Characterization of Genetic Abnormalities at Disease Progression of Chronic Myeloid Leukemia

Abstract: Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the translocation t(9;22)(q34;q11) and progresses from a relatively indolent chronic phase (CP) to an accelerated phase (AP) and finally to the more aggressive blast crisis (BC). The general aim of this thesis was to identify and characterize genetic abnormalities occurring at disease progression of CML using molecular cytogenetic, molecular genetic, and bioinformatic analyses. In the first study, multicolor fluorescent in situ hybridization (M-FISH) was used to identify possible cryptic genetic changes in CML BC and AP cases. Two novel and cytogenetically cryptic balanced translocations, t(7;17)(p15;q23) and t(7;17)(q32-34;q23), and an AML-associated translocation, t(9;11)(p21-22;q23), with concomitant rearrangement of the MLL gene, were detected, suggesting that balanced translocations are more common during CML progression than previously indicated by G-banding alone. In the second study, further characterization of the t(7;17)(p15;q23) and t(7;17)(q32-34;q23) showed rearrangement of a novel gene located in 17q23, Musashi-2 (MSI2), encoding a putative RNA-binding protein. In the case with the t(7;17)(p15;q23), a MSI2/HOXA9 fusion transcript was identified, harboring both RNA recognition motif domains of MSI2 and the homeobox domain of HOXA9, suggesting an important role in the disease progression of CML. In the third study, the mechanism of formation of ETV6/ABL1 rearrangement in CML BC, as well as the possible clinical and genetic effects of imatinib treatment, was investigated. FISH analysis revealed that a complex array of chromosomal rearrangements were required for the generation of ETV6/ABL1. Moreover, imatinib treatment resulted in a durable, albeit transient, hematologic and genetic response, lasting approximately three months. Upon disease relapse, no mutations were identified in the SH1 (kinase) domain of ABL1, but a decrease in the expression level of wild-type ETV6 was observed that may have been a contributory factor for the relapse. In the fourth and final study, a detailed mapping of the breakpoints of i(17q) - the most common structural rearrangement observed at disease progression in CML – was performed using locus-specific FISH. In silico analysis of the involved breakpoint region revealed a complex genomic architecture, characterized by large (~ 38-49 kb) palindromic, low-copy repeats (LCR), strongly suggestive of being a genomic feature implicated in the mechanism of formation of i(17q).