A gene expression prediction signature identifies new leukemias with the silenced CEBPA phenotype in an independent cohort of AML. (A) For 262 samples analyzed on Affymetrix HGU133Plus2.0 GeneChips, log-transformed (base 2) and mean-centered expression levels for 13 probe sets are depicted (left panel) for an arbitrary range from − 4 to + 4 (corresponding to 16-fold lower to 16-fold higher expression relative to the mean, respectively). The ordering of patients in the figure is arbitrary. In the right panel, these data are enlarged for 31 of these 262 leukemias, representing 3 groups: (I) the 6 cases with silenced CEBPA previously identified, with from top to bottom cases no. 2668, no. 2238, no. 3314, no. 2686, no. 3483, and no. 3491; (II) a variable selection of 10 AMLs from distinct GEP clusters for which also NOTCH1 mutational analysis and CEBPA promoter bisulfite sequencing were performed; and (III) 15 AMLs with CEBPA mutations, originating from either GEP cluster no. 4 (upper 9 samples) or cluster no. 15 (lower 6 samples). The 9 probe sets on the left side constitute the most predictive gene expression signature for group I, as determined by PAM: 216191_s_at (TRA@/TRD@ 1), 217143_s_at (TRA@/TRD@ 2), 213830_at (TRD@), 225681_at (CTHRC1), 1565809_x_at (no annotation), 1560018_at (ARPP-21), 210844_x_at (CTNNA1 1), 200764_s_at (CTNNA1 2), and 200765_x_at (CTNNA1 3). To the right, 4 additional probe sets are indicated, that is, 204039_at (CEBPA), 218902_at (NOTCH1), 202478_at (TRIB2), and 214551_s_at (CD7). Mutational data for NOTCH1 and CEBPA, and methylation status of the CEBPA promoter are depicted next to the normalized hybridization intensities of the probe sets. (B) Two hundred sixty-eight samples obtained from a second cohort of AML were hybridized to HGU133Plus2.0 GeneChips. The 9 probe set signature was used to identify leukemias with a profile similar to group I (A), resulting in the detection of group IV (from top to bottom cases no. 6376, no. 6735, no. 6947, no. 7053, no. 7076, and no. 7120).