Evaluation at single cell level of residual Philadelphia negative hemopoietic stem cells in chronic phase CML patients

In chronic myeloid leukemia, accurate determination of Ph− Hemopoietic stem cells (HSC) in peripheral blood (PB), bone marrow (BM) and leukapheresis products is important for the selection of patients for whom mobilization, collection, and autografting of Ph− HSC are envisaged. To this effect, the BCR/ABL fusion was assessed at the single cell level in 25 sets of PB and BM samples using dual-color I-FISH in immunophenotyped CD34+ cells and RT-PCR of individual CFU-GM colonies. In 15 cases found to be 100% Ph+, the respective BCR/ABL gene was absent in 30% of CD34+ cells, while the respective transcripts could not be identified in 17% of CFU-GM. The mean percentage of BCR/ABL− CD34+ cells and CFU-GM cells was higher (38% and 29%, respectively) in untreated patients than in treated patients (24% and 7%, respectively). In eight cases with cytogenetic response (CgR), the percentage of Ph− metaphases correlated with the level of BCR/ABL− colonies in BM and PB and with the proportion of BCR/ABL− CD34+ cells in the BM. Immunophenotyping and FISH was fast, easy, always informative, and quantitative for the BCR/ABL− CD34+ cells. Our results show that (a) at early diagnosis a high frequency of BCR/ABL− HSC circulate in the PB and that Ph− hematopoiesis is not completely suppressed; (b) although normal clonogenic cells decline rapidly within a few months after diagnosis, appreciable numbers of normal CD34+ cells survive in chronic phase, especially in patients with CgR.