Abstract
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by various cytogenetic and molecular abnormalities, some of which can be used as prognostic markers. Mutations in the transcription factor CCAAT/enhancer binding protein alpha (CEBPA) occur in 5–10% of AML and have consistently been associated with a favorable outcome. Three types of mutations have been described: N-terminal out-of-frame mutations, inframe mutations in the basic leucine zipper (bZIP) region, and a small variable group of remaining aberrations. Most CEBPA mutant AML cases carry two mutations, usually on different alleles (double mutant). However, there are also cases that only express a single heterozygous mutation, and thus retain a wild type allele (single mutant). It is not known whether single and double CEBPA mutations should be considered of equal biological and/or clinical importance. We applied dHPLC WAVE technology in combination with nucleotide sequencing of the entire CEBPA gene in a cohort of 598 cases of adult de novo AML. After exclusion of previously described polymorphisms, we identified 41 cases (6.9%) with at least one mutation. Of these cases, 28 carried double mutations, i.e. two different heterozygous mutations or one homozygous mutation, whereas the remaining cases carried a single heterozygous mutation. To investigate whether CEBPA mutations were associated with specific transcriptional signatures, we examined genome-wide gene expression (GEP) data of 525/598 AMLs, including 38/41 CEBPA mutant cases. Class prediction of total CEBPA mutation status based on GEP data resulted in a relatively large number of false negatives in cross-validation using the PAM algorithm (sensitivity 68%, specificity 99%). Strikingly however, all these missed cases appeared to be of the single mutant group, while the double mutants were recognized with high accuracy. In agreement, unsupervised cluster analysis of the 525 AMLs led to distinct grouping of cases with double mutations, while cases with a single heterozygous mutation did not. These observations suggested that double and single CEBPA mutant AMLs represent distinct biological entities. We next assessed the clinical relevance of this finding. In concordance with previous studies, total CEBPA mutation status associated with favorable overall survival (OS) and event-free survival (EFS) (P=0.023 and P=0.042, log rank test), which was maintained in multivariable Cox’s proportional hazards models with cytogenetic risk group, FLT3-ITD and NPM1 mutation status, age and white blood cell count (hazard ratio [HR] 0.47, 95% confidence interval [CI] 0.29–0.77; P=0.002 and HR 0.52, 95% CI 0.33–0.82; P=0.004). Surprisingly, when the double and single CEBPA mutant cases were separately analyzed, only the double mutants showed a highly favorable outcome, while the single mutants could not be distinguished from CEBPA wild type AMLs (P=0.003 versus P=0.51 (OS) and P=0.004 versus P=0.18 (EFS)). In multivariable analysis, CEBPA double mutation status remained associated with favorable outcome (OS HR 0.31, 95% CI 0.16–0.59; P<0.001, and EFS HR 0.34; 95% CI 0.19–0.61; P<0.001), contrasting with the single mutants (HR 1.18, 95% CI 0.58–2.41; P=0.64 and HR 1.65, 95% CI 0.84–3.23; P=0.15). Similarly, in multivariable analysis in the selected normal karyotype subset (n=193), CEBPA double mutations, but not single mutations, were significantly associated with OS (P=0.026 versus P=0.24) and EFS (P=0.013 versus P=0.42). In conclusion, these data demonstrate the existence of distinct transcriptional and clinical characteristics of AML cases with double CEBPA mutations and imply that it is crucial to discriminate them from single mutants to identify those patients with a favorable prognosis.
Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia
