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In vitro cytogenic studies in chronic myeloid leukemia (CML) Dubé, Ian David


In chronic myeloid leukemia (CML), neoplastic transformation occurs in a single pluripotent hemopoietic stem cell. The differentiated progeny of this clone predominate in vivo so that in most patients all the proliferating bone marrow cells bear the unique chromosomal marker of the transformed clone; the Philadelphia chromosome (Ph¹). This observation has raised questions about the fate of those pluripotent stem cells that are not involved in the leukemic clone. In this work, the hypothesis that nonleukemic stem cells exist in the hemopoietic system of most patients with CML but are suppressed from proliferating in vivo, was tested. A method was developed that allowed from two to more than 50 metaphases to be obtained from most erythroid and granulopoietic colonies harvested individually. When this technique was applied to myeloid colonies derived from progenitors in marrow and blood of patients in the chronic phase of CML, chromosomally normal, and presumably nonleukemic, hemopoietic progenitors were detected in a minority of patients. As a group, these were among those patients studied most recently after diagnosis and with lower than average white blood cell counts. It is possible that in these cases, the leukemic clone had not expanded to such a degree so as to preclude the detection of residual normal progenitors based on the analysis of reasonable numbers of colonies. The fact that chromosomally normal progenitors were observed in these patients at levels equivalent to that typical of normal individuals when there was no evidence for the proliferation of their progeny in direct preparations, suggests that the proliferation of nonleukemic progenitors was suppressed in vivo. Hemopoietic progenitors from patients in chronic phase were also cultured in long-term marrow cultures for several weeks before they were removed and assayed in methylcellulose cultures. The long-term marrow cultures selectively maintained chromosomally normal hemopoietic progenitors in most, but not all, patients. Using this assay, it was possible to demonstrate the existence of significant numbers of chromosomally normal myeloid progenitors in both newly diagnosed patients and in patients treated for over one year. The fact that progenitors in long-term cultures from a minority of patients remained exclusively Ph -positive suggests that heterogeneity exists among patients with respect to the ability of the leukemic clone to dominate hemopoiesis _in vivo. Alternatively, some marrow aspirates may, in fact, be devoid of normal progenitors . The extent to which the Ph¹-positive clone dominates hemopoiesis in the acute phase of CML was also studied. The results show persistence of the Ph¹-positive clone, even in cases where newer sub-clones containing additional abnormalities were predominant in vivo. The maturational block that characterizes the acute phase of CML was also studied in. vitro. It appeared that certain mutations, sometimes visible as gross chromosomal aberrations, were not compatible with normal myeloid differentiation in vitro. Failure to detect chromosomally normal myeloid progenitors in the acute phase of CML does not necessarily imply that normal progenitors were absent since the chemotherapy used was designed to non-selectively kill both leukemic and nonleukemic cells. The overall results demonstrate the existence of chromosomally normal myeloid progenitors in most patients in the chronic phase of CML and further illustrate how dynamic interactions between normal and leukemic progenitor populations may be studied in vitro.

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