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Multilineage and single-lineage hemopoietic precursors were studied in 102 bone marrow transplant recipients and their respective donors to determine their contribution to clinical outcome as measured by time to engraftment and survival. The patient population was heterogenous with respect to diagnosis and disease status. They included individuals with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), aplastic anemia, and a few other hematopoietic malignancies. The frequency of various clonogenic precursors in the normal donor population varied considerably. The data yielded a symmetrical distribution. In contrast, most bone marrow transplant recipients presented with significantly reduced numbers of clonogenic cells before transplantation, resulting in skewed distribution profiles. Serial studies of recipients demonstrated a significantly lower than normal level of clonogenic precursors even 3 and 4 years after transplantation. The median values and distribution profiles approximated those observed before transplantation but did not return to measurements obtained for normal donors. Patients with ALL deviated from this pattern. The median values and distribution profiles of clonogenic precursors before transplantation approximated the pattern of normal donors. The frequency of clonogenic progenitors after transplantation, however, remained significantly lower than that of their respective donor or pretransplant values. Cell cycle studies performed after normalization of peripheral blood hematopoietic parameters demonstrated for most recipients that a higher than normal proportion of multipotent cells was in S-phase (P = .011). By univariate and multivariate approaches, clonogenic precursors and clinical parameters were assessed for their contributions to clinical outcome as measured by time to engraftment and survival time. The number of nucleated cells in the transplant inoculum contributed to survival independent of other risk factors. Patients with a higher cell load had a higher probability of surviving than did patients with a lower cell concentration in the transplant inoculum (P = .042). The frequency of clonogenic precursors in the transplant inoculum altered neither survival nor time to engraftment. The time to engraftment was significantly influenced by the frequency of clonogenic megakaryocyte precursors (CFU-M) observed in recipients prior to transplantation (P = .003). Patients with high values engrafted faster than did patients with a low frequency of CFU-M. This was independent of both diagnosis and disease status of the patients at time of transplantation.

Marked patient-to-patient variation is observed when blood or marrow from AML patients is examined using colony methods in culture. Concentrations of the progenitors of colonies change with time during the course of the disease. We asked whether blast progenitor properties were more stable. We measured blast cell self-renewal and drug sensitivity (adriamycin and cytosine arabinoside) repeatedly in the courses of seven AML patients. These properties were found to be stable or slowly evolving. We conclude that capacity for renewal and sensitivities to certain chemotherapeutic drugs are heritable characteristics in leukemic clones.

The blast cell population in AML includes progenitors capable of colony formation in culture. Certain properties of these progenitors have been determined, including their capacity for self-renewal and their sensitivities to the chemotherapeutic drugs cytosine arabinoside (Ara- C) and adriamycin (Adria). Wide patient to patient variation was found in these properties, although they were stable during the course of the disease in each patient. We tested the properties, together with clinical risk factors, as attributes contributing to the variation in remission induction and survival. As univariate parameters, self- renewal and Ara-C sensitivity contributed to remission induction, but only self-renewal was related to survival. In multivariate analysis, self-renewal, age and percentage blasts in the marrow contributed to outcome variation; drug sensitivities were not significant. We conclude that self-renewal, a biological property of malignant AML clones, although measured in culture, plays a significant role in determining response to treatment and survival in AML.

Two culture methods are available for the study of the blast cells of acute myeloblastic leukemia (AML). One is an assay for clonogenic precursors; it depends on their ability to form blast colonies in culture in the presence of methylcellulose and suitable growth factors. The other assesses the growth of blast cells in suspension culture, where growth is measured by increasing numbers of clonogenic cells. We have compared the two methods as assays for the cytotoxic effects of the chemotherapeutic drug cytosine arabinoside (Ara-C). Marked patient- to-patient variation was found using either method; however, the slopes of the dose-response curves were usually greater when cells were exposed to drug in suspension rather than in methylcellulose. Control experiments showed that the difference could not be explained by drug carry-over from the suspension cultures to the methylcellulose plates when clonogenic cells in the suspensions were assessed. Further, the survival curves for Adriamycin were very similar, regardless of which assay was used. No correlation was found between D10 Ara-C values measured in suspension or in methylcellulose. However, a significant association with outcome was found between D10 Ara-C in suspension and response to treatment with a regimen in which Ara-C was the only chemotherapeutic agent used. No such association was detected when the D10 values obtained with the clonogenic assay were compared with outcome for the same group of 15 patients. Finally, a feasibility experiment was performed in which blast cells were exposed to Ara-C repeatedly during exponential growth over 238 days. A dose-related inhibition of growth was observed; no evidence was seen of emerging drug-resistant cells. Nor did the morphology of the cells change as a result of drug exposure. We conclude that drug sensitivities of AML blast cells in culture are dependent on measurement methods, even when techniques affecting cell proliferation are compared. Measurements of drug sensitivity in culture may best be interpreted when the bases of the assay systems are understood.

Blast cells from 20 patients with acute leukemia (13 diagnosed myeloblastic and 7 as lymphoblastic, using the FAB classification) were studied using antibodies to lineage-specific differentiation markers. The phenotypic findings were usually consistent with the clinical diagnosis. However, examples were encountered where individual blast cells had a cytoplasmic marker of one lineage and a surface marker of a different lineage (lineage infidelity). Six examples of intramyeloid (two different myeloid lineages in the same cell) and three examples of interlineage infidelity (myeloid and lymphoid markers in the same blast cell) were encountered. No doubly marked cells were found in control material consisting of normal marrow cells, marrow regenerating after transplantation, or multilineage colonies derived from marrow in culture. A significant trend was observed relating the presence of lineage infidelity and failure of remission-induction. The data are interpreted as support for abnormal gene expression in leukemia.

Genetic and biologic evidence suggests that the Kit receptor tyrosine kinase is important in early events in hematopoietic stem cell differentiation. Two naturally occurring isoforms of the Kit receptor, termed Kit and KitA, were originally described in mouse cells and, subsequently, in human cells. These isoforms differ by the presence (KitA) or absence (Kit) of four amino acids (Gly-Asn-Asn-Lys) that lie immediately outside the transmembrane domain. RNase protection was used to measure the levels of Kit and KitA mRNA in normal bone marrow and the blast cells from individuals with acute myelogenous leukemia (AML). Although both isoforms were present in all the AML samples tested, there was considerable heterogeneity in the relative levels of the two transcripts, with Kit to KitA RNA ratios varying from as low as 1.3 to as high as 12. In contrast, the ratio of Kit to KitA transcripts in normal bone marrow was tightly clustered between 4.4 and 5.5. Because alterations in the relative levels of expression of Kit and KitA may affect the ability of a cell to respond to the Kit ligand, Steel factor, we examined the Kit/KitA RNA ratio in AML patients that differed with respect to a number of diagnostic, prognostic, and biologic parameters. The relative levels of Kit to KitA RNA was independent of French-American-British subtype, response to therapy, and primary and secondary plating efficiencies in vitro. Thus, these data suggest that the relative levels of the two isoforms of the Kit receptor in AML are not associated with any obvious biologic or clinical parameters and, therefore, may reflect naturally occurring changes in splicing mechanisms as stem cells differentiate.