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, but some Ph− presumably normal stem cells persist in most patients. Ph− cells are relatively frequent, compared to mature 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 CD38hi and CD38lo 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-DRloCD34+ 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 CD38lo sorted CD34+ cell fractions. 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 detected in untreated CML patients and as late as 6 years after diagnosis of CML in patients treated with hydroxyurea (HU) or interferon-α (IFN-α). We conclude that whilst appreciable numbers of Ph− primitive hemopoietic progenitors are present in the 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.