Successful mafosfamide purging of bone marrow from chronic myelogenous leukemia (CML) cells

The in vitro sensitivity of human chronic myeloid leukemia-blast crisis and chronic phase (CML-BC and CML-CP, respectively) cells as well as adherent cell-depleted, T lymphocyte-depleted normal bone marrow cells (A-T-NBMC) to various concentrations of mafosfamide (ASTA Z7654), was examined by colony formation assay in the presence of IL-3 and GM-CSF, to test the possibility of purging of BMC from CML cells. Colony formation by CML cells was inhibited more efficiently than by NBMC. After the incubation with 50 micrograms/ml or 100 micrograms/ml of mafosfamide, the growth of leukemic CFU-GM was totally abrogated in 2/11 or 9/11 cases of CML-BC and in 1/7 or 6/7 cases of CML-CP, respectively. At the same time the CFU-GM arising from normal BMC were not inhibited totally with 50 or 100 micrograms/ml of the drug in any of five experiments. CML cells were still unable to form secondary colonies, while normal BMC were capable of regrowth. The CD34+ cells isolated form CML-BC and CML-CP patients were also more susceptible to mafosfamide cytotoxicity in comparison to CD34+ cells derived from NBMC. To confirm the possibility of purging, CML-BC cells were mixed with NBMC (1:1) and incubated with mafosfamide. Finally, the growing colonies were examined for the presence of bcr/abl hybrid gene by reverse transcriptase-Taq polymerase chain reaction (RT-PCR) and specific hybridization. The bcr/abl gene was not detected in the colonies growing after 100 micrograms/ml, and the signal was diminished after incubation with 50 micrograms/ml of mafosfamide, as compared to control. These results strongly suggest that high concentrations of mafosfamide may be useful for the purging of autologous BMC from CML cells.