Liquid Biopsy Analysis of the EV-Associated Micro-RNA Signature in Vulvar Carcinoma May Benefit Disease Diagnosis and Prognosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Please find my comments in the attached pdf.

Other issues: the whole paper provides only univariable analyses of the various parameters and their association with the miRNA expression. To ensure the robusteness of the conclusion, mutivariable analyses should be provided, especially for the survivial analyses

I look forward to your revised paper.

Best,

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please see attached document for comments and suggestions

General comment

The manuscript addresses a relevant topic and the overall approach is interesting. However, the lack of essential methodological and experimental results makes the study appear incomplete. In its current form, the manuscript does not fully support the scope suggested by the title. Addressing the points outlined below would significantly strengthen the work and improve its scientific rigor.

Major comments

1. Extracellular vesicle (EV) isolation and characterization

The manuscript lacks experimental evidence supporting the successful isolation of extracellular vesicles. No results are presented to demonstrate that the isolated particles correspond to vesicles within the expected size range. Please include appropriate characterization data (e.g., size distribution, concentration, or complementary validation techniques) to support that the isolated material corresponds to EVs.

2. Missing EV characterization and RNA-related results
   In addition to EV isolation, the manuscript lacks results related to EV characterization, RNA extraction from EVs and RNA synthesis

These steps are fundamental for the downstream analyses presented and should be supported by experimental results to ensure the robustness and reproducibility of the study.

3. Next-generation sequencing (NGS) analysis
   Given that NGS constitutes a central component of the study, it would be highly recommended to include a dedicated figure summarizing the NGS analysis workflow and key results. This would greatly improve clarity and help the reader understand how sequencing data supports the main conclusions.
4. Methodology for patient samples submitted to NGS
   The manuscript does not clearly describe the methodology followed for processing of patient samples prior to NGS analysis. Please include a detailed description of sample handling, processing, and preparation for sequencing to ensure methodological transparency.
5. Availability of NGS data

Studies of this nature are generally accompanied by public deposition of NGS data. The authors are encouraged to indicate whether the sequencing data will be made publicly available and, if so, provide the corresponding accession numbers.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer Report

This manuscript presents a well-designed and timely study investigating circulating EV-associated microRNAs as diagnostic and prognostic biomarkers in vulvar cancer. Given the rarity of this malignancy and the lack of validated non-invasive diagnostic tools, the work addresses a relevant clinical gap and provides novel, potentially translatable findings.

Major comments:

1. Focus of the Introduction
   The Introduction is informative; however, it could be further streamlined to focus more specifically on diagnostic challenges and biomarker development in vulvar cancer rather than on cancer-related diseases in general. This would improve clarity and strengthen alignment with the study objectives.

2. Interpretation of EV-associated miRNAs
   In the Discussion, it would be beneficial to more clearly distinguish between the value of EV-associated miRNAs as circulating biomarkers and claims related to their functional transfer or biological activity in recipient cells. While differential EV-miRNA profiles are highly informative diagnostically and prognostically, direct evidence for cytoplasmic delivery and functional engagement remains limited in the field and should therefore be interpreted cautiously.

3. Terminology and heterogeneity of extracellular vesicles
   The authors may consider briefly acknowledging that most EV preparations represent heterogeneous vesicle populations originating from different biogenetic pathways. As strict post-release discrimination between exosomes and other small EVs remains challenging, use of broader EV terminology may better reflect current consensus.

4. Use of miR-378a-3p as an internal control
   The selection of miR-378a-3p as an endogenous control based on empirical stability analysis is methodologically sound. Nevertheless, as this miRNA has been implicated in cancer-related biological processes in certain contexts, a short clarification emphasizing its use here strictly for normalization—supported by demonstrated expression stability—would enhance methodological transparency.

5. RNA quality control and potential cytoplasmic RNA contamination
   EV-associated RNA preparations are inherently susceptible to contamination by non-vesicular or cytoplasmic RNA. To further strengthen the methodological rigor, it would be valuable to explicitly report RNA quality control measures. In particular, confirmation of RNA integrity and size distribution using platforms such as a Bioanalyzer or comparable technologies would help ensure enrichment for small EV-associated RNAs and increase confidence in the robustness of the miRNA analyses.

6. Contextualization with prior exosomal miRNA liquid biopsy studies
   The Discussion could be further strengthened by referencing recent studies that have demonstrated the prognostic utility of exosomal miRNA profiling from blood samples across different cancer entities, particularly those employing stringent RNA quality control and validation strategies. Such work would help position the present findings within the rapidly expanding liquid biopsy literature.

Recommendation:

Overall, this manuscript represents a solid and original contribution with clear clinical relevance. The suggested revisions are minor, largely conceptual and methodological, and do not detract from the validity of the findings. I therefore recommend acceptance after minor revision.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for the detailed point by point responses.

My last remarks:

The kaplan meier curves need risk tables under them

Add the zenodo upload code in the data statement section.

Best,

Author Response

Please see attachement.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Please see attached file

Comments for author File: Comments.pdf

Author Response

Please see attachement.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Please see attached file

Comments for author File: Comments.pdf