EX-VIVO CYTOKINE EXPANSION OF PERIPHERAL-BLOOD 5-FLUOROURACIL-TREATEDCD-34-POSITIVE CHRONIC MYELOID-LEUKEMIA CELLS INCREASES THE SELECTIONOF PH-NEGATIVE CELLS

It has been shown that normal early progenitor or stem cells persist i n the blood and bone marrow of patients with Philadelphia chromosome ( Ph)-positive chronic myeloid leukaemia (CML); it is also known that no rmal haemopoietic progenitors can be expanded er vivo in the presence of various cytokine combinations, The selection of normal (Ph-negative ) progenitor cells from CML patients would potentially be of considera ble clinical value for ex vivo purging and autologous transplantation. To obtain these cells, CD34-positive (progenitor) cells from the peri pheral blood (PB) of CML patients were pretreated with 5-fluorouracil (5FU) 5 mu g/ml) to suppress Ph-positive cells and then grown in suspe nsion culture for 7d with a combination of cytokines. We compared two combinations of cytokines: interleukin-1 alpha (IL1 alpha) and interle ukin-3 (IL3) with either Flt3-ligand (FLT3L) or stem cell factor (SCF) , Using these two combinations, we obtained the same degree of day 14 CFU-GM expansion (3.1 +/- 0.5 and 3.4 +/- 0.7 fold expansion). FISH an alysis showed that 5FU pretreatment significantly favoured a higher fr equency of Ph-negative cells after expansion in liquid culture, Moreov er, after 5FU pretreatment, the mean (+/-SEM) percentage of Ph negativ ity was significantly greater for IL1 alpha-IL3-FLT3L compared to IL1 alpha-IL3-SCF (19.1+/-2.5% v 14.8+/-2.3%, P=0.009, n=7). The output lo ng-term culture initiating cells (LTC-IC) which could only be detected after 5FU pretreatment and the combination, IL1 alpha-IL3-FLT3L were all Ph negative by FISH analysis. Thus, a subset of Ph-negative cells was selected from CML PB by 5FU and expanded using the combination of cytokines IL1 alpha-IL3-FLT3L and IL1 alpha-IL3-SCE Ex vivo expansion of putatively normal haemopoietic progenitor cells is feasible in CML.