Detection of a dormant 20q- leukemia clone in bone marrow cultures with hematopoietic growth factors: implications for secondary leukemia post-transplant.

A patient developed secondary acute myelogenous leukemia with a 20q- marker chromosome abnormality six years following chemotherapy and radiation for Hodgkins disease (HD). Routine cytogenetics on the bone marrow which had been harvested and cryopreserved immediately following completion of initial therapy for HD showed no cytogenetic abnormality. However, a 20q- clonal marker was detected after culturing bone marrow with hematopoietic growth factors (HGF). The marrow was used successfully for an autotransplant. Post-transplant, the 20q- marker was again detected in HGF cultured samples. The patient relapsed at 165 days post-transplant with the 20q- marker and trisomy 21. These data suggest that standard cytogenetic assays may not detect abnormal clones which can cause leukemia post-transplant.

Action of antithymocyte globulin on normal human erythroid progenitor cell proliferation in vitro: erythropoietic growth-enhancing factors are released from marrow accessory cells.

Studies were undertaken to determine the mechanism of action of a horse antithymocyte globulin preparation (ATGAM) (HATG) and its control preparation of horse gamma globulin (HIgG) on the proliferation of normal human marrow and blood erythroid progenitor cells (CFU-E, BFU-E) in vitro. In preincubation studies with marrow mononuclear cells and complement, HATG did not significantly augment CFU-E or BFU-E growth greater than that expected because of removal of marrow T cells by this agent. However, direct addition of HATG but not HIgG to marrow cultures significantly stimulated CFU-E and BFU-E up to two to four times that of media or HIgG controls (P less than 0.05). The peak effect was observed at 10 to 100 micrograms/ml HATG; HATG was toxic at 1000 micrograms/ml. By contrast, the OKT3 monoclonal antibody was less stimulatory than HATG. The in vitro erythropoietic stimulatory effect of HATG was dependent on the presence of accessory cells because removal of T cells or monocytes (less than 2% to 5%) or adsorptions of HATG with T cells or monocytes reduced its stimulatory effect, highly purified BFU-E nearly devoid of accessory cells required irradiated accessory cells for demonstration of the HATG stimulatory effect, and an erythroid burst-promoting activity was released from T cells or unseparated mononuclear cells in the presence of HATG but not HIgG. The HATG enhancing effect was optimal in the first 96 hours of cultures in the presence of erythropoietin, and was reproducible with three separate lots of HATG. Up to 16% of HATG-stimulated erythroid colonies expressed nonerythroid lineage cells. Iron 59 incorporation into heme of CFU-E- or BFU-E-derived colonies was augmented equally by HATG or HIgG at 10 micrograms/ml. Erythropoietin dose-response curves and studies with antierythropoietin sera suggested that HATG also increased the sensitivity of erythroid progenitor cells to very low concentrations of erythropoietin. We conclude that HATG but not HIgG control has potent dose-dependent erythroid progenitor cell growth-enhancing effects. In addition to the ability of HATG to lyse T-suppressor cells, these findings suggest that HATG may also stimulate erythropoiesis indirectly by releasing growth-enhancing factor(s) from T cells and other marrow accessory cells, sensitizing erythroid progenitor cells to low concentrations to erythropoietin, and stimulating growth of bipotential progenitor cells. Collectively, these effects may explain the efficacy of HATG in the treatment of some patients with erythropoietic marrow failure states.

Serum interleukin-3 levels following autologous or allogeneic bone marrow transplantation: effects of T-cell depletion, blood stem cell infusion, and hematopoietic growth factor treatment.

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme-linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL-3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.

In vitro evidence for disappearance of erythroid progenitor T suppressor cells following allogeneic bone marrow transplantation for severe aplastic anemia.

In vitro coculture studies were performed in five patients with severe aplastic anemia (SAA) and their normal HLA-matched donors before and after allogeneic bone marrow transplantation (BMT) to determine whether the erythropoietic function of T cells is abnormal in this disorder. These coculture studies used fresh or cryopreserved marrow T lymphocytes with fresh or cryopreserved marrow T cell-depleted target cells. Four of five aplastic patients had little or no transfusion exposure before studies. The composite results showed that, in comparison to the erythropoietic effects of normal HLA-identical marrow T lymphocytes or engrafted T lymphocytes, T lymphocytes collected from the aplastic patients before BMT consistently suppressed or failed to support CFUE and BFUE growth optimally from autologous marrow, HLA-identical marrow, or engrafted aplastic T cell-depleted marrows. This T cell abnormality was not observed in four multiply transfused leukemics and three patients with myelodysplastic syndrome. Marker analyses of SAA marrow T lymphocytes performed before and after BMT suggested that the erythropoietic functional abnormality was due to abnormal marrow T cell composition reflecting an excess of activated Tac+, T3+, T11+ lymphocytes. Collectively, these in vitro studies provide firmer in vitro evidence implicating T cells in the pathogenesis of SAA. The erythropoietic T cells abnormalities in SAA are fully corrected by allogeneic BMT.

A rapid two-step method for elimination of bcl-2/IgH positive non-Hodgkin's lymphoma cells from human blood or marrow stem cells, employing immunomagnetic purging with streptavidin-coated ferrofluids.

BACKGROUND: Follicular lymphoma cells may contaminate blood or BM stem cell collections used for autologous transplants and contribute to relapse. We report on the development of a B-cell lymphoma-purging technique, using streptavidin-coated ferrofluids (SAFF) and a high-gradient magnetic-separation device (HGMSD). METHODS: Blood or BM samples were spiked with bcl-2 positive lymphoma cells, labeled with biotinylated B cell MAb (CD19, CD20, CD37) then purged by positive selection of CD34(+) cells, followed by negative selection with SAFF and a high HGMSD. Purging efficiency was judged by dual-labeled flow cytometry and by PCR analysis of bcl-2. RESULTS: A 6 log purge of bcl-2 lymphoma cells could be achieved by first enriching for CD34(+) cells, followed by negative selection with SAFF and HGMSD. The use of SAFF with HGMSD alone could only accomplish a 2-3 log purge. DISCUSSION: CD34 selection from blood or BM of follicular lymphoma patients, followed by negative selection of MAb-labeled cells with SAFF could achieve a 6 log purge. However, cell yields were low (10%) and purging efficacy was limited to low starting concentration of lymphoma cells (< or = 1.0%). This technique, however, may be useful for full-scale clinical purging of minimal residual disease following debulking with a cyclophosphamide or other agents.

Complications of peripheral blood stem cell harvesting: review of 554 PBSC leukaphereses.

The collection of PBSC for transplantation requires repetitive leukapheresis, typically via central venous catheters (CVC). To assess the complications of this procedure, we reviewed 75 consecutive PBSC transplant candidates requiring 554 leukapheresis on a Haemonetics V50 Plus apheresis system. CVC occlusion necessitating thrombolytic therapy or cancellation of the procedure was the most commonly observed complication, occurring among 37 patients on 86 occasions (15.9% of CVC-aided collections). Thrombolytic therapy was successful in 85%. Of the patients, 16% experienced an infectious complication during the PBSC harvesting; chemotherapy mobilization significantly increased this risk, whereas growth factor mobilization was protective (p < 0.02). Hematologic complications including anemia (median postapheresis nadir hemoglobin 8.8 g/dl) and transient thrombocytopenia (median postapheresis nadir 64,000/microliters) required transfusional support among 30.7% and 14.7% of patients, respectively. The use of chemotherapy mobilization was correlated with increased need for support of both red cells and platelets (p < 0.001). Additional complications included catheter placement problems, symptomatic citrate-related hypocalcemia, transient hypotension, and machine-related malfunctions. Although the complications of the PBSC harvests were manageable, given their frequency the decision to pursue this form of hematopoietic rescue in preference to traditional operative bone marrow harvesting must address these risks.