Ex vivo cytokine expansion of peripheral blood 5-fluorouracil-treated CD34-positive chronic myeloid leukaemia cells increases the selection of Ph-negative cells.

Abstract

It has been shown that normal early progenitor or stem cells persist in the blood and bone marrow of patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML); it is also known that normal haemopoietic progenitors can be expanded ex vivo in the presence of various cytokine combinations. The selection of normal (Ph-negative) progenitor cells from CML patients would potentially be of considerable clinical value for ex vivo purging and autologous transplantation. To obtain these cells. CD34-positive (progenitor) cells from the peripheral blood (PB) of CML patients were pretreated with 5-fluorouracil (5FU) (5 microg/ml) to suppress Ph-positive cells and then grown in suspension culture for 7 d with a combination of cytokines. We compared two combinations of cytokines: interleukin-1alpha (IL1alpha) and interleukin-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 analysis showed that 5FU pretreatment significantly favoured a higher frequency of Ph-negative cells after expansion in liquid culture. Moreover, after 5FU pretreatment, the mean (+/-SEM) percentage of Ph negativity was significantly greater for IL1alpha-IL3-FLT3L compared to IL1alpha-IL3-SCF (19.1 +/- 2.5% v 14.8 +/- 2.3%, P=0.009, n = 7). The output long-term culture initiating cells (LTC-IC) which could only be detected after 5FU pretreatment and the combination, IL1alpha-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 IL1alpha-IL3-FLT3L and IL1alpha-IL3-SCF. Ex vivo expansion of putatively normal haemopoietic progenitor cells is feasible in CML.