Ex Vivo Expansion of Normal Progenitor Cells from Acute Myeloid Leukemia Cell-Contaminated CD34 + Peripheral Blood Progenitor Cells after Mafosfamide Purging

The rationale for purging of autologous acute myeloid leukemia (AML) grafts is to eradicate contaminating leukemic cells that might contribute to relapse. However, in vitro purging generally delays post-transplant hematopoietic recovery, thus increasing treatment-related complication rates. Theoretically, this prolonged aplasia might be shortened by the additional transplantation of ex vivo-generated progenitor cells. Therefore, we investigated whether nonleukemic progenitors could be expanded ex vivo from AML cell-contaminated CD34⁺ peripheral blood progenitor cell (PBPC) preparations. Nonleukemic CD34⁺-selected PBPC and AML cells (Kasumi-1, KG-1, primary AML blasts) were cultured in cytokine-supplemented liquid culture for up to 19 days. Cells were used either unmanipulated or following in vitro purging with mafosfamide(30, 50, 75 μg/ml). Ex vivo-generated cells were assessed by flow cytometry, progenitor cell assays, and polymerase chain reaction. Without prior purging, ex vivo culture markedly amplified AML cells as well as nonleukemic CD34⁺ PBPC (day 12: Kasumi-1, 18.5 ± 0.6-fold; KG-1, 52.2 ± 2.6-fold; CD34⁺, 74.1 ± 5.6-fold). Co-culture with leukemic cells did not affect CD34⁺ cell growth and vice versa. Following in vitro purging, CD34⁺ PBPC were expanded even at the highest mafosfamide dose (day 19: 25 ± 15-fold), whereas leukemic cells were markedly depleted (approx. 1.5 log). Furthermore, normal colony-forming units (CFU) could be effectively recovered (day 19: 10 ± 3.1% of prepurging input CFU), whereas CFU-L were depleted to undetectable levels in six of seven experiments. Finally, leukemic cells were undetectable following ex vivo co-culture of purged cells (CD34⁺ PBPC plus 10% Kasumi-1 cells or primary blasts), but were clearly detectable without purging. Taken together, these data demonstrated that ex vivo expansion of normal progenitors from mafosfamide-purged AML cell-contaminated grafts might be feasible.