Ramanujachar, R.; (2009) The molecular basis of E2A-HLF induced precursor-B cell acute lymphoblastic leukaemia in childhood. Doctoral thesis , UCL (University College London).

Abstract

Translocation t(17;19)(q21-22;p13) occurs in a small subset (0.5-1%) of childhood precursor B (Pre-B) cell acute lymphoblastic leukaemia (ALL), and produces a chimeric transcription factor, E2A-HLF, which is a fusion oncoprotein. E2A-HLF expression confers a poor clinical outcome with a high risk of refractory relapse, and is associated with distinctive clinical features. Patients in the UK, currently diagnosed with this translocation are being stratified to receive “high risk” therapy protocol, including allogeneic bone marrow transplantation. Our group has identified six patients in the UK and Ireland with t(17;19)(q21-22;p13), and three from other parts of the world, in the last six years. These are in addition to the previously identified fourteen patients from a worldwide literature search. This extended patient base, coupled with poor outcome, necessitates an improved understanding of this translocation. We aimed to study the molecular aetiology of the leukaemia, and establish the molecular pathways involved. To study the molecular aetiology, we analysed six patients’ genomic DNA samples at diagnosis and/or relapse and the DNA extracted from their corresponding Guthrie cards (neonatal blood spots) by employing PCR based methods. We identified a type I genomic rearrangement in two of the patients, and a type II in one patient, at the region of the genomic fusion. Two other patients were identified with a unique type of rearrangement not classifiable as either type I or II. The mechanism of translocation in our patient samples, suggested an aberrant V(D)J rearrangement at the breakpoint region. Analysis of the Guthrie cards indicated the possibility of a prenatal origin for this translocation. We developed an in vitro inducible expression system (Tet-off) for E2A-HLF expression to understand the molecular pathways activated by this translocation. Murine foetal liver haematopoietic progenitor cells (HPC) were transduced with retroviral vectors expressing E2A-HLF in myeloid and lymphoid cultures. This allowed us to identify a gene expression profile for E2A-HLF from immortalised foetal liver HPC in methylcellulose and liquid culture. The downstream target genes of E2A-HLF identified in our study included Il15, Cd28, Kdr, Ccl9 and Ccl6. Further validation has been carried out on Il15 as a potential target of E2A-HLF.

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