear Reviewer,

Thank you for Your precise and comprehensive review,

1) Clarification of sample collection methodology  - Section "4.1 Patients" has been revised and expanded.

2) Description of DNA isolation and sequencing process has been expanded - Section "4.2. Bacterial DNA isolation"

3) Bioinformatics pipeline used for metagenomic analysis has been clarified and explained in Section "4.4 Statistical and bioinformatic analysis". Suggested citation has been added - number - 31

4)  The comprehensive summary of taxa discovered in each sample type has been delineated in the text. Supplementary Table 1, Supplementary Table 2 and Supplementary Table 3 have been attached. (lines 169 - 187)

5) Statistical tests used to compare taxa richness and diversity between sexes and cancer grades as well as significance threshold used and adjustments  for multiple comparisons has been revised.  (lines: 479 - 484)

6) Implications of smaller taxa richness in women based and potential factors contributing to these sex-specific differences and their relevance to bladder cancer progression and treatment have been expanded lines: 221 - 249

7) Bacterial diversity and richness in urine samples, particularly in relation to tumor grade and possible implications of our findings  have been discussed lines: 273 - 285

8) Differences in relative abundance of microbial communities at the family level between sexes and possible clinical implications have been discussed lines: 286 - 324

9) Potential biases and limitations has been revised: lines 383 - 394. Suggested citation has been added - number 82

10) Potential mechanistic links between microbial composition and bladder cancer progression, considering existing literature on the role of the microbiome in cancer development and immune modulation have been discussed lines:  343 - 354

11) Novel insights provided by our study according to microbiome and cancer biology have been highlithed. lines: 402-408

12)  Section Methodohogy has been revised and expanded

13) Potential avenues for future research based on our findings such as microbiome-based biomarkers for bladder cancer diagnosis and prognosis has been added: lines 371 - 382.

14) To improve clarity and readability of manuscript high resolution figures has been added, accorodingly terminology and abbreviations have been revised

Round 2

Reviewer 1 Report

Reviewer 2 Report

accept