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PubMed1995

The sensitivity of leukemic bone marrow to simvastatin is lost at remission: a potential purging agent for autologous bone marrow transplantation.

BACKGROUND: Autologous bone marrow transplantation (ABMT) is frequently used in the treatment of malignant disease but carries the risk of reintroducing tumor cells into the patient. Methods are required for removing malignant cells from harvested bone marrow (BM) without impairing hematopoietic reconstitution. We have shown that simvastatin is toxic to leukemic progenitor cells at a concentration that conserves normal BM progenitors and may be of use clinically as a novel BM purging agent. METHODS: A two-stage culture system was used to compare the effects of simvastatin on both normal BM progenitor and primary acute myeloid leukemia (AML) cells. AML cells and normal BM mononuclear cells were incubated for 18 hours in suspension culture with 10 micrograms per mL simvastatin and the numbers of surviving clonogenic progenitor cells assayed in semisolid agar culture. RESULTS: Following simvastatin treatment of 18 AML cell populations, the mean surviving fraction of progenitor cells was 21.3 +/- 4.8% ( +/- standard error of the mean [SEM]). In contrast, the mean survival of normal BM progenitors from 16 donors was 89.6 +/- 8.6% ( +/- SEM). Samples were taken from 6 AML patients before treatment and after remission of disease had been induced by chemotherapy. In 5 of these cases the AML sample was significantly more sensitive to simvastatin than the remission sample, 4 of the 5 showed > 80% difference in progenitor cell survival. CONCLUSIONS: AML progenitor cells are sensitive to a short-term exposure to simvastatin that spares normal BM hematopoietic progenitor cells. We conclude that simvastatin may be an effective in vitro purging agent in ABMT for AML.

Acute Disease↗
PubMed1994

Autologous BMT for post-remission therapy in adult ALL: an immunological approach. For The French Group of Therapy of Adult ALL.

Among patients included in the multicentric trial ALL87, 95 patients were randomly allocated to purged ABMT arm for post-remission therapy. Immunological phenotyping performed in patients at diagnosis allowed to assigned 51 patients (54%) to the B lineage and 34 patients (36%) to the T lineage. Only 64 patients (67%) actually received ABMT mainly because of early relapses. Fifty-two patients were depleted according to the protocol: 25 B-ALL were depleted with CD10+CD19 mAbs, 19 T-ALL with CD2+CD5+CD7 mAbs and 8 patients received an Asta-Z purged BMT. Among the 12 remaining patients, 4 received Asta-Z purged BMT and 8 an unpurged one. Using an intention to treat analysis, overall survival and event free survival were similar to results observed in chemotherapy group. This study emphasizes the importance of a precise phenotyping at ALL diagnosis which allows specific immunologic bone marrow purging for ABMT.

Adolescent↗
PubMed1993

Short-term liquid marrow cultures are supported by a mixture of haematopoietic cytokines but do not purge for acute myeloid or lymphoid leukemic marrow cells.

Short-term liquid marrow culture (STLMC) is a potential source for autografting in leukemia. In a preclinical setting, including candidates for autologous marrow transplantation, we have studied STLMC supported by a selected mixture of clinical available recombinant human haematopoietic growth factors. STLMC of leukemic marrow cells were prospectively performed to evaluate the purging effect. Bone marrow cells cultured and supported by the selected mixture of rhIL-3/rhGM-CSF/rhEpo revealed an increased number of day 10-12 cultured cells, parallelled by an increased proliferation rate when compared to unstimulated cultures. The median number of myeloid progenitors recognized as day 7 and day 14 granulocyte-macrophage colony-forming units (day 7/14 GM-CFU) was significantly increased in the supported STLMC to 145/305 from 105/115 per ml culture (n = 7, p < 0.01). Further addition of rhKL did not enhance the numbers of day 7 or day 14 GM-CFUs per ml culture. In no instance was the number of clonogenic cells at the end of culture greater than the input day 0, except in cultures of purified CD34-positive marrow progenitors which resulted in an expansion of late myeloid progenitors. Cytokine-supported cultures of leukemic marrow cells from acute myeloid (n = 14) and lymphoblastic (n = 7) leukemia patients were established at the time of diagnosis. In the supported cultures, the cell number increased for myeloblast but was unchanged for lymphoblast leukemic marrow cells compared to non-supported cultures. Immunophenotypic and cytogenetic studies of selected leukemic cell samples identified unchanged myeloid or slightly reduced frequencies of lymphoblastic leukemic cells at the end of culture. This preclinical study supports the idea that the addition of a mixture of clinical available haemopoietic cytokines to STLMC increases the recovery of detectable myeloid progenitors which may enhance myeloid regeneration after autografting. No substantial selective loss of myeloid leukemic cells was found in the cytokine-supported short-term culture system.

Bone Marrow↗
PubMed1991

Autologous bone marrow transplantation for acute myeloblastic leukemia in Europe: further evidence of the role of marrow purging by mafosfamide. European Co-operative Group for Bone Marrow Transplantation (EBMT).

Fifty-nine European teams have reported 919 autografts for the consolidation of acute myelocytic leukemia (AML) up to December 31, 1989. The distribution for autologous bone marrow transplantation (ABMT) was 671 in first complete remission (CR1) and 196 in CR2. Pretransplantation regimes were: total-body irradiation (TBI), 456; busulfan plus cyclophosphamide (BU-CY) 174; marrow purging with mafosfamide, 269 (corresponding to 26% of all patients in CR1 and 41% in CR2). Patients autografted in CR1 with no high risk factor (standard risk) had a leukemia-free survival (LFS) and relapse rate at 7 years of 48 +/- 2 and 41 +/- 3%, respectively. Of all the prognostic factors studied, only secondary leukemia was correlated with a poorer LFS (19 +/- 9% at 1 year) and a higher relapse rate (76 +/- 11%) (p less than 0.0001). For patients autografted in CR2, the LFS and relapse rate were 34 +/- 4 and 54 +/- 5%. With the restriction of a shorter follow-up, the results achieved with the BU-CY combinations (LFS and relapse rate at 3 years, CR1 47 +/- 6 and 45 +/- 7%; CR2, 37 +/- 9 and 50 +/- 10%) did not differ from those with TBI or other chemotherapy combinations. LFS and relapse rates were correlated with several pretransplant intervals: in CR1, patients reaching CR more rapidly (less than or equal to 40 days) had a better LFS (53 +/- 3 versus 42 +/- 3%; p = 0.03) and a lower relapse rate (46 +/- 3 versus 57 +/- 3%; p = 0.03). In patients autografted less than 3 months, 3-6 months and more than 6 months after CR, the LFS was 26 +/- 5, 49 +/- 3, and 55 +/- 4%, respectively, and the relapse rates 63 +/- 5, 38 +/- 3, and 36 +/- 4% (p less than 0.0001 for both). In CR2, patients autografted more than 18 months after the initial diagnosis had a better LFS (42 +/- 5 versus 24 +/- 5%; p less than 0.001) and a lower relapse rate (45 +/- 6 versus 65 +/- 6%; p less than 0.001). For those autografted less than 3 months, 3-6 months and more than 6 months after CR, the probability of LFS was 30 +/- 5, 30 +/- 7, and 50 +/- 9% (p = 0.06), respectively and the relapse rates 63 +/- 6, 50 +/- 8, and 36 +/- 8% (p = 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1992

Differential cytotoxicity of an ether lipid on lymphoma and bone marrow cells and its role in purging malignant lymphoid cells from remission bone marrow contaminated with tumor cells.

Four human clonogenic malignant lymphoid cell lines (CEM, Su-DHL-4, Li-A, and Raji) as well as normal human bone marrow stem cell progenitor cells were investigated for clonal in vitro growth before and after incubation with the ether lipid ET-18-OCH3 for various times (1, 4, and 18 h) and at increasing concentrations of the drug (25, 50, 75, and 100 micrograms/ml). The clonal growth of the malignant lymphoid cell lines was inversely correlated with concentrations and times of drug incubation. The antineoplastic effect of ET-18-OCH3 was further amplified by subsequent cryopreservation. In a situation of 4-h exposure to less than or equal to 50 micrograms/ml ET-18-OCH3 and subsequent cryopreservation, in which greater than 50% of the normal human bone marrow progenitor cells survived, 1-3 logs of the malignant lymphoblastoid cells were killed, indicating a potential value of this drug for bone marrow purging in lymphoid malignancy. In order to simulate the situation of autologous bone marrow transplantation (ABMT) in complete remission of the disease, we contaminated normal human bone marrow cells with malignant CEM or Su-DHL-4 lymphoid cells at a ratio of 100:1. Results show that 4 h of incubation with 75 micrograms/ml ET-18-OCH3 and subsequent cryopreservation can eliminate 2-3 logs of clonogenic cells of the malignant lymphoblastoid cell lines under conditions that allow recovery of greater than 50% of the normal human hematopoietic progenitors.

Antineoplastic Agents↗
PubMed1992

Ex vivo differentiation therapy as a method of leukemic cell purging in murine bone marrow expansion cultures.

We investigated the use of differentiation therapy as a method of purging bone marrow (BM) of leukemic cells in ex vivo murine BM expansion cultures (delta-cultures). In clonal cultures and in suspension cultures a combination of the differentiation-inducing agents granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-6, and all-trans-retinoic acid (ATRA) was found to be most effective in inducing the differentiation of the murine myelomonocytic leukemic cell line WEHI 3B D+ LacZ clone 2.8 (clone 2.8). Furthermore, we investigated the activity of a mutant form of IL-6, mutein, and found it to have a greater specific activity in cell proliferation assays and in a clone 2.8 differentiation assay than the native form of IL-6. Coculture of clone 2.8 and BM in IL-1 and kit-ligand-stimulated delta-cultures showed that the added stimuli, G-CSF, mutein, and ATRA, decreased the expansion of leukemic cells. Mice transplanted with G-CSF, mutein, and ATRA-purged BM had an increased survival time relative to nonpurged controls. The addition of G-CSF, mutein, and ATRA to delta-cultures did not result in any impairment of hematopoietic stem cells when measured 5 wk after transplantation.

Animals↗
PubMed1991

High-dose melphalan with 6-hydroxydopamine-purged autologous bone marrow transplantation for poor-risk neuroblastoma.

Long-term results are presented of 28 patients who were diagnosed with neuroblastoma at more than 12 months of age and who received melphalan 180 mg/m2 (n = 6) or 240 mg/m2 (n = 22) to consolidate remissions of Stage IV disease or to control refractory disease. Twenty-four patients also received dianhydrogalactitol 180 to 240 mg/m2, and 11 received total body irradiation 450 to 600 cGy. Autologous bone marrow transplantation (ABMT) was performed with marrow that was unpurged (n = 2) or purged ex vivo (n = 26) with 6-hydroxydopamine (6-OHDA) 20 micrograms/ml plus ascorbate 200 micrograms/ml. The median time to an absolute neutrophil count of 500/microliters was 21 days and to self-sustaining platelet counts more than 20,000/microliters, 28 days. One patient required infusion of unpurged reserve marrow. Two groups of patients underwent ABMT: (1) 17 patients (Group I) who were in first remission a median of 7 months after diagnosis; and (2) 11 patients (Group II) who had refractory disease or were in second remission. For Group I, event-free survival was 29% at 12 months and 6% at 24 months post-ABMT. All Group II patients died of disease or ABMT-related toxicity. Overall, of the 28 patients, one is a long-term relapse-free survivor; five died of ABMT-related toxicity; ten patients with tumors present at ABMT had progressive disease within 6 months of ABMT; and 12 patients with no measurable disease at ABMT relapsed 4 to 32 months (median, 12) post-ABMT. Among the latter, six relapses involved the primary site, and six were restricted to distant sites. These results--in accord with the long-term outcome in other series--suggest that for neuroblastoma high-dose melphalan cannot be relied on to ablate residual disease or to salvage patients with refractory tumors. In addition, the pattern of relapse in several patients could be explained by infusion of incompletely purged autografts; this would support recent laboratory evidence that 6-OHDA/ascorbate is a suboptimal purging method.

Adolescent↗
PubMed2000

Purging of G-CSF-mobilized peripheral autografts in acute leukemia with mafosfamide and amifostine to protect normal progenitor cells.

In the present study the in vitro growth of CFU-GM from PBPC of patients with AML (n = 11), purged with mafosfamide alone or a combination of mafosfamide and amifostine, was compared to historical controls of mafosfamide-purged bone marrow (AML CR1, n = 16). Two patients were transplanted with mafosfamide and mafosfamide/amifostine pretreated PBPC autografts. The in vitro experiments demonstrated a significantly higher resistance of peripheral blood derived CFU-GM to mafosfamide (median ID95 190 microg mafosfamide/ml) compared with bone marrow derived CFU-GM (median ID95130 microg/ml). Preincubation with amifostine significantly further increased the median ID95 to 245 microg/ml. The clinical results showed short recovery times for neutrophils >500/microl (9 and 13 days) and platelets >20 000/microl (12 and 21 days) and stable long-term engraftment with one relapse at day +118 and one patient in CR at day 760 after transplantation. The in vitro results show a significant advantage of PBPC over bone marrow-derived progenitors for purging with mafosfamide. Furthermore, a protective effect from mafosfamide of amifostine on normal progenitors could be demonstrated. The clinical results demonstrate the clinical feasibility of using mafosfamide-purged autologous PBPCT without impairing the short-term and long-term repopulating capacities of the autografts.

Acute Disease↗
PubMed1990

Effective purging of bone marrow by a combination of immunorosette depletion and complement lysis.

The effectiveness of a simple immunorosette technique for the depletion of common acute lymphatic leukemic (cALL) blasts from autologous bone marrow transplants was studied. Erythrocytes were sensitized with tetramolecular complexes consisting of rat anti-mouse IgG1 monoclonal antibodies (McAbs) that crosslink two different mouse McAbs. One of the McAbs was directed against glycophorin A, and the other was directed against marker glycoproteins of B cells and their precursors (CD9, CD10, CD19, or CD22). Immunorosettes were formed by addition of the sensitized erythrocytes to the cALL+ cells. After density-gradient separation of immunorosettes from mixtures of cALL+/terminal deoxynucleotidyl transferase-positive (TdT+) leukemic blasts and mononuclear bone marrow cells, nearly a 2-log depletion of leukemic cells was measured by flow cytometry. Clonogenic assays with two cALL+B-cell lines (Ros-17 and Nalm-16) were performed to compare the efficacy of complement-mediated cell lysis, immunorosette depletion, and a combination of both procedures. Complement-mediated cytotoxicity with the three McAbs in combination with baby rabbit complement yielded a 1- to 2-log cell kill. Immunorosette depletion resulted in a 3-log reduction of clonogenic units. Sequential application of the two methods (immunorosette depletion with CD19 McAb followed by a complement lysis with CD9 and CD10 McAbs) led to superior results in causing a 4- to 5-log purging effect. These purging procedures did not cause a loss of normal myeloid (granulocyte-macrophage colony-forming units, CFU-GM) or erythroid (erythroid burst-forming units, BFU-e) progenitors from the bone marrow. This study indicates that the combination of the two methods results in a highly efficient purging procedure for the removal of cALL+ cells from autologous bone marrow cells.

Animals↗
PubMed1988

In vitro purging of bone marrow for autologous marrow transplantation in acute myelogenous leukemia using myeloid-specific monoclonal antibodies.

Autologous bone marrow transplantation (ABMT) for acute myelogenous leukemia has gained new popularity recently due to the availability of techniques to purge the autograft of residual leukemia cells. One of the methods of purging involves the use of monoclonal antibodies (mAbs) directed to antigens expressed on the surface of leukemia cells. Several mAbs lend themselves to this use because of their high degree of reactivity with leukemia cells and their lack of reactivity with normal pluripotent hematopoietic progenitor cells. Candidate mAbs include those directed to the CD14, CD15, and CD33 molecules. This review focuses on the pre-clinical data concerning the use of mAbs to these antigens and on the results of on-going clinical trials of ABMT using mAb-mediated purging.

Antibodies, Monoclonal↗
PubMedDate not supplied

Monoclonal antibody therapy with autologous peripheral blood stem cell transplantation for non-Hodgkin's lymphoma.

BACKGROUND: With the introduction of novel monoclonal antibody products into the clinic, significant new strategies are being developed to improve upon existing treatment for non-Hodgkin's lymphoma. METHODS: Monoclonal antibodies are being used alone, in combination with chemotherapy, or as adjuncts to autologous bone marrow transplantation for the purpose of purging bone marrow of neoplastic cells. RESULTS: Monoclonal antibodies when used in vivo in conjunction with autologous bone marrow transplantation have been relatively well tolerated. Results from several trials seem to demonstrate a therapeutic benefit for the use of such combinations. CONCLUSIONS: Before these agents can be included in standard bone marrow transplantation regimen, long-term survival outcomes need to be obtained from randomized trials. We review the results from recent trials using monoclonal antibodies in conjunction with autologous stem cell transplantation for the treatment of non-Hodgkin's lymphoma.

Antibodies, Monoclonal↗
PubMed1991

Immunomagnetic removal of L1210V leukemic cells from mouse bone marrow ex vivo.

The purging of mouse bone marrow from L1210V leukemic cells was attained ex vivo by using monoclonal IgM antibody against leukemic cells and magnetic particles charged with goat anti-mouse IgM antibodies (immunobeads), followed by magnetic separation. The monoclonal antibody MoAb-16 recognizes oncofetal antigen on target cells and binds to 100% L1210V cells. The number of the remaining clonogenic leukemic cells was examined by LCFU and bioassay tests. The clonogenic capacity of the bone marrow progenitor cells was measured by NCFU test. After first treatment cycle the number of LCFU was reduced in a dose-dependent manner. After second cycle of purification a complete elimination of L1210V cells from marrow was achieved at the concentration 10 mg/ml of magnetic particles. Although at lower concentrations of immunobeads no leukemic spleen colonies were found, some residual L1210V cells were still present in the bone marrow and were able to grow and kill the recipient mice. The loss of bone marrow progenitor cells during magnetic removal procedure was about 10%.

Animals↗
PubMed1992

Predictive value of flow cytometric analyses of blast cells in assessing the phenotype of the leukemia colony-forming cell (L-CFC) population in acute myeloid leukemia.

Acute myeloid leukemia (AML) blast cells (BC) express antigens that are commonly found on their normal counterparts. The leukemia colony-forming cell (L-CFC) subpopulation, identified by its ability to form leukemia colonies in vitro, is thought to be the stem cell population that produces BC. To ascertain the association between myeloid antigens on the BC and the L-CFC from the same patient, we compared the expression of CD14, CD15, CD33, p124 and HLA class I from 17 cases of AML. These particular myeloid antigens were studied because they are suitable targets in purging bone marrow for autotransplantation. We found no significant difference in the expression of CD14, CD15, CD33, and HLA class I on the BC and L-CFC from the same patient, although we observed considerable heterogeneity among different AML cases. Analysis of the progenitor cell antigen p124 revealed significant within-patient differences on the BC and L-CFC (p = 0.007), with a greater tendency for expression on the L-CFC. This heterogeneity may be due to differences in maturation stage of the L-CFC and BC. This information is important when L-CFC phenotype is used to determine the appropriate selection of antibodies for purging of residual disease in the context of auto-transplantation.

Antigens, Differentiation↗
PubMed1990

Collagen gel cultures for detection of spared hematopoietic progenitors in Asta Z 7557 purged human marrows.

Asta-Z 7557, an in vitro active metabolite of cyclophosphamide, is used for human bone marrow purging in acute leukemias and solid tumors since it is more highly toxic to tumor than normal hematopoietic clonogenic cells. However, doses higher than 80 microM totally inhibit the growth of hematopoietic progenitors in semisolid assays, despite preservation of marrow repopulating ability in vivo. This discrepancy complicates assessment of the functional value of purged grafts. In 11 patients undergoing autologous bone marrow transplantation with 100 microM Asta-Z purged marrow, we investigated the advantages of the collagen gel culture system for detection of spared hematopoietic progenitors. This technique provides a suitable and convenient assay as compared with results with agar. In nine of 11 samples after 14 or 21 days of incubation, up to 100 colonies were observed, (64% monocytic, 27% granulocytic). Day-14 granulocytic colonies were mostly immature, suggesting that they derived from early CFU-GM or pre-CFU-GM. This development of early progenitors could account for the better sensitivity of this technique compared with agar or methylcellulose systems. The mechanism involved as well as the correlation between colony growth in collagen and clinical hematopoietic recovery is also considered.

Adolescent↗
PubMed1993

Developments in purging in autotransplantation.

High-dose therapy and bone marrow transplantation has been shown to be a potentially curative modality for patients with hematologic malignancies. Several obstacles to the use of this approach include the availability of histocompatible siblings and the increased toxicity even in HLA-matched patients owing to graft-versus-host disease and interstitial pneumonitis. The use of autologous marrow in support of high-dose therapy has lower toxicity; however, there is the issue that residual tumor cells may be reinfused into the patient. There are several laboratory studies demonstrating that residual tumor cells persist in the marrow despite histologic remission. In addition, case reports suggest that contaminated marrow has led to widespread early relapse owing to reinfusion of tumor cells. A variety of techniques have been developed that can deplete up to 5 logs of tumor cells from the marrow. These techniques include very specific immunologic as well as less specific pharmacologic purging. Both approaches have been refined so that normal hematopoietic stem cell reconstitution is relatively preserved. Although a large number of studies have been reported that have utilized ex vivo marrow purging, few have examined whether there has been an impact on disease-free survival. Although randomized studies have not been performed to date, several recent studies in ANLL and B-cell NHL strongly suggest that there is a relationship between the quality of elimination of the disease ex vivo in the marrow and disease-free survival. With further improvements in marrow treatment, whereby all detectable cells are depleted, as determined by highly sensitive molecular biologic techniques, and randomized trials involving purged and unpurged BM, the question of the impact of ex vivo marrow treatment can be better answered.

Antibodies, Monoclonal↗
PubMed1992

Pharmacological purging of syngeneic bone marrow ex vivo: effect of treatment with doxorubicin and lonidamine on normal and leukaemic cells of DBA/2 mice.

The in vivo effect of in vitro treatment with doxorubicin plus lonidamine on normal and leukaemic cells was investigated in a mouse model of syngeneic bone marrow transplantation. Different numbers of L5178Y tumour cells or normal bone marrow cells alone, or mixtures of bone marrow and leukaemic cells were incubated with doxorubicin (0.25, 0.5, 0.75, 1 microgram/ml) and/or lonidamine (50 micrograms/ml) and reinfused in DBA/2 mice. Lonidamine potentiated the cytotoxic effect of doxorubicin dependent on doxorubicin dosage and tumour cell concentration. Survival after injection of 10(4) in vitro-treated tumour cells was 42% for doxorubicin 0.75 micrograms/ml alone versus 100% for the combination with lonidamine and 50% for doxorubicin 1 microgram/ml alone versus 100% combination. Reinfusion of normal bone marrow incubated with doxorubicin alone or in combination with lonidamine in lethally irradiated mice did not occur in 12-14% of mice injected, indicating that the repopulating ability of stem cells was spared. These data suggest the potential usefulness of lonidamine in ex vivo purging of bone marrow before autologous bone marrow transplants in haemopoietic malignancies.

Animals↗
PubMed1991

[Autologous bone marrow transplantation for acute leukemia].

Autologous bone marrow transplantation (ABMT) was performed in 20 common ALL antigen (CALLA) positive acute lymphoblastic leukemia (ALL) cases. Bone marrow cells harvested in complete remission (CR) were in vitro treated with monoclonal antibodies NL-1 (IgG2a: anti-CD10), NL-22 (IgM), HL-47 (IgM) and baby rabbit complement and cryo-preserved. Three children with high risk factor such as Ph1 chromosome were transplanted in 1st CR, 5 in 2-3 CR, 8 adults in 1st CR, 2 in 2nd CR and 2 in relapse. As conditioning of ABMT, 12 cases mostly in 1st CR were treated with 4 doses of 2 g/m2 cytarabine (CA), 2 doses of 60 mg/kg cyclophosphamide (CY) and 4 doses of 3 Gy total body irradiation (TBI). 6 cases refractory to CA/CY were treated with 3 doses of 60 mg/m2 melphalan and 4 doses of 3 Gy TBI. Disease free survival (DFS) of 11 cases in 1st CR was 81.8% at 2 years from ABMT, and 9 cases are alive in CR. DFS of 5 cases in 2nd CR was 40.0%. In one Ph1 chromosome positive case, minimal residual bcr-abl mRNA was detected by polymerase chain reaction in the 1st CR bone marrow, but disappeared after purging procedures and after 3 months from ABMT. Our results indicate that ABMT with purged bone marrow is an effective mode of therapy for high risk CALLA positive ALL.

Adolescent↗