DETECTION AND CHARACTERIZATION OF PRIMITIVE MALIGNANT AND NORMAL PROGENITORS IN PATIENTS WITH ACUTE MYELOGENOUS LEUKEMIA USING LONG-TERM COCULTURE WITH SUPPORTIVE FEEDER LAYERS AND CYTOKINES

Analysis of the mitogenic activity of interleukin-3 (IL-3), Steel fact or (SF), and flt-3 ligand (FL) on acute myelogenous leukemia (AML) bla sts using the short-term endpoints of proliferation in H-3-thymidine ( H-3-Tdr) incorporation assays or methylcellulose cultures (colony assa ys) showed that greater than 90% of samples contained cells that were responsive to one or more of these cytokines. With this information, c ulture conditions that were known to support normal long-term culture- initiating cells (LTC-IC) were tested, with or without supplements of one or more of these three growth factors, for their ability to suppor t primitive progenitors from 10 cell samples from patients with AML. I n all cases cytogenetically abnormal colony farming cells (CFC) were d etected after 5 weeks when AML peripheral blood or marrow cells were c ocultured on preestablished, normal human marrow feeders (HMF) and/or SI/SI mouse fibroblast feeders and the number of CFC detected in these 5-week-old LTC maintained a linear relationship to the number of inpu t AML cells. Limiting dilution analysis, performed on 6 of the 10 samp les, showed the frequency of AML cells initiating LTC (AML LTC-IC) to be 5- to 300-fold lower than the frequency of AML-CFC in the same cell sample, whereas the average number of CFC produced per LTC-IC varied from 1 to 13. Surprisingly, in each case the concentration of cytogene tically normal ITC-IC detected in AML patient blood was at least 10-fo ld higher than that previously observed in the blood of normal individ uals. ''Mixed'' mouse fibroblast feeders engineered to produce human G -CSF,IL-3, and SF did not enhance detection of AML LTC-IC but did incr ease the output of cytogenetically normal CFC from LTC of 3 of 4 patie nt samples. Supplementation of AML LTC with IL-3 and exogenously provi ded SF and/or FL increased the output of AML-CFC from 5-week-old LTC b y greater than or equal to twofold with 5 of 9 patient samples, wherea s in one case exogenous addition of FL reduced the output of malignant CFC from LTC. These studies show that conditions that support normal LTC-IC also allow a functionally analogous but rare AML progenitor cel l type to be detected. In addition, differences in the responses of no rmal and leukemic cells to various cytokines active on normal LTC-IC w ere revealed. Further analysis of these differences may enhance our un derstanding of leukemogenesis and lead to observations that could be e xploited therapeutically. (C) 1997 by The American Society of Hematolo gy.