Allelic Methylation Levels of VTRNA2-1 Predict Outcome in Higher Risk MDS Patients Not Treated by Azacytidine.

We have recently shown that the allelic methylation level of the non-coding RNA, VTRNA2-1, predict outcome in acute myeloid leukemia (AML) (Treppendahl et al., 2012). VTRNA2-1 is located on chromosome 5q31.1, in the commonly deleted region for higher risk myelodysplastic syndrome (MDS) and de novo AML. Around 75% of the healthy Danish population carries a monoallelically methylated VTRNA2-1 promoter while the rest carries 2 unmethylated promoters. A hypermethylated promoter was only observed in patients. These interindividual differences in the methylation pattern are intriguing, and our data suggest that the gene dosage of this particular type of ncRNA may play an important role in tumor progression or response to therapy, since AML patients with hypomethylation of both alleles of the VTRNA2-1 promoter have a significantly better prognosis, while those with hypermethylation or loss of the second VTRNA2-1 copy have a poorer outcome(Treppendahl et al., 2012). Accordingly, we speculated if the allelic methylation levels of VTRNA2-1 also predict outcome in higher risk MDS patients.

Bone marrow mononuclear cells from primary higher risk MDS patients (IPSS category INT2 and HIGH risk) and peripheral blood mononuclear cells, sampled during treatment with azacytidine, were analyzed for promoter methylation by pyrosequencing and methylation specific melting curve analysis.

Bone mononuclear cells from 57 higher risk MDS patients, never treated with azacytidine, were examined for VTRNA2-1 promoter methylation. 18 (32%) cases carried an unmethylated promoter (less than 15% methylation), 31 (54%) cases carried an intermediate methylated promoter (15–41% methylation) and 8 (14%) cases carried a hypermethylated promoter (more than 41% methylation). Patients with hypomethylation of the VTRNA2-1 promoter have a considerable better prognosis than those with intermediate or hypermethylation of the VTRNA2-1 promoter (P=0.026). Interestingly, in an azacytidine treated cohort the survival benefit of having an unmethylated promoter disappeared and a tendency towards a better survival of the methylated cases were observed (N=28, P=0.180).The in vivo effect of azacytidine on VTRNA2-1 methylation were examined in a small cohort of MDS patients (N=6), showing that azacytidine can induce demethylation of the VTRNA2-1 promoter in peripheral blood mononuclear cells.

Our studies show, that the allelic methylation of VTRNA2-1 can predict outcome, not only in AML patients, but also in higher risk MDS patients not treated with azacytidine. Patients with hypomethylation of the VTRNA2-1 promoter have a considerable better outcome than those with intermediate methylation or hypermethylation of the promoter. Interestingly, in the group of azacytidine treated patients there was no difference in survival between cases with and without methylation of the VTRNA2-1 promoter These data could indicate that patients with methylation of the VTRNA2-1 promoter might have a survival benefit of the azacytidine treatment. Thus we suggest that constitutive interindividual differences in the methylation of VTRNA2-1 potentially can be used as a pretreatment marker for selecting patients who will have a survival benefit of azacytidine treatment. This is to our best knowledge the first study identifying a potential epigenetic marker for selecting patients with benefit of treatment with azacytidine before treatment start. Our study is conducted in a quite heterogeneous and small patient cohort, and it has to be verified in a more homogenously treated larger group of MDS patients, ideally in a prospective clinical trial.

No relevant conflicts of interest to declare.