Abstract

Allogeneic hematopoietic progenitor cell transplantation (HPCT) remains an established curative approach for selected patients with chronic myelogenous leukemia (CML). Patients for whom an allogeneic donor cannot be found or who are unable to tolerate allotransplantation require post-Gleevec alternatives. Autologous HPCT causes less morbidity/mortality than allotransplantation but CML cells in the autograft pose a potential risk for post-transplant relapse. An effective purging method for eliminating leukemic cells without compromising normal reconstituting activity would make autotransplantation a more attractive strategy for selected CML patients. In previous studies, we and others have shown that primitive CML cells are selectively killed after brief treatments in vitro with Mafosfamide (Maf) or Gleevec. Additionally, cultured primitive CML cells differentiate much more rapidly than their normal counterparts resulting in a selective depletion of CML stem cells after 1–2 weeks. Here we report the development of a novel autograft purging strategy that combines the use of a brief exposure of CML cells to Gleevec plus Maf followed by extended culture without cytotoxic agents but with added recombinant cytokines that favor the maintenance of normal stem cells.

Methods: CD34+ cells were isolated from the peripheral blood progenitor cell (PBPC) products of CML patients and normal donors, treated for 72 hrs with graded concentrations of Gleevec (0.5–1.0 uM) followed by 0, 30, 60 or 90 ug/ml of Maf for 30 min. Cells were then washed and cultured for 14 days in medium containing 10% FBS and 100 ng/ml each of stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (TPO). Following treatment, cells from each group were evaluated for total viable cells, BCR-ABL message by real time polymerase chain reaction (RT-PCR) assays and colony-forming unit-granulocyte macrophage (CFU-GM) content in methylcellulose assays.

Results: BCR-ABL+ cells were eliminated in the CML PBPC products from patients in chronic phase (n=3), but not blast crisis (n=1) when treated with Gleevec (0.75 uM) and Maf (60 ug/ml) followed by 2 weeks in vitro with SCF+G-CSF+TPO.

Residual Ph positive cells by RT PCR

Patient/Disease stage Culture alone Drugs alone (Gleevec 0.75 or 1uM + Maf 60ug/ml) Culture + Drugs 
1-chronic phase/Gleevec refractory Positive Positive Negative 
2-chronic phase/Gleevec refractory Positive Positive Negative 
3-chronic phase/Gleevec naïve Positive Negative Negative 
4-blastic phase/Gleevec naïve Positive Positive Positive 
Patient/Disease stage Culture alone Drugs alone (Gleevec 0.75 or 1uM + Maf 60ug/ml) Culture + Drugs 
1-chronic phase/Gleevec refractory Positive Positive Negative 
2-chronic phase/Gleevec refractory Positive Positive Negative 
3-chronic phase/Gleevec naïve Positive Negative Negative 
4-blastic phase/Gleevec naïve Positive Positive Positive 

Treatment of CD34+ PBPCs from normal donors with 0.75 uM Gleevec + 60 ug/ml Maf reduced CFU-GMs to 15% of input controls immediately post drug-exposure but these recovered to 320% of input after the additional 2 weeks in culture.

Conclusion: Triple purging of CML cells with Gleevec, Maf and 2 weeks of culture appears to eradicate chronic phase BCR-ABL+ cells while retaining most of the normal progenitor activity as assessed by in vitro clonogenic assays. This approach may be useful as a strategy for purging CML autografts for Gleevec-refractory patients and, pending confirmatory results from in vivo xenograft assays, will be tested in a clinical autotransplant trial.

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