TET2 Mutation and High miR-22 Expression as Biomarkers to Predict Clinical Outcome in Myelodysplastic Syndrome Patients Treated with Hypomethylating Therapy

Round 1

Reviewer 1 Report

The topic is of much value so, the state-of-the-art review could be improved to analyze previous and alternative solutions. The methods may be accompanied with flow charts to clarify the presentation. The number of patients may be  increased with available data in Internet. Lots of abbreviations may be avoided.

Author Response

Response to Reviewer 1 Comments

Point 1: The topic is of much value so, the state-of-the-art review could be improved to analyze previous and alternative solutions. The methods may be accompanied with flow charts to clarify the presentation. The number of patients may be increased with available data in Internet. Lots of abbreviations may be avoided.

 Response 1: First of all, thanks for your kind advice. A flow chart for the experimental method was added to the manuscript. We do not plan to conduct a meta-analysis to collect and analyze data from studies on TET2 in MDS.

Author Response File: Author Response.docx

Reviewer 2 Report

In the manuscript cimb-1276233 the authors aimed to evaluate whether TET2 mutation and miR-22 expression can be used as biomarkers to predict clinical Outcome in Myelodysplastic Syndrome Patients. After reading this work, I've found some aspects that the authors should revise from my perspective.

Why did they focus on TET2 gene when many other were also analyzed? 

Though some studies reported TET2 mutation was a predictive marker of response to HMT, this pattern response is heterogeneous? Could you explain this? Is this only because of the heterogeneity of DNA methylation?

TET2 mutations categorization is related to what? Epigenetic states, disease aggressiveness? Could you please explain?
And why categorize when TET2 seems to be not a good predictor?

Why did the authors could not distinguish whether TET2 mutation is heterozygous or homozygous?

Could the authors explain the relevance of this study when both TET2 mutation and miR-22 expression did not function as good biomarkers?

Author Response

Response to Reviewer 2 Comments

In the manuscript cimb-1276233 the authors aimed to evaluate whether TET2 mutation and miR-22 expression can be used as biomarkers to predict clinical Outcome in Myelodysplastic Syndrome Patients. After reading this work, I've found some aspects that the authors should revise from my perspective.

Point 1: In the manuscript cimb-1276233 the authors aimed to evaluate whether TET2 mutation and miR-22 expression can be used as biomarkers to predict clinical Outcome in Myelodysplastic Syndrome Patients. After reading this work, I've found some aspects that the authors should revise from my perspective.

Why did they focus on TET2 gene when many other were also analyzed?

Response 1: First of all, thanks for your kind advice. This study was inspired by the findings of Song et al. on miR-22, a TET-2 inhibiting miRNA, in MDS. The purpose of this study was to evaluate and compare the effects of TET2 mutation and miR-22 expression on the clinical characteristics of MDS, especially the outcomes of hypomethylating therapy. Genes other than TET2, such as DNMT3A, were simultaneously subjected to NGS analysis, but these were not the main subjects of this paper. Further analysis of the effects of these genes on MDS can be performed, but there are no plans yet.

Point 2: Though some studies reported TET2 mutation was a predictive marker of response to HMT, this pattern response is heterogeneous? Could you explain this? Is this only because of the heterogeneity of DNA methylation?

Response 2: We do not know why the results on the difference in responsiveness to hypomethylating therapy according to the presence of TET2 mutation are heterogenous among relevant studies. It is only our speculation that heterogenous response patterns to HMT may be related to diversity in DNA methylation patterns. If meta-analysis is performed using the results of related studies on TET2 mutation in MDS, we think some conclusions can be reached. Although not meta-analyzed, we do not believe that TET2 mutation has a significant impact on responsiveness to hypomethylating therapy, given the results of our study and other relevant studies.

Point 3: TET2 mutations categorization is related to what? Epigenetic states, disease aggressiveness? Could you please explain?

And why categorize when TET2 seems to be not a good predictor?

Response 3: We did not categorize the TET2 mutation in MDS. We think that the contents related to the categorization of TET2 mutation you mentioned probably about the research of Reilly et al.. In the study of Reilly et al., MDS patients were categorized into 5 distinct clusters according to DNA methylation pattern. In this study, they combined clusters with similar median OS into “high” (clusters B and C) and “low” (clusters A, D, and E) cluster risk groups. They reported that patients with TET2 mutations were distributed to clusters B, C, and E but enriched only in cluster D which belongs to the low cluster risk group with better survival and longer time to AML transformation than the high cluster risk group. In other words, TET2 mutation was enriched in, but not unique to, the low cluster risk group. In addition, they did not say that patients with mutant TET2 showed better prognosis than those with WT TET2.

In our study, we suggested that the TET2 mutation is not a good predictor for responsiveness to hypomethylating therapy, but it may be a good predictor for prognosis such as survival and leukemic transformation. However, it is not clear whether subclassification of TET2 mutant patients using other factors will be more helpful in predicting clinical outcomes of MDS.

Point 4: Why did the authors could not distinguish whether TET2 mutation is heterozygous or homozygous?

Response 4: The main reason we did not divide TET2 mutations into heterozygous and homozygous was because the number of patients having TET2 mutation was too small, so there was no statistical significance when analyzing them separately. Theoretically, heterozygous TET2 mutation is expected to lead to ~50% loss of TET2 function. In this situation, fortunately, we found the study of Ko et al. showing that the global levels of 5hmC were significantly reduced in patients carrying either homozygous or heterozygous TET2 mutations compared with patients bearing WT TET2. This means that even if the TET2 mutation is heterozygous, it is in a sufficient loss-of-function state. Meanwhile, in this study, it was reported that there was a tendency for global hypomethylation in the presence of TET2 mutation. However, further studies have shown that loss of TET2 leads to DNA hypermethylation, especially in enhancers.

Unfortunately, we cannot be sure that there is no difference between heterozygous and homozygous of TET2 mutation. Indeed, heterozygous TET2 knockout in mice, which led to ~50% loss of TET2 gene expression, resulted in significant but slower and less frequent malignant transformation than double-allele knockout. Moreover, AML patients with homozygous TET2 mutation showed significantly inferior Event-Free Survival and a higher relapse rate compared with those with heterozygous TET2 mutation.

Under these circumstances, we decided to revise the manuscript to reflect the above to avoid controversy.

Point 5: Could the authors explain the relevance of this study when both TET2 mutation and miR-22 expression did not function as good biomarkers?

Response 5: We think that the TET2 mutation and high miR-22 expression are not good biomarkers for predicting responsiveness to hypomethylating therapy. This is because patients with TET2 mutation or high miR-22 expression had no difference in responsiveness to hypomethylating therapy compared to their counterparts. On the other hand, we think that the TET2 mutation and high miR-22 expression may be good predictors for prognosis. This is because patients with TET2 mutation or high miR-22 expression tended to have longer survival and lower leukemic transformation compared to their counterparts. We think that these differences may be associated with chromosomal stability affected by DNA methylation. Although not meta-analyzed, we think that TET2 mutation is associated with a lower degree of chromosomal abnormality, given the results of our study and other relevant studies. Indeed, the NCCN guideline for MDS states that TET2 mutation is associated with normal chromosomes, but the reference of the content is not stated.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Authors took into consideration the suggestions of the reviewers. I have no further comments to add.