Genetic aberrations in AML with minimal differentiation (AML-M0) and their roles in leukemogenesis

Background: De novo acute myeloid leukemia (AML) is a heterogeneous group of genetically diverse hematologic malignancies. The genetic abnormalities of AML influence disease aggressiveness and responses to therapy. Two-hit model (Class I and Class II gene mutations) of leukemogenesis has been proposed. Very recently, using whole-genome technology with SNP-array analysis and whole genome sequencing has identified some novel mutated oncogenes or tumor suppressor genes in patients with myeloid neoplasms. Minimally differentiated AML (AML-M0) is a distinct subgroup of AML. Our previous study showed that AML-M0 accounted for 6% in de novo adult AML and 5% in childhood AML. We have detected several novel RUNX1 mutations in AML-M0. Their biological functions require further study. The full complement of collaboration of gene mutations and their frequencies in AML-M0 remains to be defined. Search for additional differentially genetic or genomic aberrations in AML-M0 will give more insight into the leukemogenesis of this subgroup of leukemia. Aims: In the present project, we aim (1) to analyze the mutation status of newly identified mutated genes including C-CBL, TET2, ASXL1 and IDH1/2 in patients with AML-M0 at both diagnosis and relapse; (2) to study the biological functions of the novel RUNX1 mutants we have identified in AML-M0 and to determine the leukemogenic potential of selected cooperative mutated genes with RUNX1 mutants in a mouse model; (3) to analyze comprehensively the genomic aberrations on AML-M0 samples using exome sequencing; and (4) to correlate the genetic aberrations with the clinicohematologic features to determine their diagnostic and prognostic relevance. Materials and Methods: Bone marrow samples from patients with de novo AML-M0 at diagnosis, in complete remission and at relapse will be analyzed for various gene mutations using DNA/cDNA polymerase chain reaction (PCR) followed by direct sequencing or followed by denaturing high-performance liquid chromatography with sequencing confirmation to detect the mutations. Biological functions of novel RUNX1 mutants in AML-M0 samples will be examined with luciferase reporter assay for transactivation ability, by electrophoresis mobility shift assay for the DNA-binding ability, and by immunoprecipitation assay to determine the interaction with CBF-. Whole-genome fragment libraries will be prepared using an Illumina’s genomic DNA sample preparation kit and then for target enrichment by using SureSelect Block #1, Block #2, Block #3 (Agilent Technology). The captured library will be amplified by using SureSelect GA PCR Primers. Amplified library will be used for cluster generation and subsequently placed in GAIIX illumina sequencer for 76bp paired end reads. The sequences will be compared to the novel sequence changes at diagnosis and relapse with the complete remission samples serving as germline controls. Selected mutations identified will be validated by resequencing these sites. Finally, the clinical characteristics and outcome of the patients are linked to the genetic and/or genomic abnormalities. Significance: Identification of multiplicity of genetic aberrations in patients with AML-M0 at initial diagnosis and relapse will provide more insight into the pathogenesis in the development and relapse of AML-M0. Our study holds promise to find a unique fusion or mutated gene that might permit development of effective targeted therapy against AML-M0 in the future.

Project ID:PC10202-0020
External Project ID:NSC100-2314-B182-023-MY3