Molecular Responders (<3-log Reduction) among CML Patients in Complete Cytogenetic Remission Show a Lower Number of BCR-ABL+ Hematopoietic Progenitors Compared to Non-Responders

Background. BCR-ABL+ leukaemic hematopoiesis in CML patients treated with the tyrosyn kinase inhibitor Imatinib mesylate can remarkably diminish up to undetectable levels by cytogenetic or molecular analysis. This treatment seems to target leukemic progenitors cells while sparing some quiescent BCR-ABL+ leukaemic stem cells. In this study, we investigated the number of residual BCR-ABL+ hematopoietic progenitors in bone marrow (BM) samples from CML patients who reached a complete cytogenetic remission (CCR) with Imatinib. Materials and Methods. BM samples (15–20ml) were obtained from 18 CML patients in CCR under Imatinib treatment. All of them were in 1st chronic phase when Imatinib was started. A CD34+ cell-enriched population was selected from BM mononuclear cells using a double immunomagnetic column separation. CFCs and LTC-ICs were obtained by seeding the CD34+ selected population using standard culture methods. FISH studies for the BCR-ABL fusion gene (Dual color, Dual Fusion DNA probe form Vysis, INC, Downer Grove, IL) were performed on freshly selected CD34+ cells, pooled CFCs and LTC-ICs. A total of 100–200 nuclei were scored for each sample. The normal cut-off value used for these probes (0–2%) was established by our lab validation studies. MMR were defined as BCR-ABL/ABL ratios less than 0.05% (roughly equivalent to a 3-log reduction). Real-time QRT-PCR was performed using a TAQ-Man system (ABI Prism 7700 Perkin Elmer, CA) for BCR-ABL and ABL genes. Results. The median number and purity of selected CD34+ cells was 0.51 (0.17–2.1) x 106 cells and 95% (72–99). MMR was present in 10/18 (55.5%) CML patients. The median follow-up for patients in MMR or not in MMR was 34 (14–54) and 36 (6–59) months, respectively. None of the MMR-CML showed residual BCR-ABL+ cells detected by FISH on freshly selected CD34+ cells (MMR vs not MMR P=.0039 by Fisher’s test), CFCs and LTC-ICs. Conversely, 6 out of the 8 CMLs not in MMR showed residual BCR-ABL+ CD34+ cells, CFCs and LTC-ICs. Conclusions. According to previous studies (Blood2003; 101: 4701–7), we found that CML patients in CCR under Imatinib may have residual BCR-ABL+ hematopoietic progenitors. However, the size of leukemic progenitors may differ substantially between MMR and not MMR. CML patients with more than 3-log reduction of disease show undetectable BCR-ABL+ progenitors (CD34, CFC and LTC-IC) by FISH analysis. QRT-PCR on bone marrow samples may provide a reliable estimation of the number of residual leukaemic progenitors and may identify different clusters of CML patients in CCR.