Unsupervised analysis identified 2 epigenetically distinct EVI1 AML subgroups correlating with EVI1 relative expression. (A) Unsupervised hierarchical clustering with the Pearson correlation using Ward's method revealed 2 EVI1 subclusters (ie, A and B). The cytogenetic characteristics are shown per patient; chromosome 3q26 abnormalities (red), monosomy 7 or deletion 7q (−7/7q−; blue), and 11q23 rearrangements (gray). The percentages of each characteristic are shown per cluster. (B) Median EVI1 relative expression levels and 2SD are shown per subcluster. P values were calculated using a moderated t test. (C) The volcano plot shows the methylation difference of all probe sets (n = 25 626; x-axis) comparing the methylation levels of cases in subcluster A with the cases in subcluster B with corresponding P value (−log₁₀P value–moderated t test) on the y-axis. (D) The heatmap shows the 122 probe sets (110 unique genes) differentially methylated in subcluster B, when both cluster were compared with each other using a moderated t test (P < .001 and absolute methylation difference > 1.5). All genes are hypermethylated in EVI1 AMLs from subcluster B.