A novel non-invasive monitoring assay of 5-azacitidine efficacy using global DNA methylation of peripheral blood in myelodysplastic syndrome.

Monitoring response and resistance to 5-azacitidine (AZA) is essential when treating patients with myelodysplastic syndrome (MDS). To quantify methylated DNA not only in the promoter region but also in the gene body, we established a single-molecule methylation assay (SMMA). We first investigated the methylation extent (expressed as methylation index [MI]) by SMMA among 28 MDS and 6 post-MDS acute myeloid leukemia patients. We then analyzed the MI in 13 AZA-treated patients. Whole-blood DNA from all 34 patients had low MI values compared with healthy volunteers (<0.0001). DNA hypomethylation in MDS patients was more evident in neutrophils (=0.0008) than in peripheral mononuclear cells (=0.0713). No consistent pattern of genome-wide DNA hypomethylation was found among MDS subtypes or revised International Prognostic Scoring System (IPSS-R) categories; however, we found that the MI was significantly increased for patients at very high risk who were separated by the new cytogenetic scoring system for IPSS-R (=0.0398). There was no significant difference in MI before AZA, regardless of the response to AZA (=0.8689); however, sequential measurement of MI in peripheral blood demonstrated that AZA non-responders did not have normalized MI at the time of next course of AZA (=0.0352). Our results suggest that sequential SMMA of peripheral blood after AZA may represent a non-invasive monitoring marker for AZA efficacy in MDS patients.