Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN- alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI- CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG- dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.

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We studied the clinical, hematologic, cytogenetic, and molecular biologic features of seven patients with Philadelphia (Ph1) chromosome- negative chronic myeloid leukemia (CML). In five cases the hematologic findings were indistinguishable from those of patients with classical Ph1-positive disease. Myeloid cells were studied by chromosome-banding techniques. One patient had a masked Ph1 chromosome (with translocation t(4;9;22)), one had a deletion involving chromosome 16, and one had a small minority population of 22q- cells without 9q+ but otherwise normal metaphases; metaphases from the other four patients were entirely normal. DNA prepared from the myeloid cells was digested with the restriction enzymes EcoRI, HindIII, BamHI and BglII. Southern analysis using a 0.6-kb fragment of the breakpoint cluster region (bcr) gene showed the presence in each patient's DNA of a germline fragment together with a rearranged fragment or fragments with at least one of the restriction enzymes. We conclude that genomic changes in the bcr gene characteristic of CML can be present in the absence of a Ph1 chromosome.

We investigated the effects of brief (2 h) and continuous exposure to recombinant interferon-alpha (2a) (rIFN-alpha) on the proliferation of primitive (blast colony-forming cells, Bl-CFC) and committed myeloid progenitor cells (BFU-E and GM-CFC) derived from blood and bone marrow of patients with chronic myeloid leukaemia (CML) and normal subjects. In all three clonogenic assays, rIFN-alpha suppressed colony formation in a dose-dependent manner. No differences were detected in the proliferation of CML or normal Bl-CFC and GM-CFC exposed to rIFN-alpha. Erythroid colony formation by normal, but not by CML BFU-E, was inhibited by relatively low concentrations (100 U/ml) of rIFN-alpha. However, in patients whose blood or marrow contained a mixture of Philadelphia chromosome (Ph)-positive and Ph-negative BFU-E, cytogenetic analysis of individual erythroid colonies showed no differential inhibition by rIFN-alpha. We found no difference in the sensitivity to rIFN-alpha of GM-CFC from patients whose leukaemic cells expressed BCR/ABL mRNA with the b2a2 junction and that of GM-CFC from patients with the b3a2 mRNA. We conclude that (1) rIFN-alpha does not have a significant leukaemia-specific effect on the progenitor cells detected in these assays, and (2) the sensitivity of CML GM-CFC to rIFN-alpha is independent of the type of BCR/ABL message present in the cells. The clinical efficacy of rIFN-alpha could be due to selective toxicity to cells not assayed in this study, to effects on accessory cells or to alterations induced in progenitor cell/stromal cell interactions.