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  • Laura Gramantieri 1,*,
  • Clara Vianello 2 and
  • Francesca Fornari 2,4,*
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Gramantieri et al described in this manuscript that serum miR-22 can predict TACE response. They subsequently showed that miR-22 targets WEE1 and regulates HCC growth. In general, data from clinical samples are somewhat conclusive. However, there are few concerns in cellular experiments which should be addressed.

 

Specific Comments

  1. The authors need to clearly mentioned how they measured the serum miR-22 levels. Y-axis showed numbers in several figures, however, there was no explanation for how the number was selected. For examples, the authors stated that baseline downregulation of miR-22 may predict non-response in patients subjected to TACE (line 333). The authors stress the predictability of non-response using miR-22 levels but do not discuss the predictability of response without good rationale. However, in Figure 1, patient 366 shows miR-22 overexpression but is classified as a non-responder. Please clarify.
  2. Line 354-366: The authors hypothesize that miRNA-22 increases in serum post-treatment upon tumor necrosis may predict response to TACE. This requires further explanation – how does an increase in miRNA-22 post-treatment predict therapy outcome?
  3. S1F/G: Please provide tumor volume data and explain the data in more detail (lines 425-429).
  4. 2I-J: there was no baseline control data. It is difficult to understand the results without proper control. Baseline data in all figures should be provided. It was unclear how intracellular miR-22 was measured and Y-axis.
  5. 3D: Please provide the xenograft data with these panels. Panels F-H could be moved as supplementary figures.
  6. FigureS2 should be provided in the mail text.
  7. Line 536-539 and Figure 4D – there may be a mislabeling in the western blot where it is labeled as MZIP-22, but the experiment is with pMXs-22.
  8. 5A should be removed or redo with proper magnification. It is very difficult to follow the figures with no clarity.
  9. A model depicting the resistance mechanism and WEE1/miR-22 axis in TACE-treated HCC will be helpful.
  10. Lines 653-656—the statement is too strong based on the data provided.

Author Response

Please see the attachment. Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The topic is clinically relevant, as early identification of non-responders to TACE remains an unmet need. The use of multiple independent cohorts and the integration of mechanistic data are notable strengths. However some issues must be addressed:

  1. The statistical analysis relies primarily on univariate tests and ROC curves. No multivariable modeling is presented to assess whether miR-22 independently predicts response after adjustment for clinical factors like tumor burden, AFP or BCLC stage. Without multivariable modeling or external validation or calibration (ideally) I think the study does not provide robust clinical validation of miR-22 as an independent predictive biomarker.
  2. Some patients in validation cohort 1 with new lesions were still classified as responders. This could affect the validity of the endpoint. Please clarify how these patients were classified as responders?
  3. In validation cohort 2, post-treatment samples were missing in 12/84 patients. How were patients with missing post-treatment samples handled in the analysis?
  4. The manuscript refers to miR-22 as both a predictive and prognostic biomarker. However, the response are assessed at 3 months and there are no survival analyses. Please clarify whether miR-22 is intended as a predictive biomarker of treatment response or a prognostic marker of patient outcomes.
  5. The manuscript interpret that increased circulating miR-22 reflects resistance. However, it remains unclear whether this increase reflects active secretion associated with resistance mechanisms or passive release from tumor cell death following TACE. Please clarify how these possibilities are distinguished.

Author Response

Please see the attachment. Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my concerns. 

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your responses. I have reviewed these and find them appropriate; therefore, I have no further comments.