Review Reports

Round 1

Reviewer 1 Report

The present study is aiming to develop the miRNAs predictive biomarkers of NACT in BCs. They performed the RNA-seq using patients’ plasma and bioinformatic analyses using TCGA and Matabric datasets. The primary concern would be that the miRNAs they selected did not show significantly clinical correlation. Moreover, the number of samples is limited for make any conclusion. As they claimed in the end of the Abstract section that “The results from this study suggest that miRNAs have the potential to serve as non-invasive liquid biopsy biomarkers for monitoring pCR to NACT”. This is a well-known concept documented in previous studies but not a new finding here. Together, this study did not provide any useful information but only including some meaningless data analysis.

Author Response

The present study is aiming to develop the miRNAs predictive biomarkers of NACT in BCs. They performed the RNA-seq using patients’ plasma and bioinformatic analyses using TCGA and Matabric datasets. The primary concern would be that the miRNAs they selected did not show significantly clinical correlation. The authors thank the reviewer for their concern. The microRNAs reported in this study were selected based on their very significant expression in the corresponding groups of patients with and without complete pathological response to neoadjuvant chemotherapy.

Moreover, the number of samples is limited for make any conclusion. We agree with the reviewer that the sample size was relatively small, but the expression of the microRNAs was very significant.

As they claimed in the end of the Abstract section that “The results from this study suggest that miRNAs have the potential to serve as non-invasive liquid biopsy biomarkers for monitoring pCR to NACT”. This is a well-known concept documented in previous studies but not a new finding here. The authors thank the reviewer for the comment. We have corrected the end of the Abstract and changed it as “If the miRNAs identified in this study are validated in a large cohort of patients, they might serve as predictive non-invasive liquid biopsy biomarkers for monitoring pCR to NACT in breast cancer”.

To the best of our knowledge, the significant correlation between upregulation of circulating exosomal mir-30b-5p and downregulation of mir-328-3p before the start of chemotherapy, and the positive response to neoadjuvant chemotherapy in breast cancer has not been reported.  The significant correlation between upregulated exo-mir-127-3p at baseline with pCR in TNBC is also novel. The significant correlation of miRNAs expression after the first cycle of NACT with positive and negative therapeutic response to NACT has also not been reported.

Together, this study did not provide any useful information but only including some meaningless data analysis. We respectfully disagree with this statement of the reviewer. Several recent review articles on miRNAs as biomarkers in cancer (PMID: 33485903; PMID: 30652506) indicate that currently there is a lack of consistency between the reports and further studies are needed.  We believe that our study provides a statistically significant set of miRNA for early prediction of the response to NACT, which if validated in larger cohort of patients could aid in breast cancer treatment.

Author Response File: Author Response.docx

Reviewer 2 Report

This is very important scientific work to adopt molecular techniques to early prediction of chemotherapy successes (and misses) - with broad implications for drug development and ultimately clinical practice

The science seems solid and well thought out to me and the explanations are quite clear.  

Thank you for choosing me as a reviewer for this fascinating article. 

 

Author Response

The authors thank the reviewer for the comments.

Author Response File: Author Response.docx

Reviewer 3 Report

The work is interesting in that it aims to identify biomarkers predictive of neoadjuvant chemotherapy (NCT) response for locally advanced breast cancer, and may contribute, through the identification of biomarkers, in decision-making regarding the treatment of patients undergoing neoadjuvant chemotherapy.

Suggestions/corrections

1- Include scientific basis regarding blood collection before treatment and after the first cycle of treatment.

2- Figure 1- “shows the candidate miRNAs that discriminate between the groups of patients who achieved pCR and those who did not”, this discrimination could not be identified in Figure 1. Explain the result more clearly.

3- Figure 2. include the red and blue dots in the legend.

4- Line 201-202, 223 Table 2.” After the first NACT dose, the plasma of patients with group with non-pCR showed upregulation of miR-182 and miR-183 versus baseline”. Review results versus table 2,  I have identified inconsistencies.

5- Figure 3. The results presented in figure 3, explain if the same methodology of the study was applied?

6- Line 223- Table II was not identified in the study.

7- Figure 7. line 277. “the higher expression of miR-34, miR-182 and miR-183 correlated with activation of TP53, and inhibition of NOTCH2, E2F3, CTSW, FOXP1” as already mentioned in item 4, it was not verified this increase of expression in miRs 182, 183, as well as in lines 358/359/365/371

8- the correlation between liquid biopsy and tumor tissue based on the results of TCGA miRNA expression datasets, Kaplan-Meier plots (cBioPortal), networks from the resulting IPA analysis, need to be better clarified, especially regarding data from miRs 182 and 183.

9- Difficulty in viewing the figures, the resolution needs to be improved.

Author Response

Point 1: Include scientific basis regarding blood collection before treatment and after the first cycle of treatment. We thank the reviewer for the suggestions. We have revised the text and added new information related to baseline and after initial treatment biomarkers.

Point 2:  Figure 1- “shows the candidate miRNAs that discriminate between the groups of patients who achieved pCR and those who did not”, this discrimination could not be identified in Figure 1. Explain the result more clearly. We thank the reviewer for the comment. We have revised the text and replaced the figure with a complete one.

Point 3:  Figure 2. include the red and blue dots in the legend. The red and blue dots were included in the legend.

Point 4:  Line 201-202, 223 Table 2.” After the first NACT dose, the plasma of patients with group with non-pCR showed upregulation of miR-182 and miR-183 versus baseline”. Review results versus table 2, I have identified inconsistencies. We thank the reviewer for the comment, we have corrected these inconsistencies.

Point 5:  Figure 3. The results presented in figure 3, explain if the same methodology of the study was applied?  The data are from microRNA expression in breast tumors and normal breast tissue. We have added this information in the figure legend.

Point 6: Line 223- Table II was not identified in the study. This has been corrected.

Point 7: Figure 7. line 277. “the higher expression of miR-34, miR-182 and miR-183 correlated with activation of TP53, and inhibition of NOTCH2, E2F3, CTSW, FOXP1” as already mentioned in item 4, it was not verified this increase of expression in miRs 182, 183, as well as in lines 358/359/365/371. The authors thank the reviewer for this comment. We have revised the text to correct these inconsistencies.

Point 8: the correlation between liquid biopsy and tumor tissue based on the results of TCGA miRNA expression datasets, Kaplan-Meier plots (cBioPortal), networks from the resulting IPA analysis, need to be better clarified, especially regarding data from miRs 182 and 183. We thank the reviewer for the comment. We have revised the text.

Point 9: Difficulty in viewing the figures, the resolution needs to be improved. We have increased the text size in the figures.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

This reviewer accepted the revised version. 

Reviewer 3 Report

The questions and inconsistencies identified in the first review were satisfactorily addressed.

My comment for this research is that the proposal is quite interesting, but the results are statistically inconsistent due to the small number of patients in each analyzed group.