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Characterization of hemopoietic stem cells in chronic myeloid leukemia (CML)

University of British Columbia

Much evidence indicates that the target of neoplastic transformation In chronic myeloid leukemia (CML) is a pluripotent hemopoietic stem cell, from which differentiated blood cells of the myeloid and lymphoid lineages Eire normally derived throughout adult life. CML is one of the best defined hematologic malignancies, being characterized by a consistent chromosomal and molecular abnormality, the Philadelphia (Ph1) chromosome and the BCR/ABL fusion gene, respectively. In most patients with CML, expansion of the leukemic clone is evident both at the level of mature blood cells and of their immediate precursors. Relatively little is known about the number or characteristics of the most primitive cells responsible for maintaining the leukemic clone. This has been due to a lack of suitable assays for these cells. In this thesis, I describe the development of a quantitative assay for primitive CML hemopoietic cells based on the long-term culture-initiating cell (LTC-IC) assay recently established for very primitive hemopoietic cells in normal human bone marrow. Blood samples from CML patients with high WBC counts were used as an highly enriched source of Ph1-positive leukemic progenitors. Clonogenic cell output after 5 weeks in LTC was shown to be a linear function of the number of Input blood cells, enabling this endpoint to be used as a quantitative, albeit relative, measure of CML LTC-IC. The application of limiting dilution methods allowed derivation of absolute CML LTC-IC frequencies. LTC-IC in CML blood were found to be markedly increased in proportion both to the WBC count and other clonogenic progenitors. This is in contrast to the situation in CML marrow where in the specimens examined, the frequency of leukemic LTC-IC was found to be decreased, on average >20 fold relative to normeil LTC-IC in normal marrow. Characterization of LTC-IC and clonogenic cells in CML blood and their comparison to these cells in normal marrow and blood showed some similarities and differences. Most (but not all) CML clonogenic cells were similar to clonogenic cells in normal marrow, but different from clonogenic cells in normal blood in terms of thefr apparent activation state, as measured by Rh 123 staining, HLA-DR expression, forward light scatter, and sensitivity to 4- hydroperoxycyclophosphamide. Most but not all CML LTC-IC were also found to express an activated phenotype, and thus differed from the LTC-IC in normal marrow and blood which exhibit a phenotype expected of quiescent cells. The proliferative/self-maintenance and differentiative capacities of CML LTC-IC were also studied. CML LTC-IC were not different from normal LTC-IC in terms of either the total number or different types of clonogenic cells they produced after 5 weeks. However, CML LTC-IC were defective In their self-maintenance. These results, together with the decreased quantity of these cells in CML marrow, explains why leukemic progenitor output rapidly declines in LTC initiated with CML marrow. In summeiry, my thesis demonstrates that primitive CML hemopoietic cells can be detected, quantltated, and phenotypically and functionally characterized using the LTC-IC assay recently developed for normsd marrow. The data obtained provide an explanation for the rapid and selective decline of CML progenitors observed in LTC initiated with CML marrow, but not CML blood. The phenotypic differences of leukemic and normal LTC-IC should provide Important Information for the design of new treatment strategies and for further studies into the pathogenetic mechanisms leading to the development of the neoplastic clone in CML.

Thesis/Dissertation

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2008-12-18

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http://hdl.handle.net/2429/3150

Pathology

University of British Columbia

UBCV

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