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UBC Theses and Dissertations

Clonality and cycling status of leukemic progenitors from patients with acute myeloid leukemia (AML) Guan, Yinghui

Abstract

We have detected a high concentration of cytogenetically normal long term culture-initiating cells (LTC-IC) in the peripheral blood (PB) of 12 patients with newly-diagnosed AML. To determine if these progenitors originated from normal polyclonal hematopoiesis the PCR-based human androgen receptor allele (HUMARA) assay was used to study PB cells from 5 female patients with cytogenetically abnormal AML. In 4/5 samples cytogenetics and clonality data from LTC indicate that a substantial number of normal polyclonal hematopoietic progenitors often persist in AML PB at diagnosis. However, the fifth AML sample contrasted with the others since the HUMARA assay demonstrated that LTC-derived colonies were predominantly clonal although the majority of the progenitors were cytogenetically normal. Thus, it appears that the abnormality detected on routine cytogenetics is not a reliable marker of the leukemic clone in this case. The second goal of this thesis was to characterize cycling status of different leukemic progenitors. An overnight 3H-thymidine (3H-Tdr) suicide assay was used to analyze the proliferative status of malignant progenitors detected in CFC, LTC-IC and NOD/SCID mouse leukemia initiating cell (NOD/SL-IC) assays from the peripheral blood of 15 patients with newly-diagnosed AML. FISH analysis of colonies from CFC and LTC-IC assays confirmed that most cytogenetically abnormal CFC and LTC-IC from the 15 samples as well as cytogenetically normal LTC-IC detected were actively cycling. However, the same assay has demonstrated that some AML LTC-IC and most NOD/SL-IC were largely quiescent. The growth factor responsiveness of quiescent AML cells was then studied and their functional properties compared to those of AML cells in active cell cycle. Hoechst 33342/Pyronin Y staining and FACS sorting were used to isolate Go cells in from the G1 and S/G2+M subpopulations of peripheral blood cells from 4 newly diagnosed AML patients. Go AML cells from these patients exhibited a strong tendency to enter active cell cycle when cultured even in the absence of supportive growth factors. LTC-ICs were easily detected among cycling AML cells that had been cultured for 72 hours. In contrast, the exit from Go dramatically reduced the ability of AML progenitors to engraft in mouse bone marrow.

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