MOST ACUTE MYELOID-LEUKEMIA PROGENITOR CELLS WITH LONG-TERM PROLIFERATIVE ABILITY IN-VITRO AND IN-VIVO HAVE THE PHENOTYPE CD34(+) CD71(-)/HLA-DR-/

Acute myeloid leukemia (AML) occurs as the result of malignant transfo rmation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML cells are capable of proliferation in vitro, suggesting that AML cells may be o rganized in a hierarchy, with only the most primitive of these cells c apable of maintaining the leukemic clone. To further investigate this hypothesis, we have evaluated a strategy for purifying these primitive cells based on surface antigen expression. As an in vitro endpoint, w e have determined the phenotype of AML progenitor cells which are capa ble of producing AML colony-forming cells (CFU) for up to 8 weeks in s uspension culture (SC) and compared the phenotype with that of cells w hich reproduce AML in nonobese diabetic/severe combined immunodeficien cy (NOD/SCID) mice. AML cells were fluorescence-activated cell sorted (FACS) for coexpression of CD34 and CD71, CD38, and/or HLA-DR and the subfractions were assayed in vitro and in vivo at various cell doses t o estimate purification. While the majority of primary AML CFU lacked expression of CD34, most cells capable of producing CFU after 2 to 8 w eeks in SC were CD34(+)/CD71(-). HLA-DR expression was heterogeneous o n cells producing CFU after 2 to 4 weeks. However, after 6 to 8 weeks in SC, the majority of CFU were derived from CD34(+)/HLA-DR- cells. Si milarly, the majority of cells capable of long-term CFU production fro m SC were CD34(+)/CD38(-). Most cells that were capable of engrafting NOD/SCID mice were also CD34+/CD71- and CD34(+)/HLA-DR-. Engraftment w as not achieved with CD34(+)/CD71(+) or HLA-DR+ subfractions, however, in two patients, both the CD34(+) and CD34(-) subfractions were capab le of engrafting the NOD/SCID mice. A three-color sorting strategy com bining these antigens allowed approximately a 2-log purification of th ese NOD/SCID leukemia initiating cells, with engraftment achieved usin g as few as 400 cells in one experiment. Phenotyping studies suggest e ven higher purification could be achieved by combining lack of CD38 ex pression with the CD34(+)/CD71(-) or CD34(+)/HLA-DR- phenotype. These results suggest that most AML cells capable of long-term proliferation in vitro and in vivo share the CD34(+)/CD71(-)/HLA-DR- phenotype with normal stem cells. Our data suggests that in this group of patients t he leukemic transformation has occurred in a primitive progenitor, as defined by phenotype, with some degree of subsequent differentiation a s defined by functional assays. (C) 1998 by The American Society of He matology.