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PubMed1987

Purging leukemic cells from simulated human remission marrow with alkyl-lysophospholipid.

Alkyl-lysophospholipids (ALP) are analogues of 2-lysophosphatidylcholine that have been reported to have selective antitumor activity. These compounds could potentially be useful in purging bone marrow of leukemic cells in autologous marrow transplantation in acute leukemia. To determine the efficacy of pharmacological purging by ALP, we have designed a human assay system to mimic the conditions expected in the clinical setting of autotransplantation using remission marrow. A simulated remission marrow (SRM) was prepared by mixing normal marrow cells and HL60 cells in a ratio of 1,000:1. The effect of cryopreservation on ALP-treated normal, HL60, and SRM cells was examined. In separate experiments, ALP significantly reduced the number of clonogenic HL60 cells with no effect on normal marrow progenitors. The effect of ALP was more apparent after cryopreservation. Incubation of HL60 cells with 50 micrograms/mL ALP for four hours followed by cryopreservation resulted approximately in a 3 log reduction of clonogenic HL60 cells. ALP also selectively purged the small number of leukemic cells from SRM. In SRM, the data suggested that ALP had indirect cytotoxic activity on leukemic cells by enhancing the cytotoxic activity of monocytes in addition to its direct effect. We found no evidence that clonogenic HL60 cells decreased because of induction of differentiation by ALP. These data indicated that treatment of marrow cells with ALP offers an efficient means to eliminate leukemic cells from the graft.

Bone Marrow↗
PubMedDate not supplied

Detection of occult leukemic cells in the autologous bone marrow graft of a patient with T-cell acute lymphoblastic leukemia by a highly specific and sensitive assay.

A patient with T-cell acute lymphoblastic leukemia (T-ALL) in second remission was treated with high doses of chemotherapy and radiotherapy, followed by transplantation of autologous bone marrow purged ex-vivo with an anti-CD5-saporin immunotoxin (OKT1-SAP). Prior to transplantation the bone marrow graft had been considered in complete remission, as assessed by morphology and immunophenotyping. Twenty-two days after transplantation, the disease relapsed in the bone marrow with the same phenotype as at the onset. Retrospective analysis of the transplanted marrow cells by a recently developed high sensitivity and specificity assay (HSS assay), involving immunologic fractionation and T-cell receptor rearrangement analysis, revealed a graft contamination of approximately 0.5% malignant T-cells. This finding, together with the early post-transplant leukemic relapse, strongly suggests that the bone marrow was the source of the leukemic cells. The data are discussed for their implications on residual leukemia detection by gene rearrangement studies.

Adolescent↗
PubMed1993

Removal of breast cancer cells from bone marrow by in vitro purging with ether lipids and cryopreservation.

Four cell lines from human breast cancer (HTB 19, HTB 133, IZB B, and MCF 7M) were investigated for in vitro growth before and after incubation with the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) for 4 h and at increasing concentrations (25, 50, 75, and 100 micrograms/ml), as well as with and without cryopreservation. When measured with the human tumor cloning assay in agar or methyl-cellulose the surviving fraction showed an inverse correlation with the concentrations of ET-18-OCH3. After a 4-h exposure with 75 micrograms/ml ET-18-OCH3 at a cell density of 2 x 10(5)/ml the number of colonies of HTB 19 decreased from 75 +/- 10/10(3) cells (100%) to 1 +/- 0/10(3) cells (1%), and after subsequent cryopreservation no remaining colonies were found. In order to simulate the situation in autologous bone marrow transplantation we contaminated normal human bone marrow cells with malignant HTB 19 breast cancer cells at a ratio of 100:1. After incubation with 75 micrograms/ml ET-18-OCH3 for 4 h and subsequent cryopreservation there was a considerable reduction of HTB 19 colonies whereas the majority of normal human hematopoietic progenitors recovered. We conclude that ET-18-OCH3, in combination with cryopreservation, can remove breast cancer cells from bone marrow by up to 1 log and may be useful for purging bone marrow for autologous bone marrow transplantation not only in acute leukemia and non-Hodgkin's lymphoma but also in breast cancer.

Antineoplastic Agents↗
PubMed1987

Detection of residual murine LPC-1 myeloma cells from bone marrow cell mixture after purging by 4-hydroperoxycyclophosphamide.

Mixtures of BALB/c bone marrow (5 X 10(6) and LPC-1 myeloma (2 X 10(6) cells have been purged in vitro with 100 microM 4-hydroperoxycyclophosphamide (4HC) for 30 min at 37 degrees C. Elimination of tumor cells was assessed by monitoring newly synthesized tumor-specific IgG 2a kappa in vitro and by reinjecting the cells subcutaneously into the hindlegs of mice. Drug-treated LPC-1 cells had no detectable tumor production and did not reproduce tumors. Untreated cells regrew as solid tumors and killed the host within 3-4 weeks. Bone marrow cell suspensions purged of tumor cells were then used to reconstitute lethally pretreated mice injected 24 h earlier with 300 mg/kg cyclophosphamide (CY). Mice injected with CY alone died within 10 days. Those reconstituted with bone marrow cells or with purged bone marrow-tumor cell mixture lived longer than 7 months. Mice reconstituted with untreated bone marrow-tumor cell suspension grew tumors, had detectable tumor-specific IgG 2a kappa in their serum, and died by day 44. These studies demonstrate that the success of myeloma cell purging can be determined by monitoring newly secreted tumor protein and that 4HC successfully eliminates malignant plasma cells in vitro without impairment of normal bone marrow stem cell functions.

Animals↗
PubMed1991

[Experience of multimodal therapy for advanced neuroblastoma].

The prognosis of advanced neuroblastoma is extremely poor. We treated 5 patients with advanced neuroblastoma, older than 3 years, with multimodal therapy including intraoperative irradiation and autologous bone marrow transplantation. Elevated serum NSE and ferritin level and unfavorable histology according to the Shimadas histological classification, all of which are indicators of poor prognosis, were found in all of them. N-myc oncogene was amplified in 3 cases. After preoperative intensive induction chemotherapy, delayed primary operation and intraoperative irradiation (10-15 Gy) were performed. The postoperative lethal dose chemotherapy and total body irradiation (33 Gy x 3 days) were followed by autologous bone marrow transplantation. Tumor cells were purged using immunomagnetic beads method. Two cases showed recurrence (brain; 1, bone and bone marrow; 1) and a metastatic brain tumor was extirpated completely. All of them are alive during the follow up period from 6mo. to 4y8mo. (mean; 2y5mo.) with no evidence of disease except one. It may be concluded that our multimodal therapy is effective in achieving better results for advanced neuroblastoma.

Bone Marrow Purging↗
PubMed1989

A digital image microscopy system for rare-event detection using fluorescent probes.

Instrumentation for rare-event analysis should be capable of reliably detecting infrequent cells (less than 1:10,000) while both excluding false-positive signals and including true positive cells found in multicell clumps. We have developed a digital image microscopy (DIM) system in which a cytospin of 2 million cells is scanned with an intensified video camera (ISIT) using an IBM PC AT microcomputer-controlled microscope stage. PASCAL software controls the stage and analyzes video input, storing the location of positive cells to magnetic disk. The user can then "replay" each positive cell under computer control for either visual confirmation or analysis using other fluorescent probes. The computer requires 24 min to scan a cytoprep of 2 million cells, while playback for visual confirmation by the user averages 5 min. Using Hoechst-33342 premarked cells seeded into bone marrow as a model system, we found that the DIM system reliably detects one target cell per million marrow cells. With appropriate immunological markers, this system will aid in evaluating bone marrow purged of tumor cells prior to transplantation and should also be useful for detection of minimal residual disease in blood or bone marrow from patients with leukemia or solid tumors.

Bone Marrow↗
PubMed1993

Multiple chromosome abnormalities in patients with acute leukemia after autologous bone marrow transplantation using total body irradiation and marrow purged with mafosfamide.

Cytogenetic follow-up studies such as those reported after allogeneic bone marrow transplantation are not available in patients submitted to an autologous bone marrow transplantation (ABMT). Of 114 patients with acute leukemia (69 acute myelocytic AML, 43 acute lymphocytic ALL, 2 undifferentiated) who underwent an ABMT in our institution in the period from February 1983 to December 1989, 66 had evaluable cytogenetic data post-transplant. They all received a pretransplant regimen consisting of cyclophosphamide (CY) and total body irradiation (TBI) followed by reinfusion of marrow purged with mafosfamide. Twenty patients showed chromosomal damage at some time; of these, six relapsed early post-ABMT, one died while in persisting remission at 81 months post-ABMT from overwhelming pneumococcal sepsis related to a previous splenectomy, and 13 are still alive and well at 13 to 88 months post-transplant. The bone marrow cytogenetic abnormalities were complex: they included various numbers of clonal aberrations or variations or combination of those; they affected all but the Y chromosome, with a predominance however for chromosomes 1, 3, 6, and 7; they were often transitory and in some instances became modified with time. None of these chromosomal abnormalities was connected with the initial leukemia, even in the 6 patients who relapsed early. In the other 14 patients, these abnormalities have so far had no detectable unfavourable implication. The origin of these abnormalities is unknown: both the pretransplant regimen (CY and/or TBI) and/or marrow purging with mafosfamide can be incriminated. Additional studies in patients autografted with pretransplant regimen not containing TBI and/or with unpurged marrow are necessary to discriminate between these two possibilities.

Antineoplastic Agents↗
PubMed1988

[Metastatic neuroblastoma: consolidation treatment with 2 courses of high-dose chemotherapy followed by a bone marrow autograft].

Among 62 children over 1 year of age at diagnosis who were treated for stage IV neuroblastoma, 33 entered remission after conventional therapy. They received high-dose chemotherapy and in vitro purged bone marrow transplantation as consolidation therapy. Fifteen patients received one course and 18 two courses. At present, 16/33 grafted patients are alive in CR with a median follow-up of 28 months. Toxicity of this regimen was tolerable. Under this treatment, actuarial disease free survival is improved compared with that observed under conventional therapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1985

Bone marrow macrophages and megakaryocytes express common acute lymphoblastic leukaemia antigen.

Common acute lymphoblastic leukaemia antigen (CALLA) was demonstrated on a proportion of bone marrow macrophages and megakaryocytes. CALLA was detected by two monoclonal antibodies (J5 & BA3) in a three-layer immunoalkaline phosphatase system applied to routine air-dried bone marrow smears. The J5 staining was confirmed by an indirect immunofluorescent method and the CALLA was shown to be at the surface of the macrophages and megakaryocytes by an indirect immunogold technique. The findings are discussed in relation to the known tissue distribution of CALLA and to the clinical use of anti-CALLA antibodies for bone marrow purging.

Antibodies, Monoclonal↗
PubMed1988

Selective T cell subset depletion with anti-CD4 and anti-CD8 intact ricin immunotoxins.

In the present study we have comparatively analysed the specific activity of a panel of immunotoxins (ITs) prepared by coupling Ricin to several monoclonal antibodies (MoAbs) directed against the CD3, CD4 and CD8 T cell membrane molecules. Peripheral blood and bone marrow mononuclear cells (PBMC and BMMC) were incubated with the different ITs for 2 h, in the presence of 0.1 M lactose, washed and subsequently stimulated with either phytohemagglutinin (PHA) or an anti-CD3 MoAb. Our results indicate that the proliferative response of PBMC to both stimuli was specifically inhibited (greater than 95%) by either anti-CD3 IT or a combination of anti-CD4 and CD8 ITs, at concentrations comparable to those previously used for ex vivo T cell depletion (300 ng/ml). When used individually at the same dose, anti-CD8 and anti-CD4 ITs inhibited the PHA-induced PBMC proliferative response 40 and 70% respectively. When either anti-CD4 or anti-CD8 IT-treated cells were activated with PHA and cultured for 14 days in the presence of IL-2, less than 2% of the blasts expressed the corresponding antigens as assessed by flow cytometry analysis. Similar results were observed when BMMC were treated with the different ITs. In contrast, the growth of CFU-GM was minimally affected (0-25% inhibition). Our data indicate that ITs directed against T cell subsets are highly active and specific reagents that may be potentially useful for pre-transplant bone marrow purging.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Autologous transplantation of an immunomagnetic bead purged marrow in patients with relapsed acute lymphoblastic leukemia.

In acute lymphoblastic leukemia (ALL) the expected duration of a second complete remission (CR) following conventional aggressive multi-drug treatment is rather short, and long-term survival is rarely seen. Myeloablative consolidation treatment with ABMT in second CR is thought to benefit "high risk" patients thus allowing the escalation of cytoreductive treatment into a lethal range. In three patients with relapsed ALL bone marrow was collected in the very early second remission phase. The autografts were incubated with a panel of monoclonal antibodies bound to magnetobeads (DYNABEADS M450). The pretransplant regimen consisted of total body irradiation (TBI) (14.4 Gy) and cyclophosphamide (CY) (50 mg/kg b.w. x4). Following autologous transplantation of those immunomagnetobead purged marrow grafts, complete hematologic reconstitution was seen in all three patients. They are alive and disease-free for +26, +24 and +17 months post transplant, which is also proven by the absence of detectable gene rearrangements after transplantation. In two of them disease-free survival exceeds the duration of the preceding remission y 20 and 12 months respectively, which is a definitive parameter for therapy effectiveness. Therefore, "high dose" consolidation treatment in very early second CR followed by autologous transplantation of purged bone marrow seems to allow long-term disease-free survival. The technique of ex vivo immunomagnetic bead purging, i.e. the "direct" and "indirect" method of binding monoclonal antibodies to magnetobeads is described.

Antibodies, Monoclonal↗
PubMed1994

Fractionated total-body irradiation, etoposide, and cyclophosphamide plus autografting in Hodgkin's disease and non-Hodgkin's lymphoma.

PURPOSE: High-dose etoposide was incorporated into a regimen of fractionated total-body irradiation (FTBI) and high-dose cyclophosphamide before autologous transplant with the goal to enhance the antitumor effect of the myeloablative regimen in poor-risk lymphoid malignancies. PATIENTS AND METHODS: Ninety-six patients, 24 with recurrent or refractory Hodgkin's disease and 72 with poor-risk non-Hodgkin's lymphoma (NHL), were treated on this study. Cytoreduction with conventional therapy was attempted before administration of the preparatory regimen. The preparatory regimen consisted of 12 Gy total-body irradiation administered in 10 1.2-Gy fractions on day -8 through day -5, etoposide 60 mg/kg on day -4, and cyclophosphamide 100 mg/kg on day -2. Patients with NHL received bone marrow purged with a panel of monoclonal antibodies and complement on day 0, while patients with Hodgkin's disease received peripheral-blood stem cells alone or with unmanipulated bone marrow. RESULTS: The major morbidities of transplant were mucositis and skin toxicity. Eight patients (8.6%) died of regimen-related toxicities within 100 days of transplant. Engraftment was related to the rescue product; the median time to a neutrophil count more than 500/microL was 10 days for patients with Hodgkin's disease and 16 days for NHL patients. With a maximum follow-up duration of longer than 5 years, the 3-year actuarial survival rate is 57%. At 3 years, the actuarial freedom from progression (FFP) rate is 55% and the event-free survival rate is 47% for patients with Hodgkin's disease, while the respective figures for NHL patients are 60% and 53%. Among 32 patients with intermediate- and high-grade lymphoma transplanted subsequent to first relapse, 70% are free of lymphoma and 60% are event-free at > or = 3 years. CONCLUSION: The preparatory regimen consisting of FTBI, etoposide, and cyclophosphamide demonstrates relative efficacy in patients with Hodgkin's disease and NHL selected for high-dose therapy. Longer follow-up duration is needed to determine the rate of cure and to assess late complications. Major remaining challenges for high-dose therapy are a more inclusive strategy for all poor-risk patients and the need to reduce posttransplant relapses.

Adolescent↗
PubMedDate not supplied

[Autologous bone marrow graft in the initial treatment of follicular lymphomas in young high risk subjects. A new therapeutic approach].

Low grade malignant follicular lymphoma is characterized by its slow course over many years. However, despite a median survival of 4 to 8 years the cure rate is lower than 10 percent and even nil for some authors. The best therapeutic approach of the disease is unknown, and many teams of oncologists are in favour of a more intense chemotherapy. We present a study of 10 patients selected for their young age and for the presence of detrimental prognostic factors (index 3 of Coiffier's classification in 8/10 patients). Nine patients received BCNU, cytosine arabinoside, etoposide and melphalan, followed by reinjection of autologous bone marrow purged in vitro by mafosfamide in the adjusted dose CFUGM LD 95. Eight of these 9 patients are in complete, unmaintained remission 15 to 43 months after the bone marrow transplantation (including 3 patients in a more than 2 years' remission). The 10th patient had autologous bone marrow transplantation in 1979; after treatment with heavy TACC chemotherapy followed by reinjection of unpurged bone marrow, he remained in complete remission for 9 years, then relapsed; he is now alive with a progressive tumour. Although the follow-up was relatively short for a particularly slow disease, this study shows that, owing to autologous bone marrow transplantation as early as the first complete remission, one of the heaviest types of chemotherapy can be delivered in patients with non-Hodgkin's lymphoma, unless precluded by toxicity. At the moment, this protocol is experimental and can be used only in young subjects at high risk. Further studies on larger series of patients and with a longer follow-up are needed to evaluate the effectiveness of this new type of treatment compared with conventional chemotherapies.

Adult↗
PubMed1987

Regrowth of granulocyte-macrophage progenitor cells (GM-CFC) in suspension cultures of bone marrow depleted of GM-CFC with 4-hydroperoxycyclophosphamide (4-HC).

The hematopoietic capacity of bone marrow treated with 4-hydroperoxycyclophosphamide (4-HC) was studied using long-term suspension cultures. It was shown that, in the presence of a previously established marrow adherent cell layer (ACL), the 4-HC treated bone marrow, deprived of GM-CFC, generates GM-CFC in long-term suspension cultures in vitro. The kinetics of GM-CFC reappearance in in vitro cultures indicate their origin from more primitive progenitors rather than from surviving GM-CFC. In the presented modification, the suspension culture system is useful in evaluating the transplantation potential of bone marrow purged in vitro with chemotherapeutic or biologic agents.

Bone Marrow↗
PubMed1995

Bone marrow transplantation: the City of Hope experience.

Allogeneic BMT from histocompatible-related and -unrelated donors can provide curative therapy for a variety of hematologic, oncologic, immunologic and hereditary diseases. Obstacles to successful outcome of allogeneic BMT include GVHD, disease relapse, opportunistic infection and toxicity of the conditioning and GVHD prophylaxis regimens. As these problems are surmounted, the number of BMT survivors is expected to steadily increase and the QOL post-BMT will become as important as the duration of survival in evaluating the outcome of allogeneic BMT. High-dose chemotherapy or chemoradiotherapy, followed by ABMT or autologous peripheral blood stem cell transplant has been shown to produce long-term, disease-free survival in patients with relapsed, refractory and poor-risk HD and NHL. Despite the success of ABMT and PBSCT in treating these diseases, the short- and long-term transplant-related mortality continues to be a major concern and mandates exploration of new approaches to reduce acute and delayed fatal toxicities. ABMT as post-remission therapy for adult AML has yielded results similar to those achieved with allogeneic BMT. The role of bone marrow purging and posttransplant immunomodulation in preventing disease relapse after ABMT is being investigated.

Adult↗
PubMed1988

Prediction of the ability to purge tumor from murine bone marrow using clonogenic assays.

The development of potential purging regimens for autologous bone marrow transplantation has been limited by the inability to predict the antitumor activity of these regimens at doses which will allow engraftment. We describe an in vitro model which estimates the in vivo efficacy of potential purging regimens in mice. The log kill of clonogenic L1210 cells after in vitro incubation with graded doses of 4-hydroperoxycyclophosphamide and vincristine (alone or in combination) was linearly related to the incubation dose of drugs. Clonogenic assays could only directly demonstrate about three logs of cell kill. However, the log linear dose-response allowed the extrapolation of cell kill for doses of drugs whose kill could not be determined directly. The extrapolated cell kill accurately predicted the in vitro activity of the drugs as established by determining the survival of B6D2F1 mice given injections of the drug-treated L1210 cells. Lethally irradiated B6D2F1 mice were given injections of mixtures of syngeneic bone marrow and L1210 cells purged with a combination of 4-hydroperoxycyclophosphamide and vincristine. Combining the results of in vitro granulocyte-macrophage colony-forming unit and clonogenic L1210 sensitivities to this drug combination predicted the survival of mice and, therefore, the effectiveness of the purging regimen.

Animals↗
PubMed1995

Successful in vitro purging of leukemic blasts from marrow by cortivazol, a pyrazolosteroid: a preclinical study for autologous transplantation in acute lymphoblastic leukemia and non-Hodgkin's lymphoma.

An important new approach to curative treatment of leukemia is vigorous treatment of autologous marrow to remove residual abnormal cells in vitro prior to reinfusion. This in vitro 'purging' must destroy the malignant cells while preserving sufficient normal precursors to allow re-establishment of normal in vivo function. We have confirmed that cortivazol (CVZ), a glucocorticoid with an unusual structure, can kill dexamethasone (DEX)-resistant leukemic cells and have examined its ability to purge DEX-sensitive (CEM-C7) and -resistant (ICR-27) human leukemic blasts artificially mixed with normal marrow mononuclear cells in vitro. By carefully defining time and dose, we established a 'therapeutic window' that allowed 'cure' of the artificial remission marrow. A sufficient number of viable normal myeloid precursor cells (CFU-GM) remained to suggest that normal marrow precursors adequate for successful marrow transplantation could survive such treatment. CVZ could be a useful drug for in vitro purging of bone marrow for autologous transplantation in patients with acute lymphoblastic leukemia or non-Hodgkin's lymphoma, sensitive or resistant to standard glucocorticoid.

Antineoplastic Agents, Hormonal↗
PubMedDate not supplied

Mouse L1210V leukemia as a model to analyse efficiency of leukemic cell elimination by immunotoxin, antibody and complement, and cytostatic agents.

In an attempt to eliminate selectively mouse L1210V leukemic cells from infiltrated bone marrow, the immunotoxin MoAb-16-RTA composed of monoclonal antibody recognizing oncofetal antigen on L1210V cells and the ricin A chain was prepared. The immunotoxin exhibited an antigen-specific, dose-dependent cytotoxic effect in vitro. One hour's exposure of leukemic cells to 10(-6) M of immunotoxin appeared to be sufficient to kill all leukemic cells. In the experimental conditions applied, a complete elimination of L1210V cells from leukemic bone marrow was achieved. MoAb-16 antibody and complement in the same experimental protocol, the bone marrow purging was not complete. The surviving leukemic cells were still able to grow and kill recipient mice. The in vitro exposure to immunotoxin or to antibody and complement was not toxic to normal bone marrow progenitors. The exposure of leukemic cells to selected cytostatic agents of the oxazaphosphorine group appeared to be effective in the elimination of leukemic cells, but doses required for killing leukemic cells were highly toxic to normal bone marrow progenitors.

Animals↗