Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reviews the diagnostic and prognostic potential of microRNAs (miRNAs) in breast cancer, with emphasis on circulating/exosomal miRNAs within a liquid biopsy framework. The topic is timely and clinically relevant, and the review highlights well-known candidates (e.g., miR-21, miR-155, miR-200 family) and important translational barriers (pre-analytical variability, RNA isolation, normalization). The narrative is generally comprehensive in scope, and the manuscript provides useful background sections on miRNA biology and mechanisms.

However, the current version has major methodological and reporting limitations that substantially affect clinical reliability and reproducibility of the conclusions. The Materials and Methods section lacks sufficient detail for a PRISMA-aligned review, the study selection appears very limited (15 papers from >7000 hits) without clear justification, and several sections contain language/formatting issues and text-encoding artifacts. I recommend major revision focusing on methodological transparency, evidence grading, and careful editing.

 

Major comments

-The authors state PRISMA 2020 was followed, but essential elements are missing: full search strategy for each database (exact search strings), dates of searches, screening process, number of reviewers, conflict resolution, and inclusion/exclusion criteria.

-Please clearly define whether this is a systematic review, scoping review, or narrative review with systematic search. If it is not a fully systematic review, PRISMA language should be revised accordingly.

-From 7415 results, selecting only 15 studies suggests very strict criteria, but these criteria are not described.

-Please provide explicit criteria (population, sample type: plasma/serum/exosome; assay platform: qPCR/NGS/ddPCR; endpoints: diagnosis/prognosis/therapy response; study design; minimum cohort size; validation requirement; etc.).

-Consider expanding the included set or explaining why key evidence (meta-analyses, larger multi-cohort validations) was excluded.

-For diagnostic/prognostic biomarker literature, study quality and bias (selection bias, case-control designs, overfitting, lack of external validation, pre-analytical confounding like hemolysis) strongly influence conclusions.

-Please include a structured appraisal (e.g., QUADAS-2 for diagnostic accuracy studies; QUIPS for prognostic factors; or at minimum a transparent qualitative assessment).

-Several statements report very high AUC/sensitivity/specificity for miRNA panels. These metrics often come from case-control discovery cohorts and can be inflated without proper external validation and calibration.

-Please consistently state whether results are from discovery vs. validation cohorts; whether analyses were blinded; and whether panels were externally validated.

-Where possible, provide ranges across studies and highlight variability and failure to replicate.

-A dedicated table summarizing pre-analytical variables and recommended controls would improve clinical reliability.

-The manuscript’s section numbering becomes inconsistent (e.g., “5. Conclusions” appears after “11. Future Directions”). Please correct section numbering to match journal style.

-Ensure that Tables/Figures are cited in order and are present with appropriate captions.

-There are multiple typographical issues and text-encoding artifacts (e.g., “u􀄴erly”, “pa􀄴ern”, “a􀄴ractive”, “se􀄴ings”), which must be corrected throughout.

-Some sentences are awkward or repetitive; streamline and reduce redundancy, especially in Sections 8–11.

Author Response

Thank you for the generosity of your time and your valuable comments and recommendations. They have allowed us to significantly improve our manuscript.

Comment 1: The authors state PRISMA 2020 was followed, but essential elements are missing: full search strategy for each database (exact search strings), dates of searches, screening process, number of reviewers, conflict resolution, and inclusion/exclusion criteria.

Response 1: Thank you for this observation, indeed our methods do not contain sufficient detail. The methods section has been updated. 

Comment 2: Please clearly define whether this is a systematic review, scoping review, or narrative review with systematic search. If it is not a fully systematic review, PRISMA language should be revised accordingly.

Response 2: This was a scoping review, however we used the PRISMA guidelines to ensure a more rigorous selection process. We have updated the methods section. 

Comment 3: From 7415 results, selecting only 15 studies suggests very strict criteria, but these criteria are not described.

Response 3: The methods section has been updated.

Comment 4: Please provide explicit criteria (population, sample type: plasma/serum/exosome; assay platform: qPCR/NGS/ddPCR; endpoints: diagnosis/prognosis/therapy response; study design; minimum cohort size; validation requirement; etc.).

Response 4: The methods section has been updated.

Comment 5: Consider expanding the included set or explaining why key evidence (meta-analyses, larger multi-cohort validations) was excluded.

Response 5: Results of meta-analyses and other larger studies have been mentioned and referenced in the main text through-out, but they were not included in the selection of individual studies.

Comment 6: For diagnostic/prognostic biomarker literature, study quality and bias (selection bias, case-control designs, overfitting, lack of external validation, pre-analytical confounding like hemolysis) strongly influence conclusions.

Response 6: The methods section has been updated.

Comment 7: Please include a structured appraisal (e.g., QUADAS-2 for diagnostic accuracy studies; QUIPS for prognostic factors; or at minimum a transparent qualitative assessment).

Response 7: Since this was a scoping review, this step was not performed at the moment of paper review and it was also not possible to perform and add at this time, due to lack of time in the revision process.

Comment 8: Several statements report very high AUC/sensitivity/specificity for miRNA panels. These metrics often come from case-control discovery cohorts and can be inflated without proper external validation and calibration.

Response 8: The methods section has been updated.

Comment 9: Please consistently state whether results are from discovery vs. validation cohorts; whether analyses were blinded; and whether panels were externally validated.

Response 9: The methods section has been updated.

Comment 10: Where possible, provide ranges across studies and highlight variability and failure to replicate.

Response 10: The methods section has been updated.

Comment 11: A dedicated table summarizing pre-analytical variables and recommended controls would improve clinical reliability.

Response 11: The methods section has been updated.

Comment 12: The manuscript’s section numbering becomes inconsistent (e.g., “5. Conclusions” appears after “11. Future Directions”). Please correct section numbering to match journal style.

Response 12: This has been corrected.

Comment 13: Ensure that Tables/Figures are cited in order and are present with appropriate captions.

Response 13: This has been corrected.

Comment 14: There are multiple typographical issues and text-encoding artifacts (e.g., “u?erly”, “pa?ern”, “a?ractive”, “se?ings”), which must be corrected throughout.

Response 14: The manuscript has been review and these issues corrected.

Comment 15: Some sentences are awkward or repetitive; streamline and reduce redundancy, especially in Sections 8–11.

Response 15: The text has been reviewed and corrected.

Reviewer 2 Report

Comments and Suggestions for Authors Biomedicines/MDPI Jan 18 This report relates to use of what are termed ‘miRNA biomarkers’ for assessing the success of  breast cancer therapy. The report relates to an analysis of the current literature and points out potential pitfalls in data acquisition. A total of 15 literature reports were selected for the review. It is indicated (Table 2) that miRNA can have varying roles, ranging from ‘promotes resistance’ to ‘provides resistance’. It is noted that the proposed tests might be used to monitor the success of therapy. The authors also note (Section 10) that protocols can vary, that standard methods have yet to be identified, that there are many variables in miRNA detection and that tumor heterogeneity can alter results.  It would be helpful if this report was to be evaluated by someone currently involved in the treatment of breast cancer. What may appear to be plausible to someone who is not involved, could be less plausible to someone who is. In the Conclusion, the authors first claim that microRNAs provide ‘significant advantages’, but then indicate that ‘large-scale validation studies’ are needed. What should be indicated is that such studies could be useful but the evidence is apparently not yet there. It is proposed that biopsies can be inadequate because of tumor heterogeneity. Is is known whether ‘blood-based miRNA assays’ could also be inadequate if miRNAs are not all shed into the circulating blood?  Literature reviews are useful for readers who are thereby spared the task of doing individual reviews. Material just before Section 8 identifies many possible elements that can provide misleading data. It is proposed that ‘international consortia and multi-center trials’ are needed. Who is going to promote and support such studies?     

Author Response

We greatly appreciate your comments and your valuable time in reviewing our work. We have taken your recommendations into account and have made changes to the manuscript.

Comment 1: In the Conclusion, the authors first claim that microRNAs provide ‘significant advantages’, but then indicate that ‘large-scale validation studies’ are needed. What should be indicated is that such studies could be useful but the evidence is apparently not yet there. It is proposed that biopsies can be inadequate because of tumor heterogeneity. Is is known whether ‘blood-based miRNA assays’ could also be inadequate if miRNAs are not all shed into the circulating blood? 

Response 1:

A significant barrier to the clinical reliability of liquid biopsies is the potential for shedding discordance. While blood-based assays are often proposed to overcome the limitations of tissue heterogeneity, they are subject to their own physiological constraints. Specifically, the presence of a miRNA in the tumor does not guarantee its presence in the circulation; miRNAs are released into the blood through selective active secretion (via exosomes) or passive leakage (via necrosis/apoptosis). If a high-priority diagnostic miRNA is not efficiently shed, or if its concentration falls below the limit of detection during early-stage oncogenesis, a blood-based assay may yield a false-negative result. Consequently, the 'circulating secretome' may provide only a partial and potentially biased snapshot of the total tumor burden.

Comment 2: Literature reviews are useful for readers who are thereby spared the task of doing individual reviews. Material just before Section 8 identifies many possible elements that can provide misleading data. It is proposed that ‘international consortia and multi-center trials’ are needed. Who is going to promote and support such studies?     

Response 2: 

We agree that calling for large-scale trials is only useful if the pathways for their support are identified. Such initiatives are typically spearheaded and funded by three key entities: governmental and transnational agencies, public-private partnerships or non-profit foundations.

Reviewer 3 Report

Comments and Suggestions for Authors

The title matches the content.

In the introduction. Please,  indicate what other reviews have been given on this topic and how your work differs from them.

In the introduction, add a description of the increased MicroRNA level in other cancers.

The purpose should be more specific, with specific treatment methods indicated. - ”in every aspect of breast cancer management”. In addition, this review demonstrates the features of mRNA in the development of etiopathogenic factors in patients with breast cancer and does not study the features of this biomarker for the choice of treatment method.

Please indicate specifically what type of review was presented in the Materials and Methods section.

“No formal protocol was registered for this review” Why was the protocol not registered in advance to eliminate bias?

In my opinion, this review is narrative in nature. If we are talking about a systematic review, then the requirements are completely different.

In Table 15, add a reference before each author and a column with the research results.

In table 2, indicate the reference directly in the table.

 

“Future Directions” and  “Limitations and Challenges “ . These sections are disproportionately long and can be shortened. I suggest the authors provide data on the points.

 

The sections are written in a very informative and original format. However, the manuscript lacks illustrations to help readers understand such a complex topic.

I think this topic is very relevant, especially when diagnosing early forms of breast cancer. A section dedicated to this issue needs to be added.

Conclusions should be drawn from the results obtained, with brief recommendations for insufficiently studied aspects and for further research in this area.

The list of references is not formatted according to the journal guidelines.

Author Response

Thank you for your thorough analysis and your valuable comments. You have generously helped and allowed us to improve our work.

Comment 1: In the introduction. Please,  indicate what other reviews have been given on this topic and how your work differs from them.

Response 1: We added the requested information in the Introduction. 

 

Comment 2: In the introduction, add a description of the increased MicroRNA level in other cancers.

Response 2: We added the requested information in the Introduction. 

Comment 3: The purpose should be more specific, with specific treatment methods indicated. - ”in every aspect of breast cancer management”. In addition, this review demonstrates the features of mRNA in the development of etiopathogenic factors in patients with breast cancer and does not study the features of this biomarker for the choice of treatment method.

Response 3: This review aimed at integrating data regarding microRNAs in breast cancer (from diagnostic potential to clinical reliability) and we were not looking for specific treatment-related miRNA changes. The methods section was updated. 

Comment 4: Please indicate specifically what type of review was presented in the Materials and Methods section.

Response 4: This was a scoping review. This aspect was added to the methods section.

 

Comment 5: “No formal protocol was registered for this review” Why was the protocol not registered in advance to eliminate bias?

Response 5: Since this was a scoping review, registration was not necessary.

Comment 6: In Table 15, add a reference before each author and a column with the research results.

Response 6: These changes have been added.

Comment 7: In table 2, indicate the reference directly in the table.

Response 7: We have added this in the main text.

 

Comment 8: “Future Directions” and  “Limitations and Challenges “ . These sections are disproportionately long and can be shortened. I suggest the authors provide data on the points.

 Response 8: We admit that these sections are quite long, but we found them to be important aspects related to this topic as miRNA in breast cancer is a new method with such great potential. It is difficult to clearly explain the multitude of challenges that come with this line of research and moreover, the potential directions of future research are essential for other researchers that aim to improve this line of work. We graciously ask to maintain these sections at their current length as we do not wish to limit our ideas on these aspects.

 

Comment 9: The sections are written in a very informative and original format. However, the manuscript lacks illustrations to help readers understand such a complex topic.

Response 9: We completely agree that illustrations would have been a great addition, unfortunately at the given time we were not able to undertake this task due to limited graphic resources.

Comment 10: I think this topic is very relevant, especially when diagnosing early forms of breast cancer. A section dedicated to this issue needs to be added.

Response 10: Section 4 is detailing the diagnostic potential of miRNAs in breast cancer. We are currently working on a paper on the early diagnosis of breast cancer through miRNAs and other similar methods and have not provided a more detailed description in this manuscript. Current methods for non-invasive early diagnosis comprise of numerous methods, that go well beyond the scope of this review and also cannot be justly described briefly.

Comment 11: Conclusions should be drawn from the results obtained, with brief recommendations for insufficiently studied aspects and for further research in this area.

Response 11: The conclusions of the manuscript have been updated.

Comment 12: The list of references is not formatted according to the journal guidelines.

Response 12: The reference list has been corrected.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

The authors have addressed all of my concerns thoroughly and responded to each comment in a clear and satisfactory manner.

Therefore, I recommend acceptance of the manuscript in its current form.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have produced a much-improved manuscript, with added clarity and crucial information that strengthens the overall work. The enhanced introduction, materials, results and revised conclusions have significantly elevated the paper's impact and significance, directly addressing all previous concerns.