No abstract

CCAAT/enhancer binding protein alpha (CEBPA) mutations in AML are associated with favourable prognosis and are divided into N-and C-terminal mutations. The majority of AML patients have both types of mutations. We assessed the prognostic significance of single (n ¼ 7) and double (n ¼ 12) CEBPA mutations among 224 AML patients. Double CEBPA mutations conferred a decisively favourable overall (P ¼ 0.006) and disease-free survival (P ¼ 0.013). However, clinical outcome of patients with single CEBPA mutations was not different from CEBPA wild-type patients. In a multivariable analysis, only double -but not single -CEBPA mutations were identified as independent prognostic factors. These findings indicate heterogeneity within AML patients with CEBPA mutations.

IntroductionKaryotype abnormalities detectable at diagnosis in approximately 50% of acute myeloid leukemia (AML) patients are considered the most important prognostic factor. 1-3 The 5-year overall survival (OS) of AML patients with a normal karyotype (NK) is between 35% and 45%, 1,3 but individual outcome of such patients may vary greatly. Therefore, additional molecular markers, such as mutations in the genes encoding CCAAT/ enhancer binding protein-␣ (CEBPA), FLT3, NPM1, IDH1 and IDH2, C-KIT, RAS, or WT1, have been reported to identify subgroups among NK-AML patients. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] In the hematopoietic system, the transcription factor CEBPA is expressed in myelomonocytic cells and specifically up-regulated during granulocytic differentiation. 19,20 Expression of CEBPA in myeloid precursor cells can trigger terminal differentiation. [20][21][22] Remarkably, cebpa knockout mice exhibit a selective block in neutrophil differentiation at the myeloblast level, whereas other blood cells develop normally. 23 Dominant-negative CEBPA mutations are observed in up to 15% of AML patients, preferentially in NK-AML patients. 4,5 Leukemic cells from AML patients with CEBPA mutations express distinctive gene expression signatures at the RNA 24-26 and the miRNA level. 27 Favorable prognosis of CEBPA mutations in AML is restricted to patients with double CEBPA mutations. [5][6][7][8]28,29 The expression of the AML1-ETO fusion protein suppresses CEBPA at the mRNA level, 30 and CEBPA expression and function can be inhibited by fms-like tyrosine kinase 3 (FLT3) mutations. 31,32 The CBFB-SMMHC fusion protein selectively blocks CEBPA translation through induction of the mRNA binding protein calreticulin. 33 Finally, CEBPA can be silenced by promoter hypermethylation, and forced expression of TRIB2 is blocking CEBPA wild-type protein by physical interaction, ultimately inducing proteasomal-dependent degradation. 26 As various karyotype abnormalities target CEBPA function, deficient CEBPA function can also be present in NK-AML, such as in patients with CEBPA mutations. We therefore hypothesized that various levels of CEBPA function in NK-AML patients might be of prognostic significance. Methods PatientsFicoll separated mononucleated cells from bone marrow samples of untreated consecutive de novo AML patients from a single center (University Hospital, Berne, Switzerland) were collected at diagnosis. Clinical characteristics and course of all patients are given in supplemental Tables 1 and 2 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Informed consent was obtained according to the Declaration of Helsinki. A total of 20% of the patients received SAKK 30/95 chemotherapy, 56% were treated within SAKK 30/00, and 24% underwent SAKK 30/01 chemotherapy. 34,35 Approval of all studies was obtained by decisions of the local ethics committee of Berne, Switzerland. Stable cell linesLeukemic U937 cells stably transfected with the tetracycline induc...

The myeloid transcription factor CEBPA is crucial for normal differentiation of granulocytes. In addition, genomic mutations and deregulated expression of CEBPA have been reported in specific subsets of patients with acute myeloid leukemia (AML). We here investigated the prognostic significance of CEBPA mRNA, CEBPA protein, and CEBPA function in 105 consecutive de novo AML patients with a particular focus on AML patients with a normal karyotype. We found that the DNA binding activity of CEBPA in normal karyotype AML patients as determined by an ELISA-based assay conferred significant prognostic information: normal karyotype AML patients with suppressed CEBPA function showed a better OS (p=0.0068) and DFS (p=0.0138) compared to patients with conserved CEBPA function. In addition, suppressed levels of the 42kDa CEBPA protein in these patients tended to predict favorable outcome, whereas differences in p30 CEBPA protein levels and in mRNA expression did not affect the outcome. Finally, AML patients with suppressed CEBPA function had more frequently FLT3-ITD and an unmutated nucleophosmin status identifying CEBPA function as an independent prognostic marker for normal karyotype AML. This is the first study comprehensively assessing CEBPA transcription factor function in leukemic cells from AML patients. Our data suggest that suppressed CEBPA function is associated with favorable prognosis in normal karyotype AML patients independently of other molecular markers, and we propose assessment of CEBPA binding activity to be integrated into clinical practice. Moreover, our results highlight the importance of posttranscriptional mechanisms of CEBPA modulation in AML.