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PubMed1989

Efficacy of ex vivo purging for autologous bone marrow transplantation in the treatment of acute nonlymphoblastic leukemia.

We used an in vitro measure of drug activity to predict the efficacy of ex vivo purging of leukemic cells from autologous bone marrow grafts. We previously found that the myeloid progenitor cell (CFU-GM) content of the marrow grafts after ex vivo purging with 4-hydroperoxycyclophosphamide (4-HC) correlates with time to hematologic recovery after autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia. We observed that variable red blood cell concentration of the bone marrow incubation mixture results in differential cytotoxic activity of 4-HC. The CFU-GM content of the graft after the ex vivo treatment is a measure of this 4-HC activity. We analyzed the disease-free survival of 45 patients with acute nonlymphoblastic leukemia undergoing autologous bone marrow transplantation with 4-HC purged grafts. Patients who relapsed after transplantation had 4.2 +/- 1.1% of graft CFU-GM surviving the ex vivo purge, compared with 1.1 +/- 0.4% for patients who achieved a sustained remission (P = .06). Twenty-three patients with a CFU-GM content after 4-HC purging of greater than 1% of the pretreatment value had an actuarial disease-free survival of 12%, compared to 36% for 22 patients with a less than or equal to 1% CFU-GM content after purging (P = .006). Therefore, percent CFU-GM survival as a measure of 4-HC cytotoxicity identified a group of patients with insufficient purging. Although no randomized clinical trials have documented the need for ex vivo purging, our results suggest that effective bone marrow purging is important for the optimal application of autologous transplantation in the treatment of acute nonlymphoblastic leukemia.

Age Factors↗
PubMed1994

Successful purging of murine plasmacytoma by mafosfamide (ASTA-Z).

Mafosfamide (ASTA-Z) is a chemotherapeutic agent currently in use for in vitro purging of tumor-bearing human BM cells prior to autologous bone marrow transplantation (ABMT). We tested the efficacy of ASTA-Z against mouse plasmacytoma cells MOPC-315 (MOPC), a model of human multiple myeloma. BALB/c mice were injected intraperitoneally with different doses of MOPC preincubated with ASTA-Z. All control mice receiving > or = 10(4) MOPC intraperitoneally (ip) died within 23 days. All recipients of ASTA-Z pretreated MOPC remained healthy for > 180 days. To simulate the clinical situation, BALB/c mice received lethal doses of 10(3) MOPC ip prior to ABMT. Subsequently, mice were treated with cyclophosphamide 200 mg/kg one day prior to syngeneic BMT with 10(7) BMC containing 10(6) MOPC; 90% of the mice receiving unpurged syngeneic BMC died within 45 days whereas all mice transplanted with ASTA-Z-treated BMC/MOPC mixtures remained disease-free for > 100 days. Our results suggest that a similar approach may be successful in patients with multiple myeloma and residual disease prior to cryopreservation of their BM for ABMT. Bone marrow purging with ASTA-Z is effective and under certain conditions could be critical for prevention of relapse following ABMT, provided that effective elimination of residual disease in the host can be achieved by the conditioning regimen prior to ABMT.

Animals↗
PubMed2003

Purging of leukemia-contaminated bone marrow grafts using suicide adenoviral vectors: an in vivo murine experimental model.

Autologous bone marrow transplantation is an alternative therapeutic option for acute myeloid leukemia patients lacking a compatible donor. However, bone marrow from these patients may contain residual leukemic cells that should be ideally eliminated prior to the infusion of the graft. With the aim of developing more efficient protocols of graft purging, adenoviral-mediated gene transfer protocols have been conducted. We studied whether suicide adenoviral vectors expressing the cytosine deaminase gene (AdCD) could be used for selectively killing leukemic WEHI-3B cells. The AdCD transduction followed by the 5-fluorocytosine exposure abrogated the growth of WEHI-3B cells in vitro, with a minimal effect on normal hematopietic progenitors. To test the efficacy of the purging protocol in vivo, bone marrow cells were mixed with syngenic WEHI-3B cells and this chimeric cell population was transduced with AdCD vectors. Infected cells were injected into myeloablated Balb-c mice, which then received a 5-fluorocytosine treatment for 4 days. All mice transplanted with unpurged bone marrow developed leukemia and died. However, 90% of recipients receiving the purging treatment were healthy up to 9 months post-transplantation and had a perfectly re-established hematopoietic system, without any signal of leukemic cell presence. In conclusion, suicide adenoviral vectors are proposed as a tool for the purging of Adenoviral-susceptible myeloid leukemia cells contaminating autologous bone marrow grafts.

Adenoviridae↗
PubMed1997

Role of cytoprotective mechanisms in the photochemical purging of autologous bone marrow grafts.

The molecular basis of the differential sensitivity of normal hematopoietic stem cells and of leukemia, lymphoma, and neuroblastoma cells to merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) is not yet completely understood. While the capacity to bind dye molecules appears to be the major determinant of a cell's susceptibility of MC540-mediated PDT, we here present evidence that under certain experimental conditions a cell's capacity to repair MC540-mediated photodynamic damage is also an important factor. Two parameters, temperature and intracellular glutathione (GSH) content, were varied to investigate the role of cellular defense mechanisms in the dye-sensitized photoinactivation of normal murine granulocyte/macrophage progenitors (CFU-GM) and K562, L1210, and melphalan-resistant L1210/L-PAM1 leukemia cells. When exposed to MC540 and light at room temperature, the three leukemia cell lines bound similar amounts of dye and accumulated similar amounts of lipid hydroperoxide (LOOH) but differed markedly in their sensitivity to MC540-mediated PDT. Performing MC540-mediated PDT at 4 degrees C instead of at room temperature reduced dye binding and LOOH generation and enhanced cytotoxicity in some but not all cell lines. A brief (< or = 120 minutes) incubation at 37 degrees C immediately following MC540-mediated PDT accelerated the decay of LOOH in all leukemic cell lines and reduced cell kill by about 2 log in both CFU-GM and leukemia cells. The effect of post-PDT incubation at 37 degrees C on LOOH decay was most pronounced in K562 and least pronounced in L1210/L-PAM1 cells, whereas its effect on cell survival was less pronounced in L1210 cells than in the remaining cell types. L1210/L-PAM1 cells whose GSH content had been reduced from 8.2 to 1.6 micrograms/mg protein by incubation with buthionine sulfoximine recovered from potentially lethal photodynamic damage as rapidly as untreated L1210/L-PAM1 cells and more rapidly than wild-type L1210 cells with a GSH content of 4.5 micrograms/mg protein. Thus, with regard to capacity of L1210/L-PAM1 cells to recover from photodynamic damage, the cells' enhanced capacity to synthesize GSH appeared more decisive than intracellular GSH levels per se. Taken together, these data suggest that temperature-dependent cellular defense mechanisms are significant determinants of a cell's susceptibility to MC540-mediated PDT. The data emphasize the need for temperature control during and immediately after the photochemical purging of autologous bone marrow or peripheral blood stem cells.

Animals↗
PubMed1985

Monoclonal antibody-mediated cytotoxicity of human myeloid leukemia cells: an in vitro model for estimating efficiency and optimal conditions for cytolysis.

Monoclonal antibodies (MoAbs) to myeloid differentiation antigens have a potential use in purging bone marrow of leukemia cells in autologous bone marrow transplantation (ABMT) therapy for acute myelogenous leukemia (AML). Because the efficiency of purging by MoAb and complement (C) is important to the success of ABMT, we have designed an assay to determine optimal conditions for leukemia cell lysis. In order to mimic the conditions of remission bone marrow, normal buffy coat cells were mixed with cells from the HL-60 promyelocytic leukemia cell line at a concentration that approximated the normal-leukemia cell ratio found in remission marrow. The cell mixture was treated at variable times and temperatures in the presence of C and PM-81, an IgM MoAb that reacts with both normal granulocytes and monocytes as well as with HL-60 cells. PM-81 binds to the majority of cells from 90% of patients with AML yet does not react significantly with normal stem cell populations. Because of the potential use of PM-81 in ABMT, it seemed especially important to show that the antibody was capable of mediating cytotoxicity of HL-60 cells in the presence of an excess of antigen-positive cells. A clonogenic assay that permitted the growth of HL-60 cell colonies but not normal progenitor cells in methylcellulose cultures was used to measure the efficiency of HL-60 cell lysis. We found that under certain conditions, PM-81 was capable of removing the small percentage of HL-60 clonogenic cells admixed with normal buffy coat cells. This information was useful for determining the optimal conditions for purging bone marrow of leukemia cells for ABMT.

Antibodies, Monoclonal↗
PubMed1997

[Purging of hemopoietic progenitor cells in autologous transplantation].

The authors describe the results of purification of bone marrow and peripheral progenitor cells (PBPC) for clinical transplantations. Vepeside was used to purify in 1990-1995 a total of 41 bone marrows of adults and children. Of these 23 were transplanted. Maphosphamide was used bone marrow purging in two patients; transplantation was performed in one case. By a combination of Vepeside with methylprednisolone haematopoietic cells of 24 patients were purged, transplantations were performed in 10. Three-day cultivation of haematopoietic cells in the presence of Desferal was used for purging cells of 22 patients with neuroblastoma; transplantations were performed in 10 patients. The authors give the values of nucleated cells, haematopoietic colonies of CFU-GM and CD34 positive cells obtained after purification calculated per kg body weight of the patient and the percentage yields.

Adult↗
PubMed1999

Immunocytochemical assessment of bone marrow aspirates for monitoring response to chemotherapy in small-cell lung cancer patients.

Recent reports have suggested that tumour cell immunodetection in bone marrow of small-cell lung cancer patients is by far more frequent than found cytohistologically and may have clinical relevance. This study evaluates primarily the efficacy of chemotherapy as method of in vivo purging, but also the relationship of marrow involvement with survival. A total of 112 bone marrow aspirates from 30 chemo-naïve patients were stained twice using anti-NCAM antibodies, first at diagnosis and then after chemotherapy (24 patients) or at disease progression (six patients). Marrow contamination was associated with lower survival (P = 0.002), and was also detected in 7/17 patients conventionally staged as having limited disease. At multivariate analysis, marrow involvement was an independent factor of unfavourable prognosis (P = 0.033). The amount of tumour contamination, before and after chemotherapy, remained unchanged also in responders and even in the subset of patients with apparent limited disease. Following chemotherapy, bone marrow became tumour negative only in 25% of initially positive responders and in none of non-responders. Our results indicate that (i) chemotherapy is not effective in purging bone marrow even in chemo-responsive patients and (ii) a subset of patients with limited disease and negative bone marrow aspirates might have a more favourable prognosis.

Aged↗
PubMed1995

Activated lymphocytes in the marrow cell suspension decrease the mafosfamide-induced CFU-GM cytotoxicity.

The aim of the study was to assess whether other cells, besides erythrocytes, may influence the cytotoxic effect of mafosfamide (maf) during ex vivo bone marrow purging from residual tumor cells before autologous transplantation. It was shown that the presence of normal granulocytes, blast cells from acute myeloid leukemia-patients (AML) and lymphoma cells from patients with chronic lymphocytic leukemia (CLL) during maf incubation did not change the maf-induced growth inhibition of CFU-GM. Similar observation was made in experiments with resting lymphocytes. However, when phytohaemagglutinin- and pokeweed mitogen-preincubated lymphocytes were present in the marrow cell suspension, significant decline of the maf-related CFU-GM cytotoxicity was observed. These results suggest that besides erythrocytes also the activated lymphocytes in the marrow mononuclear suspension may change the final effect of maf purging.

Antineoplastic Agents↗
PubMed1985

Effect of specific anti-human leukaemia immunotoxins on the colony formation by human haematopoietic progenitors and by human leukaemia cells.

The specific cytotoxic activity of anti-human T cell leukaemia immunotoxins (IT) was investigated for their effects on in vitro colony formation of leukaemic cells and normal haematopoietic progenitors, i.e., CFU-GM, CFU-E and CFU-GEMM. These IT were prepared by conjugating ricin A chain with the monoclonal antibodies SN1 and SN2. These antibodies define two unique human T cell leukaemia antigens. This study reveals that while these IT are only marginally cytotoxic to normal haematopoietic progenitors, they strongly suppress colony formation of human T leukaemia cells. The latter suppression is augmented by the presence of 10 mM NH4Cl which results in total suppression. The present results indicate the therapeutic usefulness of these IT for in vitro eradication of leukemia cells in the bone marrow of patients with T cell leukaemia. Potentially, such a purged bone marrow can be used in autologous transplantation in leukaemia patients.

Antibodies, Monoclonal↗
PubMed1993

The peanut-agglutinin (PNA)-binding surface components of malignant plasma cells.

Plasma cells within bone marrow aspirates from multiple myeloma patients have been shown to be reactive with the lectin peanut agglutinin (PNA). This has been recently exploited by using PNA for purging bone marrow of malignant cells in autotransplantation therapy of the disease. The purpose of this investigation was to isolate and characterize the PNA-binding proteins of myeloma cells. We used the malignant plasma cell-derived line Karpas-620 (K620) as a model, and showed by affinity chromatography, SDS-PAGE, and immunoprecipitation that, among several PNA-binding proteins, a major one is an incompletely sialylated form of CD44. CD44 is a well-known homing receptor protein which is rich in carbohydrate and usually completely sialylated so that it does not react with PNA. We have then examined the PNA reactivity of myeloma cells from different patients and showed a clear difference in the profile of PNA-binding proteins from case to case. Moreover, in contrast to K620 cells, some of the patient plasma cells tested did not have a PNA-binding form of CD44. In conclusion, therefore, we have shown that a number of different proteins participate in PNA binding by malignant plasma cells. Moreover, we have demonstrated a novel, incompletely sialylated form of CD44 on a myeloma cell line. It is known that the level of glycosylation of CD44 and other proteins may affect their function, but how this relates to the malignant behaviour of plasma cells remains to be determined.

Bone Marrow↗
PubMed1997

[Study of magnetic microspheres for purging cancer cells from bone marrow. II. Preparation of polystyrene magnetic microspheres. ].

Magnetic microspheres (MS) used to purge tumor cells from human bone marrow were prepared with a two step method. Main factors that can influence the magnetization result were discussed. The MS were coated with polyacrolein which has active aldehyde group. The magnetic material content and the magnetic response of MS were determined and showed that the magnetic material y-Fe(2)O(3) by X-ray diffraction.

Acrolein↗
PubMed1992

Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia.

The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte-macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA-compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

Adult↗
PubMed1991

The biology of long-term bone marrow cultures and its application to bone marrow transplantation.

Long-term bone marrow cultures have been applied to the study and to the treatment of hemopoietic disease. The incidence of murine primitive (stem) cells with the ability to repopulate irradiated bone morrow stroma is similar to that of cells able to repopulate the hemopoietic system of potentially lethally irradiated mice. In human bone marrow, cells with in vitro repopulating ability have been estimated as 0.5% to 1% of the CD34+ cells. The increase in primitive cells in the circulation seen after administration of growth factors has been exploited by harvesting those cells for use together with bone marrow in autologous transplants. The ability of primitive cells (harvested after granulocyte colony-stimulating factor plus chemotherapy) to reconstitute irradiated marrow stromas indicates that their repopulation capacity in vitro is at least as good as that of bone marrow. This is confirmed by experimental data in mice showing that small volumes of peripheral blood (after granulocyte colony-stimulating factor administration) are capable of long-term repopulation. The regulator capacity of marrow stroma has been exploited to purge bone marrow cultures from patients with acute myeloblastic leukemia in first remission and from treated patients with chronic myelogenous leukemia before using the cultured cells for autologous transplant. The results to date, although preliminary, indicate a role for this methodology in the treatment of leukemia.

Animals↗
PubMed1996

The potential role of amifostine (Ethyol) in haematological malignancies.

Autologous bone marrow transplantation (ABMT) has considerably developed in the past 20 years. In AML, the beneficial role of purging the graft with cyclophosphamide derivatives (4-HC or mafosfamide) has been strongly suggested by retrospective studies from the European Cooperative Group for Blood and Marrow Transplantation and by single institution studies. Also, gene marking experiments have clearly shown that tumour cells infused with unpurged marrow indeed recirculate and in some instances, induce or contribute to tumour recurrence. Amifostine protects normal progenitor cells without concomitantly protecting colony forming unit leukaemic progenitors (CFUL). In comparative in vitro studies, we have shown that pre-incubation of normal marrow contaminated by leukaemic progenitors with amifostine followed by mafosfamide, results not only in a protection of the more mature progenitors (CFUGM, BFUE), but also sensitises leukaemic progenitors, so that in the end, the therapeutic index of mafosfamide is increasing by 6 logarithms. In the clinical field, it has been shown in patients with breast cancer autografted with protection by amifostine results in a shortening of the duration of aplasia of about 10 days. A European randomised study evaluating amifostine in the context of autografting for acute leukaemia has just started.

Acute Disease↗
PubMed1993

Phase I trial of intravenous cyclosporine to induce graft-versus-host disease in women undergoing autologous bone marrow transplantation for breast cancer.

PURPOSE: We investigated if graft-versus-host disease (GVHD), which is associated with an antitumor effect, could be induced in women with advanced breast cancer by treatment with cyclosporine (CSA) following reinfusion of purged autologous marrow after treatment with high-dose chemotherapy and defined the toxicities of this approach. PATIENTS AND METHODS: Fifty-one women with advanced breast cancer responding to therapy were treated with escalating doses of CSA (1.0, 2.5, or 3.75 mg/kg/d) for 28 days following high-dose chemotherapy and autologous bone marrow transplantation and monitored for induction of GVHD and toxicity of therapy. RESULTS: GVHD was induced in a dose-dependent fashion in 14%, 68%, and 92% of patients at each dose level, respectively, a median of 15 days following autologous marrow reinfusion. GVHD was clinically mild and limited to skin. Toxicity was acceptable, with two deaths within 50 days of marrow reinfusion. Statistically significant increases in maximum creatinine and bilirubin levels were seen at all dose levels when compared with similarly treated historic controls who did not receive CSA. Time to last platelet transfusion was significantly delayed in patients treated at the highest dose. CONCLUSION: GVHD can be safely induced by treatment with CSA in women with advanced breast cancer who are receiving high-dose alkylating agents and autologous bone marrow transplantation. The toxicity of this approach is acceptable. Evidence of antitumor efficacy awaits further investigation.

Adult↗