EFFICIENT LONG-TERM MAINTENANCE OF CHRONIC MYELOID LEUKEMIC COBBLESTONE AREA FORMING CELLS ON A MURINE STROMAL CELL-LINE

Stroma-supported long-term cultures (LTC) of chronic myeloid leukemia (CML) progenitor cells have previously revealed differences between no rmal and malignant stem cells with respect to their maintenance and ad hesive properties. Using the cobblestone area forming cell (CAFC) assa y and LTC, we have examined the frequencies of stem cell subsets, thei r ability for long-term progenitor cell production and the relative fr equencies of malignant and normal progenitor cells before and after a 5-6 week culture period. Cells were obtained from bone marrow (BM) and peripheral blood (PB) samples of patients in chronic phase CML. CD34- enriched cells were sorted by FACS on the basis of CD34 and CD38 expre ssion and overlaid on confluent stromal layers of murine FBMD-1 cells. The presence of the bcr/abl chimeric gene was detected by fluorescent in situ hybridization (FISH) using differently labelled bcr and abl-s pecific probes. In the CD34(pos)/CD38(pos) subset of CML-PB, represent ing 64-95% of CD34(pos) cells, CAFC frequencies at week 1 (wk-1) were much higher than those of CAFC wk-5 (1.10(4)/10(5) cells vs 1.10(3)/10 (5)). In contrast, in the CD34(pos)/CD38(neg) subset, representing 2-3 % of CD34(pos) cells, the frequency of CAFC wk-1 was only 1.10(2)/10(5 ) cells, but a high CAFC frequency (10(3)-10(4)/10(5)) was detected af ter 5 weeks of culture. CAFC frequencies in the CD34(pos) subset obtai ned from CML-BM were 10- to 100-fold lower than those from CML-PB, but displayed a similar distribution over CD38(pos), CD38(dim) and CD38(n eg) cells. Analysis of the percentage of Philadelphia chromosome-posit ive (Ph(+)) and Ph(-) cells by FISH on freshly sorted cells revealed t hat normal cells were not enriched in any CD34(pos)/CD38 subset. In ad dition, Ph(-) as well as Ph(+) cells were maintained with similar effi ciency throughout 5 week LTC. These results demonstrate that immature normal and malignant stem cells in CML have a comparable distribution on the basis of CD34 and CD38 expression. The ability to maintain imma ture normal and malignant hemopoietic cells with similar efficiency in LTC provides a model enabling a direct comparison of differential eff ects of cytokines or drugs on either normal or malignant immature stem cells in CML.