Selection of BCR/ABL-negative stem cells from marrow or blood of patients with chronic myeloid leukemia

Abstract

Philadelphia (Ph) or BCR/ABL-negative cells with immature phenotype (CD34-positive, DR-negative) can be recovered from patients with chronic myeloid leukemia (CML) in chronic phase. We used the technique described by Berardi et al (Science 1995; 267: 104–108) to select stem cells from marrow or blood of CML patients at diagnosis or during treatment with alpha-interferon. Mononuclear cells (MNC), and in some experiments CD34⁺ cells, were maintained for 7 days in the presence of 5-fluorouracil (5-FU), stem cell factor and interleukin-3. The number of viable cells recovered after culture was between 7.4 and 70.2 for 10⁶ cells plated. These cells exhibited the following phenotype: CD34⁺, CD117⁺, CD38⁻, lineage−, and were able to generate cobblestone areas and secondary colonies in long-term culture (LTC), with a frequency similar to that of cells selected from normal marrow. Study by fluorescence in situ hybridization of LTC cells or secondary colonies showed no evidence of BCR/ABLrearrangement. Reverse transcriptase polymerase chain reaction studies on pooled LTC cells or secondary colonies were also negative. By contrast, LTC cells or secondary colonies obtained from CML CD34⁺ cells without culture in the presence of 5-FU were always positive for BCR/ABL rearrangement. Finally, 5-FU selected cells were able to engraft NOD/SCID mouse, as human cells were detected in blood and marrow 10 weeks post transplantation, which were BCR/ABL negative by RT-PCR. This method of culture makes it possible to select constantly BCR/ABL-negative cells with capacities of development in LTC assay and of NOD/SCID mouse engraftment.