Autologous bone marrow transplantation (ABMT), whereby the patient's own marrow is used as the source of stem cell rescue, is potentially curative in patients with acute myeloid leukemia (AML). However, the presence of residual occult viable leukemic cells in the marrow autograft limits the therapeutic effectiveness of ABMT in AML. To overcome this problem, methods of ex vivo treatment ('purging') of autologous marrow must be developed that optimally eradicate AML cells but spare normal hematopoietic stem cells. The proposed preclinical collaborative research between the All-Union Cancer Research Center (AUCRC), Moscow, USSR and The Johns Hopkins Oncology Center (JHOC), Baltimore, MD, will evaluate novel ex vivo marrow purging regimens by comparison of the effects of incubation with graded concentrations of immunologic and/or pharmacologic agents on the growth of normal marrow and leukemic cells, using an authentic animal model of human AML (the LBN hybrid rat). In vitro clonogenic assays for normal hematopoietic progenitors (colony-forming-unit, granulocyte-macrophage; CFU-CM) and AML cells (colony-forming-unit, leukemia; CFU-Leuk), which we have shown are predictive for hematopoietic engraftment and AML log-kill in rats given syngeneic marrow and/or AML cells incubated ex vivo with selected chemotherapeutic agents, will be used in these dose-response studies. Suspensions of normal LBN marrow, AML cells maintained in sus- pension cultures in vitro, and 10:1 mixtures of normal marrow:AML cells (to approximate an early marrow relapse of AML) will be incubated with complement and graded concentrations of anti-AML monoclonal antibodies (MoAbs) AP64, RM124, and RM152, or with bruneomycin (an antineoplastic antibiotic with potential utility as a marrow chemopurging agent), or the alkylating agent 4-hydroperoxycyclophosphamide (4HC). After incubation, cells will be plated for CFU-GM and CFU-Leuk assays to determine survival of committed hematopoietic progenitors and log-kill of AML cells, and optimal conditions for antileukemic effect with stem-cell sparing. Variables to be examined include agent concentrations, incubation duration and temperature, cell density, sequence of treatment, and number of cycles of in vitro treatment. Finally, results of the above studies will be used to examine the effects of combined immunopharmacologic incubation with MoAbs (singly and in combination) plus bruneomycin and/ or 4HC on the survival of CFU-GM and CFU-Leuk. These In vitro investigations of immunopharmacologic purging in an authentic model of human leukemia are relevant to the elucidation of basic mechanisms of normal and neoplastic hematopoiesis and will provide the basis for subsequent collaborative AUCRC/JHOC studies of clinical ABMT in patients with AML.
