Immunologic purging of autologous peripheral blood stem cell products based on CD34 and CD133 expression can be effectively and safely applied in half of the acute myeloid leukemia patients.

PURPOSE: Several studies have shown survival benefit by autologous stem cell transplantation in acute myeloid leukemia (AML) after purging of grafts. This has, however, not been confirmed in randomized studies due to high toxicity of purging modalities for normal progenitor/stem cells. In this study, we investigated whether positive selection for CD34+ and/or CD133+ cells, which results in high recovery of normal progenitor/stem cells, is applicable for purging AML grafts. EXPERIMENTAL DESIGN: Positive selections of normal stem cells using CD34 and/or CD133 can be done if one or both markers are absent or have dim expression and remain so during the course of the disease. Marker expressions in newly diagnosed AML were measured with flow cytometry using a cutoff value for positivity of 1%. Stability of marker expression was studied by pairwise comparison of material at diagnosis and relapse. Leukemia associated phenotype expression was used to measure the efficacy of tumor cell reduction. RESULTS: In newly diagnosed AML (n = 165), we found no CD34 and/or CD133 expression in 32% of the cases and dim expression in 20% of the cases. No increase in the percentage of CD34+ cells (n = 44) and CD133+ cells (n = 29) was found in corresponding relapses. Positive selection using grafts contaminated with AML blasts, showing either no or dim expression of CD34 or CD133, resulted in a 3 to 4 log tumor cell reduction (n = 11) with median 50% recovery of normal stem cells. CONCLUSIONS: Purging by positive selection of CD34+ and/or CD133+ cells can safely, effectively, and reproducibly be applied in about 50% of AML cases.

Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts.

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.

Function of the ABC transporters, P-glycoprotein, multidrug resistance protein and breast cancer resistance protein, in minimal residual disease in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Relapse is common in acute myeloid leukemia (AML) because of persistence of minimal residual disease (MRD). ABC-transporters P-glycoprotein (Pgp) and multidrug resistance protein (MRP), are thought to contribute to treatment failure, while it is unknown whether breast cancer resistance protein (BCRP) does so. However, whether up-regulation of pump activity or selection of subpopulations with higher pump activity occurs during chemotherapy is unclear. The aim of this study was to elucidate whether ABC-transporter function changes during the course of disease. DESIGN AND METHODS: MRD cells were identified using leukemia-associated phenotypes combined with a fluorescent probe assay with substrate/modulator: Syto16/ PSC833 (Pgp), calcein-AM/probenecid (MRP) and BODIPY-prazosin/Ko143 (BCRP); efflux profiles were directly compared with blasts at diagnosis and relapse from the same patient. RESULTS: At diagnosis BCRP activity was undetectable in AML blasts from 23/26 cases, while Pgp activity was present in 36/45 and MRP activity in 26/44 of the cases. Furthermore, no subpopulations of blasts with considerably higher drug efflux capacities were found. Overall, no consistent changes were observed at follow-up [during chemotherapy (n=20), MRD (n=37), relapse (n=26))] in forty-five patients, the mean activities (as percentages of values at diagnosis) were 97% (Pgp), 103% (MRP) and 102% (BCRP). INTERPRETATION AND CONCLUSIONS: Emergence of MRD is thus not accompanied by either upregulation of ABC-transporter function during or after chemotherapy or by selection of pre-existing highly resistant subpopulations. The prognostic value of Pgp and MRP is, therefore, likely related to drug efflux capacity homogeneously distributed in the whole blast population, while BCRP probably has a limited function in drug efflux-related resistance in AML.