In vitro colony growth of acute myelogenous leukemia.

Colony formation in vitro by marrow cells from patients with untreated acute myelogenous leukemia (AML) and from patients in AML relapse is infrequent using the standard Robinson assay. A newly developed culture system has been described in which marrow from AML patients in these disease stages form leukemic cell colonies. In this in vitro system, phytohaemagglutinin is the essential stimulator for colony formation. The leukemic origin of the colonies has been proven by ultrastructural morphology and cytogenetics. It appears that colony formation by leukemic cells in this system is predominantly independent from the leukocyte factor which is the main stimulator in the Robinson assay for growing colonies of marrow cells from haematologically normal individuals. Bone marrow cells in untreated acute myelogenous leukemia (AML) demonstrate abnormal growth in vitro in the Robinson assay (Robinson et al., 1971; and Bull et al., 1973). Characteristically, there is a near total failure of colony formation; predominantly clusters are formed containing 20 cells or less (Bull et al., 1973; Greenberg et al., 1971; Moore et al., 1973 and 1974, and van Bekkum et al., in press). The absence of colonies has been shown to be due to a marked decrease of the normal myeloid precursor cell population in untreated AML. The small agregate formation of AML cells has been attributed to the suboptimal response of leukemic cells to the leukocyte stimulation factor. Because this poor proliferation in vitro might not represent the maximal in vitro and in vivo proliferation potential of the leukemic cells, we studied a number of modifications of the in vitro culture system. A number of factors were studied which may have some influence on cell proliferation in general, notably phytohaemagglutinin (PHA), which induces lymphocyte colonies in vitro (Rozenszajn et al., 1974), and endotoxin which has been demonstrated to increase the labelling index of leukemic cells in vivo (Golde et al.). In this paper an in vitro system is described in which marrow cells from untreated AML and AML in relapse were stimulated by phytohaemagglutinin (PHA) to form leukemic cell colonies in soft agar. These (similar) cells predominantly formed small aggregates (20 cells or less) in the presence of the normal leukocyte feeder layer alone. Moreover, in the course of the experiments, it appeared that by adding low concentrations of endotoxin to the cultures, the stimulating effect of PHA could be amplified.