Abstract SY38-01: Genetic and epigenetic changes contributing to leukemogenesis and prognostic impact in acute myeloid leukemia
Authors: Chen Sai-Juan 
Published: August 2012

Abstract
Acute myeloid leukemia (AML) is a group of heterogeneous diseases with considerable diversities in terms of clinical consequences and biological implications. Abnormalities in genes that encode transcription factors and tyrosine kinases represent two classes of the most frequently detected genetic events in human AMLs. Recently, a new category of gene mutations associated with epigenetic regulation have also been implicated in leukemogenesis. For example, the mutations of IDH1, IDH2 and TET2 might result in a hypermethylation phenotype with impairment of hematopoietic differentiation. Chromosomal translocations involving MLL which actually encoding a histone methyltransferase are mostly found in acute monocytic leukemia (or AML-M5). Discovery of these epigenetic events has raised the possibility of a new class of leukemogenic genes.
Individuals with AML-M5 usually have a poor prognosis associated with hyperleukocytosis and extramedul[[Unsupported Character - Codename &shy;]]lary involvement. Abnormalities in MLL, NPM1, FLT3 and NRAS have been reported in this disease. However, these genetic and/or epigenetic changes occur only in some of AML-M5 cases. AML subtypes are clonal hematopoietic diseases caused by somatic alterations of genomic information. Genome analysis on these distinct subtypes allows not only a better under[[Unsupported Character - Codename &shy;]]standing of leukemogenesis but also the identification of new biomarkers and/or drug targets. To gain new insight into leukemogenesis and the molecular basis underlying the clinical heterogeneity of AML-M5, we carried out exome sequencing and subsequent Sanger sequencing analysis in a large series of individuals with this leukemia. Recurrent somatic mutations of DNMT3A (encoding DNA methyltransferase 3A) were identified in 23 of 112 (20.5%) cases. Among the six different mutations affecting Arg478, Gly543, Arg882 and Val897 in our case series, those affecting Arg882 were the most frequent. DNMT3A mutations were present with NPM1 mutations in the same sample in 16 cases, whereas mutations of NPM1 did not overlap with MLL abnormalities. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. We also carried out experiments to explore the possible epigenetic consequences of DNMT3A mutations. Indeed, gene expression pro[[Unsupported Character - Codename &shy;]]files and DNA methylation patterns showed some differences between patients with and without DNMT3A mutations. Notably, we found that members of HOXB genes were extensively upregulated at mRNA level in individuals with mutations of DNMT3A, and we detected hypomethylation of certain CpG islands in the HOXB locus. DNMT3A mutations enabled 32D cells to acquire growth and survival advantage even without growth factor. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. These data suggest a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases. The identification of genes whose expressions are specifically modulated by DNMT3A inactivation and the mechanism by which mutations in DNMT3A enable the pathogenesis of monocytic leukemia is on going.
Genomic sequencing researches in AML and other malignancies have greatly facilitated identification of new oncogenic mutations. With the accumulation of more new data, decision making of risk-stratified therapy will be possible and should be integrated into the individualized treatment of AML. We performed the study to systemically investigate the frequencies and the prognostic relevance of previously known genetic events and newly established molecular markers in a large series of adult AML patients. In particular, we intent to stratify the “ambiguous” AML patients who lacked cytogenetic prognostic markers into appropriate prognostic groups by using these genetic markers. We examined the gene status for both fusion products such as AML1 (CBFα)-ETO, CBFß-MYH11, PML-RARα and MLL rearrangement and point mutations in genes including FLT3, C-KIT, N-RAS, NPM1, CEBPA, WT1, ASXL1, DNMT3A, MLL, IDH1, IDH2, and TET2 in 1,185 AML patients. Clinical analysis was mainly carried out among 605 cases without recognizable karyotype abnormalities except for 11q23. 452 out of these 605 patients (74.7%) were found to have at least 1 mutation, and the relationship of gene mutations with clinical outcome was investigated. Notably, we revealed 13 novel DNMT3A mutations, and a correlation pattern among NPM1, DNMT3A, FLT3, IDH1 and IDH2 mutations; CEBPA and TET2 mutations were discovered. Multivariate analysis identified DNMT3A and MLL mutations as independent factors predicting inferior overall survival (OS) and event free survival (EFS), while bi-allelic CEBPA mutations or NPM1 mutations without DNMT3A mutations conferred a better OS and EFS in whole group and younger patients aged less than 60 years. In summary, we have established a new stratification system with which to subclassify the prognosis of AML without cytogenetic prognostic factors according to the mutation patterns of 4 genes. Evaluation of molecular markers in AML, especially through detection of DNMT3A, MLL, NPM1, and CEBPA mutations, can therefore be recommended. Further studies will be focused on using different treatment strategies according to AML genotypes.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY38-01. doi:1538-7445.AM2012-SY38-01