Schematic overview of dHPLC analysis, gene expression profiling analysis, and survival estimates. (A) Schematic representation of the CEBPA gene and location of amplicons a, b, and c for polymerase chain reaction, used for dHPLC analysis. Functional regions are depicted: 2 transactivation domains (TAD1 and TAD2) in the N-terminal part, and the bZIP region in the C-terminal part. Nucleotide (nt) position is indicated relative to the main translation start site. Amino acid (aa) numbering and the alternative translation start site at position nt 358 (aa 120) are also depicted. (B) Representative profiles of dHPLC analysis of 1 of the 3 investigated fragments (ie, amplicons b) in a random selection of approximately 90 samples. Heteroduplexes (various colors) are released earlier than homoduplexes (green) and can therefore be recognized as distinct peaks. Time is depicted on the x-axis, and absorbance on the y-axis. (C) A gene expression prediction signature for CEBPA^mut AML (irrespective of single- or double-mutant status) was derived in a dataset of 524 AMLs, including 38 CEBPA^mut cases. Prediction accuracy for each of the 38 CEBPA^mut cases was estimated using repeated 10-fold cross-validation, as detailed in supplemental data. The proportion of correct predictions for the selected 38 CEBPA^mut specimens is indicated (top panel). Mutation status is color coded (CEBPA^single-mut, blue; CEBPA^double-mut, red). The heatmap in the bottom panel depicts the 19 probe sets in the resulting CEBPA^mut gene expression classifier (Table S2, probe set information). Intensity values (log2) were mean centered over the cohort of 524 AML cases; and for visualization purposes, the genes were hierarchically clustered (Euclidian distance, average linkage). Cells represent relative log 2 expression values and have been color coded on a scale ranging from bright green (−3) to bright red (+3), with black indicating no change relative to the mean. (D) Kaplan-Meier estimates of overall survival among CEBPA^mut and CEBPA^wt AML (log rank test, P = .027). (E) Overall survival among CEBPA^double-mut versus CEBPA^wt AML (P = .004) and versus CEBPA^single-mut AML (P = .005; pooled P = .012). (F) Event-free survival (EFS) among CEBPA^double-mut and CEBPA^wt AML (P = .005) and versus CEBPA^single-mut AML (P = .004; pooled P = .008). The cumulative proportion of survival at the intercept (the point where a line crosses the y-axis) reflects the proportion of patients reaching complete remission. Analyses similar to those depicted in panels D-F were performed after splitting the group of CEBPA^wt AMLs into those with favorable cytogenetics and those with other cytogenetics. These additional analyses can be found in Figure S4.