Methylation of the Proximal, Distal and Core Promoter of CEBPA in 574 Cases with Normal Karyotype AML and 44 with t(8;21) Disclosed Different Frequencies but No Impact on Prognosis

Loss of CEBPA function due to mutations is thoroughly explored in AML. Recently epigenetic modifications such as promoter methylation have gained increasing interest as additional mechanisms for transcriptional regulation of cancer related genes. In this context, the clinical impact of aberrant CEBPA promoter methylation (PM) in AML is controversially discussed. The aim of this study was to clarify the frequency and the significance of aberrant CEBPA PM with regard to clinical features in a large cohort of de novo AML patients. The study comprises the CEBPA core promoter as well as the distal and proximal CEBPA promoter region as it has been shown that these upstream located regions also bear promoter activity. Patients:CEBPA PM was analyzed in a cohort of 574 de novo AML patients with normal karyotype (NK-AML) and without CEBPA mutations. The cohort was composed of 268 females and 306 males. Age ranged from 20.0 to 89.6 years (median: 63.7). In addition, methylation status was assessed in 48 patients with biallelic (n=10) or monoallelic (n=38) CEBPA mutations to exclude coincidence with CEBPA PM. As the fusion transcript RUNX1-RUNX1T1 is known to down-regulate CEBPA mRNA and protein levels in AML, we also analyzed CEBPA distal PM status in 44 RUNX1-RUNX1T1 positive AML patients to evaluate a possible correlation between CEBPA distal PM and RUNX1-RUNX1T1 induced down-regulation of CEBPA expression. All patients were intensively treated using standard AML protocols. Methods: Proximal PM and distal PM were analyzed in the total cohort using Sanger sequencing. Core PM was screened by methylation specific PCR in a subcohort of 326 CEBPA unmutated cases. Methylation data was correlated to clinical outcomes and to the presence of FLT3-ITD (n=175/568), NPM1 (n=256/565), RUNX1 (n=91/275), MLL-PTD (n=76/567), IDH1G105 (n=47/353), IDH1R132 (n=38/371), IDH2R140 (n=65/332) and IDH2R172 (n=12/342) molecular mutations. In addition, CEBPA mRNA expression levels were assessed by quantitative real time PCR (Taqman^®, Life Technologies, Carlsbad, CA) in 39 cases with CEBPA distal PM and in 8 cases of the NK-AML cohort tested negative for CEBPA distal PM. CEBPA expression was normalized against the expression of the control gene ABL1. Results: The CEBPA distal promoter was methylated in 54/574 cases (9.4%) whereas CEBPA proximal PM was found in none of the 574 cases. Methylation of the CEBPA core promoter was detected in only 8 of 326 cases (2.5%). As CEBPA proximal and core PM seem to be rare events in AML, they were excluded from further analysis. None of the 48 CEBPA mutated cases revealed any PM and thus aberrant CEBPA PM and mutation status were mutually exclusive. Surprisingly, analysis of CEBPA mRNA expression level revealed no difference between CEBPA distal PM positive and CEBPA distal PM negative cases (mean ± SD 145 ± 97.9 and range 2.7–474.2 vs. 141.4 ± 85.3 and 23.1–259.2, n.s.) suggesting that CEBPA distal PM has no influence on CEBPA mRNA expression in NK-AML. In contrast, we observed a significantly higher frequency of CEBPA distal PM in patients with RUNX1-RUNX1T1 positive AML (n=17/44; 38.6%) compared to the NK-AML cohort (n=55/572; 9.4%) (p<0.001) indicating a correlation between RUNX1-RUNX1T1 induced down-regulation of CEBPA expression and CEBPA distal PM. In the NK-AML cohort, there was no correlation between CEBPA distal PM and age, sex, white blood cell count or Hb levels at diagnosis compared to unmethylated cases. We also were not able to detect a significant correlation between the presence of CEBPA distal PM and the other molecular mutations except for the frequency of IDH2R140 mutations which was significantly lower in CEBPA distal PM positive compared to CEBPA distal PM negative cases (21/267; 7.9% vs. 13/65, 20%, p=0.010). In addition, CEBPA distal PM was not related to overall survival, event free survival or incidence of relapse in NK-AML. Also in subcohorts that were defined by specific molecular mutations (FLT3-ITD, NPM1, RUNX1, MLL-PTD, IDH1, or IDH2) no prognostic impact of CEBPA distal PM could be shown. Conclusion:CEBPA PM in NK-AML was detected in 11.9% of all cases and seems to be mainly restricted to the distal promoter (9.4%). In contrast to RUNX1/RUNX1T1 positive AML no impact of CEBPA PM on CEBPA mRNA expression levels was detected in NK AML. We also conclude that the presence of aberrant CEBPA PM has no clinical relevance in NK-AML and therefore is negligible as prognostic marker.

Fasan:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.