C/Ebpa Gene Mutation and C/Ebpa Promoter Hypermethylation in Acute Myeloid Leukemia with Normal Cytogenetics.

Abstract 1570

Poster Board I-594

Acute myeloid leukemia with normal cytogenetics (CN-AML) represents approximately 40-50% of de novo AML cases and constitutes the single largest cytogenetic group of AML. CN-AML is composed of a heterogeneous group of AML considered to be more or less in the intermediate prognostic category. Stratified prognostic determinants are required to predict which patients in this heterogeneous category have an increased risk of relapse, resistance to therapy or long term disease outcomes. The CCAAT enhancer binding protein alpha (C/EBPA) is a key transcription factor involved in regulation of granulocytic differentiation and myelopoiesis. In the past few years, various studies have suggested that C/EBPA is negatively regulated in certain AML patients. Impairments in C/EBPA signaling such as gene mutation, transcriptional dysregulation, as well as epigenetic modification via promoter hypermethylation have been identified in CN-AML and these alterations may play an important role in pathogenesis and may predict prognosis in these patients. In the current study, we investigated C/EBPA gene mutations and promoter hypermethylation in a series of 53 patients with CN-AML. In addition, we also analyzed two other frequent mutations (FLT3/ITD and NPM1) in these patients and correlated them with C/EBPA gene alterations. Genomic DNA was isolated from diagnostic bone marrow and peripheral blood samples. The C/EBPA gene mutations were detected by PCR amplification followed by direct DNA sequencing while the promoter hypermethylation was characterized by methylation-specific PCR analysis. The FLT3/ITD mutation and NPM1 mutation were detected by using multiplex PCR followed by capillary electrophoresis. The study included 28 female patients and 25 male patients, all adults, with a median age of 49 years. 13/53 (24.5%) patients were FLT3/ITD+/NPM1-, 11/53 (20.8%) patients were FLT3/ITD+/NPM1+, 9/53 (17.0%) patients were FLT3/ITD-/NPM1+, and 20/53 (37.7%) patients were FLT3/ITD-/NPM1-. Four out of the 53 cases (7.5%) displayed C/EBPA mutations, whereas 49 (92.5%) cases had only C/EBPA wild-type alleles. Of the 4 positive cases, three patients had N-terminal mutations only, while one patient had mutations in both the N- and C-terminal region. Two of the 4 positive cases also harbored both FLT3/ITD and NPM1 mutation simultaneously, while the other two patients had neither FLT3/ITD nor NPM1 mutations. In addition, 7 (13.2%) of the 53 cases displayed C/EBPA promoter hypermethylation. Interestingly they were all in CN-AML cases without FLT3/ITD or NPM1 mutations. None of the 7 patients with C/EBPA promoter hypermethylation showed C/EBPA mutation. In conclusion, C/EBPA mutation and promoter hypermethylation can be detected at a relatively low frequency in de novo CN-AML patients, suggesting they may contribute to leukemogenesis. C/EBPA mutation appears to be frequently seen “high-risk” AML (FLT3/ITD+/NPM1+; FLT3/ITD+/NPM1- or FLT3/ITD-/NPM1-), while C/EBPA hypermethylation appears to be more common in AML with FLT3/ITD- /NPM1- and is not associated with C/EBPA mutation. The clear prognostic relationship of C/EBPA alteration and FLT3/ITD, NPM1 mutation needs to be further validated.