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Our data suggest that ex vivo bone marrow purging using monoclonal antibodies (MoAbs) and sheep-anti-mouse immunobeads (SAM beads) prior to autologous bone marrow transplantation (ABMT) allows satisfactory tumor cell reduction without critical stem cell losses. Nevertheless, there is only a reduction but no elimination of tumor cells. The consequences will have to be clinically discussed.
A group of 56 patients with immunophenotyped acute lymphoblastic leukemia had their bone marrow purged using monoclonal antibody (MAb) and complement. Mononuclear marrow cells were treated with panels of MAb directed against T cells (n = 19), CD10-CALLA (n = 16), and B cells (n = 21), resulting in median specific cell depletions of 96.50, 97.59, and 96.97%, respectively. The resulting CFU-GM recoveries were 60.15, 110.6, and 72.9%. Long-term bone marrow cultures showed normal formation of adherent cell layers in 17 of 23 cases, and failure to form an adherent layer correlated with poor hematological reconstitution. A total of 32 patients (aged 4-40 years) received purged marrow grafts containing a median of 1.32 x 10(7) mononuclear cells/kg and 1.64 x 10(4) CFU-GM/kg; 7 patients required backup marrow. Time to engraftment of 500 granulocytes, 1000 leukocytes, 20,000 platelets, and 50,000 platelets was 37, 38, 39, and 57 days, respectively. A total of 16 patients (50%) remain alive and in complete remission 313-913 days posttransplant (median 565 days); 13 patients (41%) relapsed between 50 and 365 days posttransplant (median 100 days), and 3 patients (9%) died from transplant-associated complications.
Alkyl-lysophospholipid compounds which are selectively cytotoxic to neoplastic cells and relatively sparing of normal marrow progenitor cells are appealing as purging agents to rid remission marrows of residual leukemic cells. A multi-institutional phase II study was conducted in 57 patients with acute leukemia (50 AML and 7 ALL) in which remission marrows were purged in vitro and reinfused after ablative chemotherapy. The median time for granulocyte recovery to 500/microliter was 33 days and for platelet recovery to 25000/microliter was 46 days. The overall DFS and survival was 37% and 46% respectively. Transplantation in first remission gave a better survival than transplant in a subsequent remission.
Recent data suggest that tumor cells contaminating reinfused bone marrow may contribute to relapse in patients undergoing autologous bone marrow transplantation. Purging strategies that are able to remove these contaminating tumor cells need to be developed. This study describes how electroporation (EP) can be used to improve intracellular delivery of synthetic antisense oligodeoxynucleotides (ODNs), thereby enhancing their ability to suppress a target protein. Antisense ODNs that were introduced into cells by EP led to immediate suppression of targeted c-myc protein; this was associated with rapid cell death in the diffuse histiocytic lymphoma, U937; Burkitt's lymphoma, ST486; breast carcinoma, MCF-7; and Ewing's sarcoma, CHP-100, cell lines. Electroporation was found to have little or no detrimental effect on cells responsible for murine hematopoietic long-term reconstitution as determined from in vivo competitive repopulation studies. Using human c-myc-directed antisense ODNs as a model for the application of this approach to bone marrow purging, selective killing of human lymphoma U937 cells relative to normal human bone marrow cells was shown in cell mixing studies. In vivo studies were performed in which a survival advantage was shown for athymic mice that were inoculated with antisense-treated U937 cells as opposed to control cells. These studies suggest that EP of bone marrow may be of use in enhancing intracellular delivery of a variety of molecular/pharmaceutical agents. Taken together, these data suggest that the use of electroporation to enhance delivery of antisense ODNs is a promising new approach towards ex vivo bone marrow purging.
An immunomagnetic method was developed to purge human bone marrow of malignant T cells, for use in conjunction with autologous bone marrow transplantation in patients with acute lymphoblastic leukemia and lymphoma. Three monoclonal antibodies anti CD2 (BH1), CD5 (BB8) and CD7 (BF12) were used. In model experiments employing MOLT4 cells it was found that with 50-fold excess of immunobeads relative to antigen-positive cells, the use of each antibody alone resulted in a 3.3-3.6 log tumor cell depletion, as assessed in a sensitive and reproducible clonogenic soft agar assay. When all three antibodies were used in a mixture, a purging efficacy of 5 logs was achieved. Two treatment cycles improved these figures to about 4 logs and more than 5 logs. When MOLT4 cells were mixed 1:10 with fresh bone marrow cells the antibody mixture yielded 3.1 and more than 5 log tumor cell depletion with one and two treatment cycles, respectively. This procedure resulted in only an insignificant reduction of the number of CFU-GM and CFU-GEMM progenitor cells. In two patients autotransplanted with purged marrow, the loss of CFU-GM was 37% and 48%, and no tumor cells could be detected by immunocytochemistry after purging. Rapid and sustained engraftment was achieved and both patients remain in complete remission after more than 20 months.
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One of the principal challenges of cancer chemotherapy is the relative inability of most anticancer drugs to distinguish between normal and neoplastic tissues. Consequently, a broad range of toxicities are experienced by patients, especially myelosuppression. Amifostine, a phosphorylated aminothiol, increases the selectivity of specific anticancer drugs for neoplastic cells by protecting normal tissues. One potential application of this protector is during bone marrow purging to selectively remove contaminating cancer cells. This study took normal or leukemic marrow from human subjects and evaluated the ability of amifostine to selectively protect normal bone marrow progenitor cells versus leukemic progenitor cells from the cytotoxic effect of mafosfamide. The dose response of mafosfamide amifostine on leukemia colony-forming units or normal marrow progenitor cells was determined and the LD95 was calculated. Amifostine pretreatment resulted in a statistically significant protection of granulocyte-macrophage colony-forming units and erythroid blast-forming units from the toxicity of mafosfamide (P = .031). Thus, amifostine protection of normal marrow progenitor cells allows a higher LD95 concentration of mafosfamide to be used in ex vivo purging. In contrast, amifostine pretreatment increased the cytotoxicity of mafosfamide on the fresh human leukemia progenitor cells (P = .006). The dual effect of amifostine protection of normal marrow progenitor cells coupled with amifostine-induced sensitization of the leukemia cells increases the possible cell-kill of leukemic stem cells. With amifostine pretreatment, at the LD95 concentrations of mafosfamide for marrow progenitor cells, there was an estimated 6 log increase in cell-kill of the leukemia cells. This selective cell-kill offers the potential for lowering the incidence of leukemic relapse, while preserving more normal stem cells for autologous transplantation.
6-Hydroxydopamine(6-OHDA), a specific neurotoxin against sympathetic nerve cells, is a drug already used for purging of bone marrow from neuroblastoma cells before autologous bone marrow transplantation. However, we could not detect significant differences in the toxicity of 6-OHDA against neuroblastoma and other tumor cells under the purging conditions clinically used. In contrast, bone marrow stem cells were much more resistant. The unspecific toxic effect of 6-OHDA is caused by H2O2 or H2O2-derived products which are generated by auto-oxidation in the incubation medium before a significant amount of 6-OHDA is taken up by the cells. Withdrawal of oxygen during the incubation period and subsequent incubation with an oxygen containing medium led to a more specific destruction of neuroblastoma cells which can take up 6-OHDA selectively.
Autologous bone marrow transplantation (ABMT) offers a therapeutic alternative for children with poor prognosis acute lymphoblastic leukemia (ALL) who lack an HLA-matched sibling donor. The most common cause of treatment failure after ABMT in these patients is leukemia relapse. We have developed an ex vivo autologous marrow purging program for children with ALL using an immunomagnetic method. BM purging has been performed in 37 children with ALL (31 B-lineage ALL and 6 T-lineage ALL) following an indirect method, using panels of mouse monoclonal antibodies (MAbs) directed against B or T cell antigens, Dynabeads M-450 (Dynal) coated with sheep antimouse (SAM) antibodies, and the MaxSep Magnetic Cell Separator (Baxter). Purging efficiency has been assessed by flow cytometry. Considering the limit of detection of target cells 0.1%, the median depletion was 2.0 log (range 0.8- > 2.8 log) for the B-lineage ALL and 2.7 (range 2.2- > 2, 9 log) for the T-lineage ALL patients. Twenty-seven patients have been autografted (6 in first complete remission, CR, 13 in second CR, and 8 in third or subsequent CR). Engraftment has been satisfactory in all of them, reaching levels of 500 neutrophils/mm3 and 20,000 platelets/mm3 after a median of 17 (range 12-39) and 30 (range 13-96) days post-ABMT, respectively. In summary, our results show that this immunomagnetic procedure achieves high levels of target cell depletion and can be safely applied to bone marrow purging in childhood ALL patients.
OBJECTIVE: To observe the clinical effectiveness of IHW2 purged autologous bone marrow transplantation (ABMT) in the treatment of AML patients. METHODS: Large volume of bone marrow was purged in vitro by IHW2 and then reinfused to patients for ABMT. RESULTS: All of the three patients transplanted with IHW2 purged autologous bone marrow successfully reconstituted hemopoiesis. Two patients got continuous complete remission for 36+ and 34+ months, respectively. The karyotype of one of the two patient was 46,XY,del(16)(q22) before ABMT and changed to 46,XY in 2 months after transplantation. The third patient relapsed after ABMT 19 months. CONCLUSION: The preliminary study suggests that IHW2 may have certain purging effect on leukemic cells and is safe for clinical application.
The effectiveness of adoptive immunotherapy in eliminating minimal residual disease in tumour-bearing mice after bone marrow transplantation was tested. This model mimics the human clinical condition when autologous bone marrow was purged ex vivo of leukaemia with mafosfamide or was not purged, and stored in liquid nitrogen before transplantation. Animals with minimal residual disease were prepared with marrow-ablative but leukaemia-noncurative doses of cyclophosphamide (CY) and total body irradiation followed by bone marrow transplantation. The next day after transplantation the recipients were injected with splenocytes immunized against the leukaemia cells (Imm-SPL) or monoclonal antibody (mAb). All the control mice died from leukaemia relapse, but 51% of purged bone marrow recipients, which received Imm-SPL, were cured. In similar conditions mAb did not exert a therapeutic effect. Imm-SPL were not able to eradicate minimal residual disease in the recipients of nonpurged bone marrow. Thus, in an animal model, we demonstrated that purging of bone marrow before grafting seems to be indispensable for successful adoptive immunotherapy of minimal residual disease (MRD) after autologous bone marrow transplantation.
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Relapse is a major concern in autologous bone marrow transplantation (BMT). Therefore, purging of bone marrow to reduce the amount of tumor cells reinfused into the patient is widely used. Immunologic effector cells such as lymphokine activated killer (LAK) cells are attractive for purging of bone marrow since these cells might have an additional in vivo effect on tumor cells in contrast to other purging protocols. In patients with chronic myelogenous leukemia (CML), LAK cells can only be used in some patients for purging bone marrow since LAK cells possess no or only limited cytolytic activity against autologous CML tumor cells in most cases. In this study, we investigated the effect of autologous and allogeneic cytokine-induced killer (CIK) cells on tumor cells from patients with CML. CIK cells have been generated from peripheral blood lymphocytes by incubation with interferon-gamma on day 0, interleukin-1, interleukin-2 and a monoclonal antibody against CD3 on day 1. In contrast to LAK cells, CIK cells were able to lyse both autologous and allogeneic cells from patients with CML as determined by a 51Cr release and a tumor colony assay. The cytotoxicity of CIK cells against CML cells was confined to the CD56+ population. CIK cells showed no major toxic effect on hematopoietic progenitor cells when tested in CFU-GM assays. CIK cells eliminated three orders of magnitude of K562 cells and less than one order of magnitude of progenitor cells (25% reduction). This represents a differential effect of CIK cells on tumor and progenitor cells.(ABSTRACT TRUNCATED AT 250 WORDS)
To study effects of a hammerhead ribozyme on chronic myelogenous leukemia (CML) cells and bone marrow purging in vitro, a bcr-abl specific ribozyme gene was introduced into CML and normal bone marrow cells using retroviral transduction. The effects of the ribozyme on primary cells from CML patients were detected by hematopoietic progenitor cell assays, flow cytometry and immunocytochemical methods. Then a model of remission was built up, and the effects of ribozyme on bone marrow purging were detected by leukemia colony assay and nest-PCR after transduction of this model by using the ribozyme. The results showed that ribozyme significantly suppressed the clonogenic growth and the expression of p210 protein in primary cells from CML patients, but did not affect the growth of normal hematopoietic progenitor cells. In the model of remission, ribozyme eliminated the proliferation and the expression of bcr-abl mRNA in residual K562 cells, but did not affect the expression of abl mRNA. The results suggest that anti-bcr-abl ribozyme might be used for bone marrow puring of CML cells.
The number of autologous bone marrow transplants done for solid tumours, particularly breast cancer, has risen steadily over the last ten years. The role of bone marrow or peripheral blood progenitor cell purging in transplantation is incompletely understood. Theoretically, the reinfusion of untreated bone marrow containing tumour cells might result in relapse in some patients treated with high-dose chemotherapy and hematopoietic support. Therefore, safe and effective purging techniques may increase long-term, disease-free survivorship. In this study, hyperthermia was evaluated for its ability to purge CAMA-1 breast cancer cells from normal human bone marrow. Between two and nine trials of a range of temperatures (42-45 degrees C) and durations of treatment (1-4 h) were performed. The effect of hyperthermia on normal bone marrow alone and in mixes with breast cancer cells was also evaluated. Hyperthermia (45 degrees C, 4 h) produced > 5 logs of CAMA-1 cell kill. Exposures of 45 degrees C for 2 h and 44 degrees C for 4 h resulted in approximately three logs of cell kill, corresponding to < 1% survival of clonogenic cells. Normal bone marrow was considerably more vulnerable to heat treatments, however, with approximately 1% of progenitors remaining clonogenic after exposure of 43 degrees C for 2 h and 44 degrees C for 1 h. Therefore, although hyperthermia is able to achieve adequate CAMA-1 breast cancer cell kill, it remains more toxic to normal bone marrow as a purging method. To make hyperthermia useful in purging systems, mechanisms to selectively alter thermal sensitivity must be pursued.
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BACKGROUND: An effective purging technique plays an important role in autologous hematopoietic stem cells transplantation. Photodynamic therapy (PDT) provides a novel approach for this purpose. This study dealt with the purging effects of di-sulfo-di-phthalimidomethyl phthalolcyanine zinc (ZnPcS2P2)-based photodynamic therapy (ZnPc-PDT). METHODS: Fluorescence intensity of cell extracts was measured using a fluorescence spectrophotometry. The proliferative potency of K562 cells and HL60 cells was detected using MTT colorimetric assay, Typan blue dye exclusion method, colony formation test. The proliferative potency of normal hematopoietic cells was evaluated using mixture colony-forming unit (CFU-Mix), granulocyte-macrophage colony-forming unit (CFU-GM), and erythrocyte colony-forming unit (CFU-E) assays. K562 cells were mixed with normal mononuclear cells (MNCs) at ratios of 1:100 and 1:1000 for creating the model of simulated remission bone marrow. Colony formation test and nested-RT-PCR were carried out to detect the residual K562 cells in cell mixture. RESULTS: After a 5-hour incubation with ZnPcS2P2, the content of ZnPcS2P2 in normal MNCs was the lowest value. At the same time, the content in K562 cells and HL60 cells was very high. Therefore, the time point was selected as the optimal one for irradiating the cell suspensions. ZnPc-PDT could significantly kill proliferative K562 cells and HL60 cells in a dose-dependent manner. At the concentration of 1.0 microg/ml, the inhibitory rate of ZnPc-PDT on the colony formation was 100% for K562 cells, 89.7% for HL60 cells. 0.25 microg/ml ZnPc-PDT could completely photoinactivate residual K562 cells in the simulated remission bone marrow. Under an identical condition, the inhibitory rates of CFU-Mix, CFU-GM, CFU-E were 18.0%, 18.6%, and 17.8% respectively. CONCLUSION: ZnPc-PDT appears to be a promising approach for bone marrow purging.
Ex vivo purging of contaminating tumor cells may reduce the incidence of relapse in patients undergoing bone marrow transplantation. In this study we demonstrate that resveratrol, a phytoalexin with anti-oxidant and chemopreventive activity, exhibits anti-leukemic activity against mouse (32Dp210, L1210) and human (U937, HL-60) leukemic cell lines by inhibiting cell proliferation. Long-term exposure to resveratrol also inhibits the clonal growth of normal hematopoietic progenitor cells but at a higher IC50 of resveratrol than that for most of the leukemia cell lines tested. The inhibitory effect of resveratrol on hematopoietic progenitors is partially reversible, whereas the effect on leukemia cells is largely irreversible. The inhibition of leukemia cells by resveratrol involves nucleosomal DNA fragmentation (apoptosis). On the other hand, resveratrol does not induce or enhance spontaneously occurring apoptotic death in normal hematopoietic progenitor cells. In vivo experiments performed with untreated and resveratrol-treated bone marrow showed comparable hematopoietic reconstitution in lethally irradiated mice (10 Gy) as determined by survival, hematologic recovery, and the number of hematopoietic progenitor cells present in the marrow of reconstituted animals. Taken together, these results indicate the potential use of resveratrol for ex vivo pharmacological purging of leukemia cells from bone marrow autografts without significant loss in the hematopoietic activity of progenitor cells.