Leukemic burden in subpopulations of CD34+ cells isolated from the mobilized peripheral blood of α-interferon-resistant or -intolerant patients with chronic myeloid leukemia

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of α- interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph⁺, whereas cells from four of five CP MPB were Ph^-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph⁺ cells, while AP/BC MPB contained 39.2% Ph⁺ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34⁺ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph⁺ cell frequency as determined by FISH within the CD34⁺Thy-1⁺ Lin^- and CD34⁺Thy- 1^-Lin^- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph⁺ cell frequencies of 59.2% and 72.7% for the CD34⁺Thy- 1⁺Lin^- and CD34⁺Thy-1^-Lin^- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34⁺Thy-1⁺Lin^- cells as compared with 1 of 133 for the Thy-1^- fraction indicating a higher frequency of primitive progenitor cells in the Thy-1⁺ subpopulation. CD34⁺ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph⁺. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34⁺Thy-1⁺Lin^- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.