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To develop new purging regimens for ABMT the ability to predict potential for purging of tumor cells from BM is important. Since the sensitivity of human B cell lymphoma to hyperthermia is not known, we examined its effect on the growth of B cell lymphoma cell lines (Raji and Daudi) in vitro to evaluate potential for purging clonogenic tumor cells from normal marrow by heat, using a limiting dilution assay to measure log depletion of tumor cells in a 20-fold excess of normal BM. When exposed to heat (42-43 degrees C) for 120 min, both clonogenic Raji and Daudi cells were dramatically reduced (a 4-to-6 log reduction) with time, whereas at 42 degrees C over half and at 43 degrees C 10% of normal granulocyte-macrophage progenitor cells survived for the same time period. This high level of lymphoma cell depletion by heat correlated with that of immunologic and pharmacologic studies. In addition, these survival curves during heating were found to correlate with the Gompertz-Makeham formula--a law of human mortality. This formula may be useful in predicting the purging effect of heat. These results suggest that in vitro hyperthermia could be applied effectively for the elimination of residual, clonogenic lymphoma cells in autologous marrow grafts before ABMT.

Alpha-interferon (IFN) may inhibit the proliferation of human leukemic progenitor cells (L-CFU) in vitro and enhance the anti-tumor effects by heat. In this study, the combined effects of IFN and hyperthermia on the growth of L-CFU and human granulocyte-macrophage progenitors (CFU-GM) were examined to determine if this combination resulted in a greater selective killing of L-CFU than that obtained by heat treatment alone. The survival of normal CFU-GM without IFN decreased at elevated temperatures (42-44 degrees C). However, IFN added during heating (42 and 43 degrees C) appeared significantly to protect against the hyperthermic killing of CFU-GM in vitro leaving over 50% of CFU-GM surviving. The optimal dose to protect CFU-GM in vitro dropped to a rather low dose (100 U/ml). On the other hand, the addition of IFN to leukemic cell suspensions enhanced the hyperthermic killing of myeloid leukemic cell lines (HEL and KG-1) as well as a T lymphoblastic cell line (CEM) in a dose-related manner. In addition, similar results were observed in the study of L-CFU from patients with acute myelogenous leukemia. These results suggest that IFN can be used to broaden the difference between surviving fractions of CFU-GM and L-CFU by heat. Thus, this combination could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous remission marrow graft before autologous bone marrow transplantation.

Normal tissue toxicity is a major concern in cancer therapy. Since the hyperthermic killing of normal human hematopoietic progenitor cells including multilineage progenitor cells (CFU-mix) other than committed granulocyte-macrophage progenitor cells (CFU-GM) is not known, we have studied the thermal sensitivity of various types of human hematopoietic progenitor cells: early and late erythroid progenitors (CFU-E and BFU-E), CFU-GM and CFU-mix using semisolid clonogenic assays. When these progenitor cells were exposed at 42-44 degrees C for 2 h, their thermal sensitivity always appeared to be in the order of CFU-E greater than BFU-E greater than CFU-GM greater than CFU-mix; the CFU-mix was the most tolerant to heat damage. In addition, the survival of human hematopoietic progenitor cells decreased exponentially in exposure time- and temperature-dependent manner. At 44 degrees C human hematopoietic progenitor cells including CFU-mix dropped dramatically in number and therefore were unable to form colonies in vitro after exposure for 2 h. This suggests that 44 degrees C may be the critical temperature for the CFU-mix to survive in vitro. Thus temperatures selected for hyperthermia used to purge residual leukemic cells in remission marrow grafts should be 43 degrees C or lower.