Marked promoter DNA hypomethylation is achieved after 15 days of treatment with 5AC. (A-C) Unsupervised clustering analysis (COA, PCA, and hierarchical clustering) of 9 MDS cases for which we obtained pretreatment and posttreatment (day +15) paired samples. (D) Plot of DNA methylation difference between baseline MDS and day +15 after treatment (x-axis) versus statistical significance (y-axis). Red points mark probe sets that reached both criteria for differential methylation on our analysis [P < .0005 and absolute fold change in log(HpaII/MspI) > 1.5], whereas blue points mark probe sets that reached statistical significance but did not have a change in log(HpaII/MspI) greater than 1.5. (E) Two-dimensional hierarchical clustering of genes differentially methylated between pretreatment and day +15 paired samples identified by supervised analysis is shown with a heatmap. Cases are represented in the columns and probe sets in the rows. (F) Density plots representing the relative frequency (y-axis) of promoters with different degrees of methylation (x-axis). Baseline (black) distributions are compared with day +15 of treatment with 5AC (red) and day +29 (blue). For 8 of 9 cases a clear shift toward more hypomethylated values is observed at day 15, and this shift persisted at day 29 for the cases for which we also had samples at that time point.