Genetic aberrations in de novo myelodysplastic syndrome and their roles in disease progression

Project: Ministry of Health and WelfareMinistry of Health and Welfare Grants Research

Project Details

Abstract

Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia, and a tendency to progress to acute myeloid leukemia (AML). The molecular pathogenesis of MDS and the molecular basis of the progression of MDS to AML remained largely undefined. Previous studies on genetic alterations of MDS were carried out either at initial presentation or only at AML transformation. Moreover, most of the previous studies focused on chemotherapy- or irradiation-related MDS. Systematic study comparing the genetic abnormalities in the matched paired samples at both MDS and AML phases have not been reported. In the present proposal, we will analyze the paired samples at both MDS and AML phases in same individuals to get more insight into the pathogenesis of the progression from MDS to AML. Our aims are (1) to explore the multiplicity of genetic alterations including mutations of receptor tyrosine kinase (RTK)/tyrosine kinase/Ras signaling pathways and hematopoietic transcription factors, altered tumor suppressor genes (mutation or aberrant methylation), overexpression of oncogenes and MLL amplification in patients with de novo MDS at initial presentation and also to determine the role of genetic aberrations involved in the progression of MDS to AML; (2) to define the spectrum and patterns of cooperating mutations in both phases of disease; (3) to correlate the genetic abnormalities with the cytogenetic findings, clinicohematologic features and outcome; (4) to study the biological functions of novel mutants identified and the potential of cooperating mutations in leukemogenesis. Bone marrow samples from adult patients with de novo MDS at diagnosis and at AML transformation have been freshly frozen since 1991, which allow us to examine their genetic abnormalities in a systematic and expeditious way. DNA/cDNA polymerase chain reaction (PCR) followed by direct sequencing or followed by denaturing high-performance liquid chromatography with sequencing confirmation will be used to detect the mutations. Quantitation of aberrant methylation of target genes or promoters of tumor suppressor genes will be carried out by bisulfite-pyrosequencing analysis. MLL amplification will be analyzed by FISH, and real-time quantitative PCR assay will be used for measuring the expression levels of EVI-1 and HOX genes. Biological function of novel AML1 mutants will be examined with luciferase reporter assay for transactivation ability, with yeast two-hybrid assay for interacting protein analysis and further confirmed by electrophoresis mobility shift assay. The leukemogenesis of the novel mutations detected and cooperating mutations of AML1 and N-Ras will be determined by retroviral transduction/transplantation. Identification of multiplicity of genetic aberrations in patients with MDS at initial diagnosis and at AML transformation will provide more insight into the pathogenesis in the development and progression of MDS. Detection of the genetic alterations serves as potential targets for identifying patients to different targeted therapies in the future. The results of this study are expected to have immediate clinical implications and to provide a valuable resource for additional biological investigations in leukemogenesis.

Project IDs

Project ID:PG9804-0128
External Project ID:NHRI-EX98-9711SI
StatusFinished
Effective start/end date01/01/0931/12/09

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