ALBERT

Description: The origin of marrow stromal cells post allogeneic bone marrow transplantation (BMT) was studied. Two groups of patients receiving HLA- identical marrow grafts from sex mismatched siblings were included in the study: the first group (eight patients) received conventional marrow grafts and the second group (ten patients) received stromal cell and T cell depleted grafts. All patients showed hematopoietic engraftment with donor cells. Marrow aspirates obtained from these patients were used to establish stromal layers in long-term marrow cultures (LTMC) for 4 to 6 weeks. In both groups, karyotype analysis of nonhematopoietic cultured stromal cells showed host origin even as late as day 760 posttransplantation. Immunofluorescence methods using monoclonal antibodies against components of fibroblasts, macrophages, and endothelial cells, showed that the composition of stromal layers was similar to those obtained from normal controls. Our data indicate that marrow stromal progenitors capable of proliferation are nontransplantable and do not originate from a hematopoietic-stromal common progenitor.

Description: The promising antineoplastic agent diaziquone is associated with prolonged aplasia and rare instances of bone marrow necrosis, but only mild extramedullary toxicity. To explore the drug's potential as a myeloablative agent prior to bone marrow transplantation, we compared its effects on hematopoietic versus marrow stromal cells. After short- term (one to six hours) or prolonged (three to seven days) exposure to the drug, marrow was assayed for hematopoietic (CFU-Mix, BFU-E, CFU-GM) and stromal (CFU-F) colony-forming cells and studied in long-term marrow culture (LTMC). One- and three-hour treatments produced little cytotoxicity, even at 5000 ng/mL. After six-hour treatments with this dose, marrow was depleted of CFU-Mix, BFU-E, and CFU-GM, but produced CFU-GM in LTMCs, indicating an ongoing input of CFU-GM from a surviving pre-CFU-Mix population. In contrast, elimination of the latter may be inferred from the absence of CFU-GM in LTMCs exposed for three to seven days to diaziquone at only 150 ng/mL. Under these conditions, CFU-F recovery was 40% and adherent stromal layers in LTMCs were similar to untreated controls regarding rate of development and cellular composition. Our in vitro pre-CFU-Mix-ablative regimen correlates with clinical data that show prolonged but reversible myelosuppression at steady-state diaziquone plasma levels of 101 +/- 10 ng/mL (mean +/- standard error of mean) during 7-day constant infusions. In conclusion: hematopoietic cells are more sensitive than marrow stromal cells to the dose- and highly time-dependent cytotoxicity of diaziquone, a direct drug-induced noxious effect on the marrow microenvironment is an unlikely cause of the isolated episodes of marrow necrosis after the use of diaziquone in vivo, and prolonged infusion of diaziquone represents an attractive means for achieving myeloablation in selected clinical situations.

Description: The origin of marrow stromal cells post allogeneic bone marrow transplantation (BMT) was studied. Two groups of patients receiving HLA- identical marrow grafts from sex mismatched siblings were included in the study: the first group (eight patients) received conventional marrow grafts and the second group (ten patients) received stromal cell and T cell depleted grafts. All patients showed hematopoietic engraftment with donor cells. Marrow aspirates obtained from these patients were used to establish stromal layers in long-term marrow cultures (LTMC) for 4 to 6 weeks. In both groups, karyotype analysis of nonhematopoietic cultured stromal cells showed host origin even as late as day 760 posttransplantation. Immunofluorescence methods using monoclonal antibodies against components of fibroblasts, macrophages, and endothelial cells, showed that the composition of stromal layers was similar to those obtained from normal controls. Our data indicate that marrow stromal progenitors capable of proliferation are nontransplantable and do not originate from a hematopoietic-stromal common progenitor.

Description: An evaluation of the effects of VP-16 on normal human marrow cells and representative lymphoma-leukemia cell lines was performed to assess this agent's applicability to ex vivo marrow purging. Tumoricidal dose curves were defined using malignant lymphoid (SK-DHL2 and Reh) and myeloid (HL-60) cells admixed with a 20-fold excess of irradiated marrow cells to simulate a borderline remission marrow. One-hour treatments yielded ID50 of less than 5 mumol/L of VP-16 for clonogenic units from each cell line; rare-to-zero clonogenic units survived exposure to 50 to 100 mumol/L. CFU-Mix, BFU-E, and CFU-GM were equal in their sensitivity to VP-16 (ID50s25 to 30 mumol/L). Marrows treated with 75 mumol/L were completely depleted of these colony-forming cells but produced CFU-GM in one-stage long-term marrow cultures (LTMCs). This dose had little adverse effect on the proliferative capacity of marrow stromal progenitors, as measured by CFU-F (ID50 271 mumol/L) and by the unperturbed development of adherent layers in LTMCs. Furthermore, these stromal layers were able to support hematopoiesis as well as controls in co-culture experiments with autologous marrow cells (two-stage LTMCs). In conclusion, doses of VP-16 that cleanse marrow of lymphoma-leukemia cells spare hematopoietic and stromal progenitors as demonstrated by LTMCs. These data favor the use of VP-16 in the clinical autotransplant setting.

Description: An evaluation of the effects of VP-16 on normal human marrow cells and representative lymphoma-leukemia cell lines was performed to assess this agent's applicability to ex vivo marrow purging. Tumoricidal dose curves were defined using malignant lymphoid (SK-DHL2 and Reh) and myeloid (HL-60) cells admixed with a 20-fold excess of irradiated marrow cells to simulate a borderline remission marrow. One-hour treatments yielded ID50 of less than 5 mumol/L of VP-16 for clonogenic units from each cell line; rare-to-zero clonogenic units survived exposure to 50 to 100 mumol/L. CFU-Mix, BFU-E, and CFU-GM were equal in their sensitivity to VP-16 (ID50s25 to 30 mumol/L). Marrows treated with 75 mumol/L were completely depleted of these colony-forming cells but produced CFU-GM in one-stage long-term marrow cultures (LTMCs). This dose had little adverse effect on the proliferative capacity of marrow stromal progenitors, as measured by CFU-F (ID50 271 mumol/L) and by the unperturbed development of adherent layers in LTMCs. Furthermore, these stromal layers were able to support hematopoiesis as well as controls in co-culture experiments with autologous marrow cells (two-stage LTMCs). In conclusion, doses of VP-16 that cleanse marrow of lymphoma-leukemia cells spare hematopoietic and stromal progenitors as demonstrated by LTMCs. These data favor the use of VP-16 in the clinical autotransplant setting.

Description: The promising antineoplastic agent diaziquone is associated with prolonged aplasia and rare instances of bone marrow necrosis, but only mild extramedullary toxicity. To explore the drug's potential as a myeloablative agent prior to bone marrow transplantation, we compared its effects on hematopoietic versus marrow stromal cells. After short- term (one to six hours) or prolonged (three to seven days) exposure to the drug, marrow was assayed for hematopoietic (CFU-Mix, BFU-E, CFU-GM) and stromal (CFU-F) colony-forming cells and studied in long-term marrow culture (LTMC). One- and three-hour treatments produced little cytotoxicity, even at 5000 ng/mL. After six-hour treatments with this dose, marrow was depleted of CFU-Mix, BFU-E, and CFU-GM, but produced CFU-GM in LTMCs, indicating an ongoing input of CFU-GM from a surviving pre-CFU-Mix population. In contrast, elimination of the latter may be inferred from the absence of CFU-GM in LTMCs exposed for three to seven days to diaziquone at only 150 ng/mL. Under these conditions, CFU-F recovery was 40% and adherent stromal layers in LTMCs were similar to untreated controls regarding rate of development and cellular composition. Our in vitro pre-CFU-Mix-ablative regimen correlates with clinical data that show prolonged but reversible myelosuppression at steady-state diaziquone plasma levels of 101 +/- 10 ng/mL (mean +/- standard error of mean) during 7-day constant infusions. In conclusion: hematopoietic cells are more sensitive than marrow stromal cells to the dose- and highly time-dependent cytotoxicity of diaziquone, a direct drug-induced noxious effect on the marrow microenvironment is an unlikely cause of the isolated episodes of marrow necrosis after the use of diaziquone in vivo, and prolonged infusion of diaziquone represents an attractive means for achieving myeloablation in selected clinical situations.