A unique methylation profile distinguishes CEBPA^sil from CEBPA^mut AML. (A) Principal component analysis of DNA methylation data using the HELP assay on 8 CEBPA^sil and 8 CEBPA^mut AML cases revealed that the cases were readily segregated into 2 clusters, which matched exactly with CEBPA status. (B) Heatmap representation of the 4 probe sets annotated to the CEBPA locus on the HELP microarray; cases are clustered according to their methylation status. CEBPA^sil cases cluster together (left node), and all show higher levels of methylation for at least 3 of the 4 probe sets. (C) Representation of the positioning of the 4 probe sets relative to the genomic localization of the CEBPA locus and its CpG island on chromosome 19. HELP methylation values for each leukemia case are represented in one row; the y-axis represents centered log2 (HpaII/MspI) ratios. Positive values correspond to hypomethylated fragments, whereas a negative deflection reflects a methylated fragment. The first 8 rows correspond to the CEBPA^sil cases (in red), and the remaining rows to the CEBPA^mut cases (in blue). (D) Heatmap representing the DNA methylation status at 5 different regions of the CEBPA locus. Percentage cytosine methylation was determined at these regions for all cases using MassARRAY EpiTyper. (E) Two-dimensional hierarchical clustering of genes differentially methylated between the 2 leukemia subgroups, illustrated by a heatmap. Supervised analysis identified 567 HpaII-amplifiable fragments (P < .001 and absolute difference in methylation > 2). Cases are represented in the columns; probe sets are represented in the rows. CEBPA^sil cases are clustered in the left node and display high methylation levels for 563 HpaII-amplifiable fragments. (Right) Heatmap representation of the 4 probe sets that displayed the opposite behavior, ie, relative hypomethylation in CEBPA^sil leukemia. (F) A plot of methylation difference between CEBPA^sil and CEBPA^mut cases (x-axis) versus statistical significance (y-axis) shows the marked asymmetry of the 2 branches, illustrating the overall tendency to higher methylation levels in the CEBPA^sil cases. Red points represent probe sets that reached both criteria for differential methylation on our analysis (P < .001 and absolute methylation difference > 2).