BCR/ABL-negative progenitors are enriched in the adherent fraction of CD34(+) cells circulating in the blood of chronic phase chronic myeloid leukemia patients

Philadelphia chromosome-positive (Ph+) hemopoietic cells predominate in patients with chronic myeloid leukemia (CML) in chronic phase, bur some Ph- presumably normal stem cells persist in most patients. Ph- cells are relatively frequent, compared to nature cell populations, in primitive hemopoietic cell populations from CML patients. We have purified CD34(+) cells from chronic phase CML blood and separated them into two fractions on the basis of adherence or non-adherence to tissue culture plastic. We also separated CD34(+) CML cell populations into HLA-DRhi and HLA-DRlo fractions and CD38(hi) and CD38(lo) fractions by flow cytometry. The CD34(+) cells that adhered to plastic were predominantly CD33(-), CD38(-) and HLA(-)-DR; cells with these phenotypic properties were significantly rarer in the CD34(+) non-adherent cell population (P = 0.008-0.02). Expression of p210 BCR/ABL mRNA by adherent, non-adherent, HLA-DRhi and HLA-DR(lo)CD34(+) cell subpopulations was demonstrated by RT-PCR. Using fluorescence in situ hybridization (FISH) in conjunction with BCR and ABL probes we detected Ph+ and Ph- cells in both adherent and non-adherent CD34(+) cell fractions of 15/15 patients studied and in the HLA-DRlo or CD38(lo) sorted CD34(+) cell tractions, The concentration of Ph- cells in the adherent CD34(+) cell fraction was three-fold higher than in the non-adherent fraction (P = 0.001). Ph- adherent cells were defected in untreated CML patients and as late as 6 years after diagnosis of CML in patients treated with hydroxyurea (HU) or interferon-alpha (IFN-alpha). We conclude that whilst appreciable numbers of Ph- primitive hemopoietic progenitors are present in;he circulation in untreated patients and also in treated patients in late chronic phase, the majority of cells expressing CD34 but not CD33, CD38 or HLA-DR antigens, are part of the CML clone.