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PubMed1990

Immunomagnetic purging of bone marrow grafts for autologous transplantation in neuroblastoma.

Bone marrow grafts of 5 patients with stage IV neuroblastoma and one patient with stage IV retinoblastoma were harvested during complete remission and in vitro processed for autologous transplantation. Because of the risk to reinfuse metastatic cells, the grafts were immunomagnetically purged. After preparation of the buffy coat fraction, mononuclear cells (mnc) and progenitor cells (CFU-GM) were enriched by density centrifugation and incubated successively with a cocktail of five tumour reacting monoclonal antibodies and magnetic microparticles. Target cells were removed by a set of samarium cobalt magnets. The purged grafts were stored in liquid nitrogen. After the complete procedure, an average of 16% nucleated cells of the initial harvest could be recovered. This corresponds with a mean number of 0.5 X 10(8) mnc and 2.4 X 10(4) CFU-GM per kg body weight being available for grafting. So far, 5 patients received their purged marrow graft and showed a sustained take.

Bone Marrow Cells↗
PubMed1988

Concentration of bone marrow progenitor cells by separation on a Percoll gradient using the Haemonetics model 30.

To perform an optimal ex vivo bone marrow purge, it is necessary to concentrate the bone marrow progenitor cells and to eliminate both the red blood cells and the polymorphonuclear leucocytes. To achieve this goal which cannot be accomplished by using the Haemonetics model 30 alone, we used the Haemonetics model 30 and a density gradient together in a two-step procedure. In the first step we obtained the buffy coat from original bone marrow grafts and in the second we reintroduced these buffy coats into the Haemonetics bowl followed by Percoll, adjusted to 1.079 g/ml, at 5 ml/min in order to recover the light density mononuclear cells. After this second step, the mean volume of the marrow and the RBC and nucleated cell contaminations were reduced to 9%, 0.96% and 16% of their original values the unseparated bone marrow, respectively. This was far better than the values obtained after the first step (volume: 19%; RBC: 10%; nucleated cells: 54%). The CFU-GM recoveries after the first and second steps were 71% and 70% of the original samples, respectively. The entire procedure lasted between 75 and 150 min. At this time, 17 of the 24 patients whose bone marrow was separated using Percoll gradient in the Haemonetics bowl have been grafted. Thirteen of these 17 patients had an evaluable haematological recovery which was complete and rapid for all but one patient with acute myeloid leukaemia. These results demonstrate that the introduction of a density gradient into the Haemonetics model 30 bowl is possible and effective. The reduction in total volume and cell number permits ex vivo purging, without decreasing the grafting capability.

Bone Marrow↗
PubMed1991

Molecular analysis of retroviral transduction in chronic myelogenous leukemia.

We have developed a polymerase chain reaction (PCR) assay for detection of integrated retroviral transgenomes containing the neo G418 resistance gene in colonies (40 cells or more) grown in G418 selection after exposure to the neo-positive retrovirus LNL6. This assay also provides for simultaneous characterization of these colonies as belonging to a chronic myelogenous leukemic (bcr-abl positive) or nonleukemic population (bcr-abl negative). Using these techniques, we assessed transduction of the LNL6 retrovirus into the normal and leukemic cells of a blast-crisis chronic myelogenous leukemia (CML) patient. This work was designed to support the use of the LNL6 retroviral marker to help identify the origin of relapse after autologous marrow infusion. The data from these experiments show that the majority of CML blast crisis cells that, following exposure to the LNL6 virus, produce colonies under rigorous G418 selection are indeed transduced by the virus, as shown by the presence of the neo retroviral gene. Most of these colonies are also shown to be leukemic by PCR detection of the bcr-abl RNA. This demonstrates the feasibility of the study of CML marrow for retroviral marking. These procedures will be of use in establishing if relapse arises from leukemic blasts which contaminate purged autologous bone marrow infused following intensive therapy for leukemia.

Base Sequence↗
PubMed1993

Autologous bone marrow transplantation for leukemia: monoclonal antibody-mediated purging of multidrug-resistant leukemia.

Nine drug-resistant variants of human leukemic/lymphoma cell lines were evaluated for the expression of antigens which are commonly targeted in antibody-dependent purging protocols used for autologous bone marrow transplantation. Flow cytometry revealed that most drug-resistant variants differ in antigen expression from parental lines. Both epipodophyllotoxin-selected T-ALL variants tested expressed lower levels of CD5 than parental counterparts. In contrast, other antigens (CD7, CD3, CD4, CD8, CD38, CD9, CD10, CD24, CD19, CD20) were not consistently altered in drug-resistant variants. Drug-resistant cells were lysed as effectively as drug-sensitive cells by murine antibody and baby rabbit complement, but only when particular monoclonal antibodies or cocktails of monoclonal antibodies were utilized. Two of the nine drug-resistant variants proved to be more resistant than parental cells to antibody-dependent cell-mediated cytolysis (ADCC).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

[CD34+ cells in the autotransplant of bone marrow and peripheral blood].

Identification of a glycoprotein expressed on 1.2% of normal bone marrow cells, including progenitors of all hematolymphopoietic lineages and pluripotent stem cells, has allowed the production of several monoclonal antibodies directed against the same structure and included in the new differentiation cluster CD34. Availability of these antibodies coupled with techniques of positive selection of normal progenitors has opened an interesting and new alternative for purging bone marrow. Two of these techniques (avidin-biotin immunoadsorption on column and paramagnetic microspheres) have found clinical application and recently data on the first series of patients transplanted with CD34+ cells enriched marrows have been published. In the area of peripheral blood stem cells transplantation, detection by flow cytometry of CD34+ cells in the peripheral blood should replace the poorly standardized CFU-GM assay, allowing the best timing of apheretic procedures and the easy quantification of stem cells number in a harvest. Combination of negative (tumor cell killing) and positive (hemopoietic stem cell purification) selection might result in a significant improvement of the purging procedure and in a larger application of autologous hematopoietic stem cell transplantation for hematological and non-hematological malignancies.

Antibodies, Monoclonal↗
PubMed1989

Malignant leukemic cell separation by iron colloid immunomagnetic adsorption.

We describe an immunomagnetic assay applicable to bone marrow purging of leukemic patients before an autologous bone marrow transplantation. An iron colloidal suspension with a CD10 monoclonal antibody (MoAb) against the common acute lymphoblastic leukemia antigen (CALLA) covalently bound to the surface of the particles has been used. NALM-6 cells, a pre-B leukemic cell line expressing the CALLA, were labeled with supravital DNA dye (Hoechst 33342), mixed with peripheral blood, and incubated with the MoAb coated to iron particles. Using this reagent, at a cell concentration of 2000/microliters, a purging effect greater than 3.5 logs is observed with 0.1 mg of coated particles. Three successive rounds of treatment with the same coated particles at the same dose showed approximately the same depletion after each treatment. The recovery of clonogenic myeloid progenitors (granulocyte-macrophage colony-forming units; CFU-GM) is around 75%. No depletion was observed when the iron particles were not attached to the CD10 MoAb, or when they were attached to a MoAb not recognizing CD10+ cells. A comparison with another commercially available magnetic support was also performed in order to evaluate the performance of our assay, which appears simple, efficient, cheap, and capable of handling large volumes of cells in sterile conditions and minimal time.

Adsorption↗
PubMed1988

Metastatic retinoblastoma successfully treated with immunomagnetic purged autologous bone marrow transplantation.

There are no documented cases of long-term, disease-free survival in retinoblastoma (RB) metastatic to the bone marrow. The following study details successful outcome in a 3-year-old child with extensive marrow replacement 7 months postenucleation in an otherwise untreated group V RB. Therapy consisted of combinations of vincristine, doxorubicin, and cyclophosphamide for 3 months. After demonstrating cross-reactivity by a panel of six monoclonal neuroblastoma (NB) antibodies with the patient's RB cells, her marrow was purged by using microsphere-linked monoclonal antibodies, and then reinfused as rescue therapy after ablative doses of etoposide and cyclophosphamide. The authors conclude that short-term induction therapy followed by marrow ablative combination chemotherapy and immunomagnetically purged autologous marrow rescue can (1) effect successful outcome in widely metastatic RB, and (2) eliminate the risk of therapy-induced second malignancies.

Antibodies, Monoclonal↗
PubMedDate not supplied

Factors leading to the appearance, after BMT, of cytotoxic antibodies against T, B and activated T lymphocytes: critical appraisal.

In order to investigate the role of lymphocytotoxic antibodies, acquired after allogeneic and autologus bone marrow transplantation, we studied 309 sera from 42 transplanted patients (16 adults and 26 children). We tried to correlate antibody elicitation towards T, B and activated T lymphocytes with the following parameters: genetic (recipient's and donor's sex, HLA profile), clinical (recipient's primary disease, GvHD, transplant outcome) and technical (bone marrow purging, auto-or allotransplant). There is evidence that anti-T and -B cytotoxic antibodies appear earlier than anti-activated T antibodies. Anti-HLA specific antibodies seem to be produced by the transfusional stimulus: they appear early after BMT and wane after the first year. Humoral responsiveness seems to be age related (adults are more responsive than children) and conditioned by GvHD (the level of cytotoxic antibodies decreases when GvHD is prevented by bone marrow purging). The level of cytotoxicity is significantly lower in the sera of autotransplanted patients compared with the allotransplanted ones. It appears that anti-activated T antibodies are produced by cell activation at different times in adults and children: in adults this occurs during GVHD and in children during the relapse of disease.

Adolescent↗
PubMed1993

Busulfan/etoposide--initial experience with a new preparatory regimen for autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia.

Current intensive chemotherapy for acute nonlymphoblastic leukemia (ANLL) results in a complete remission in the majority of patients. Unfortunately, the duration of remission is short and most of the patients will experience a relapse of their underlying disease. Autologous bone marrow (BM) transplantation is being explored as a treatment modality designed to improve relapse-free survival. We have conducted a phase II trial exploring the combination of busulfan (16 mg/kg) and etoposide (60 mg/kg) in an attempt to improve antitumor efficacy using this novel preparative regimen. To date, 50 patients (48 with ANLL and 2 patients with biphenotypic acute leukemia) have been treated. The first 20 patients received unmanipulated BM; 28 patients subsequently received 4-hydroperoxycyclophosphamide (4-HC) (60 micrograms/mL)-purged bone marrow, and 2 patients with biphenotypic acute leukemia received both 4-HC (60 micrograms/mL) and etoposide (5 micrograms/mL)-purged BM. Thirty-four patients were in first complete remission (CR1), 12 patients in second complete remission (CR2), and 4 patients in relapse. The median time from first complete remission to BM harvest was 3 months (range, 0.8 to 4) compared with median time of 2 months (range, 1.5 to 5.0) for patients in second complete remission. The median time from harvest to transplant was 1 month for both groups (range, 0.4 to 36). A median of 0.7 x 10(8) (range, 0.2 to 1.4) mononuclear cells were infused. Patients achieved an absolute neutrophil count of > or = 500/microL at a median of 26 days (range, 13 to 96), an untransfused platelet count > or = 20,000/microL at a median of 56 days (range, 15 to 278) and a sustained hematocrit > or = 30% at a median of 50 days (range, 19 to 116). Twenty-six patients are alive and in continued CR. Follow-up of the surviving patients ranged from 6 months to 66 months with a median follow-up of 31 months. Patients receiving purged BM have an actuarial disease-free survival of 57% with a relapse rate of 28% compared with patients receiving unpurged BM whose actuarial disease-free survival is 32% with a relapse rate of 62% (P = .06 for relapse rate). The most significant extramedullary toxicities for this regimen are hepatic and cutaneous (including mucositis). The BU/VP-16 regimen is associated with a significant proportion of patients surviving disease free, especially in the group receiving purged BM. Whether this regimen offers a substantial improvement in disease-free survival over currently used regimens will require a prospective randomized study.

Adult↗
PubMed1987

Evaluation of a monoclonal IgM antibody for purging of bone marrow for autologous transplantation.

A monoclonal antibody of the IgM class, reacting with the CD9 (p24) antigen is described. The antibody (FMC27) is cytotoxic against cells of the common type of acute lymphoblastic leukaemia (c-ALL), giving killing at higher dilutions than an IgG antibody (FMC8) against the same antigen. FMC27 and FMC8 recognise different epitopes, and FMC27 may thus be used in a cocktail together with FMC8 and an antibody against the c-ALL antigen, WM21. Furthermore, the IgM antibody can be coated directly onto magnetic microparticles for magnetic purging, unlike the IgG antibody which must be used in a two-layer procedure.

Antibodies, Monoclonal↗
PubMed2002

Autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide purging for acute myeloid leukaemia beyond first remission: a 10-year experience.

Between January 1987 and January 1997, 69 eligible patients with acute myeloid leukaemia (AML) in either second (CR2) or third (CR3) complete remission (CR2 = 60, CR3 = 9) underwent 4-hydroperoxycyclophosphamide-purged autologous bone marrow transplantation (BMT) at the Johns Hopkins Oncology Center. The patients' median age was 27 years (range 1-62) and all received busulphan and cyclophosphamide as their preparative regimen. The probability of event-free survival (EFS) at 5 years was 30% [95% Confidence Interval (CI): 19-42%] for CR2 patients and 22% (3-51%) for those in CR3, with a median follow up of 8 years in the surviving group. The median time to an absolute neutrophil count of 0.5 x 109/l was 45 d (range 20-185). Relapse was the major cause of failure with a relapse rate of 55% in CR2 and 44% in CR3, while the non-relapse, transplant-related mortality rate was 15% in CR2 and 33% in CR3. In univariate analysis, patient age, cytogenetics, white blood cell count at presentation, CR1 duration and the sensitivity of clonogeneic leukaemia (CFU-L) in the graft to 4HC were all prognostic for EFS. Using each of these significant variables in multivariate modelling, patient age and sensitivity of CFU-L to 4HC were determined to be predictors of EFS. 4HC-purged autologous BMT produced results similar to allogeneic BMT for AML patients beyond first remission.

Acute Disease↗
PubMed1997

Optimization of purging of autologous bone marrow grafts for children with precursor B acute lymphoblastic leukemia.

In our laboratory, a two-step procedure is used for purging precursor B ALL from autologous bone marrow grafts of children in second bone marrow remission. An immunorosette depletion method with CD19 and CD22 MAbs is followed by one cycle of complement-mediated cell lysis with CD9 and CD10 MAbs. The aim of the present study was to determine if the efficacy of this procedure could be further enhanced by including CD20 and CD72 MAbs in the current protocol. Leukemia-contaminated remission bone marrow was simulated by mixing cell line cells and normal bone marrow cells. The efficacy of purging of malignant cells was determined by culturing the cells in a limiting dilution assay. The effect of including CD20 and CD72 in the immunorosette depletion was limited. In contrast, when these MAbs were added during complement-mediated cell lysis, a significant increase in depletion of tumor cells was observed. This was true when complement lysis was carried out alone (0.4 versus 3.0 log depletion for Ros cells) and when it was preceded by immunorosette depletion (2.7 versus 4.1 log depletion for Ros cells). The loss of hematopoietic progenitor cells was not greater than with the current purging protocol.

Antibodies, Monoclonal↗
PubMed1990

Relative efficiency of leukemic cell depletion using anti-murine-IgG1(Fc) or anti-murine-IgG coated immunomagnetic microbeads.

Microbeads for immunomagnetic bone marrow purging, to which sheep anti-mouse-IgG1(Fc) antibodies have been linked, and beads linked with antibodies against whole murine immunoglobulin were compared. Competitive binding studies, in which Fc fragments and Fab fragments were titrated onto the microbeads, followed by incubation with 125I-labeled whole mouse Ig, revealed that the beads linked with anti-mouse-IgG1(Fc) specifically bound the Fc region of the murine immunoglobulin molecules. The total amount of antibody of either IgG1 or IgG2 isotype that would adhere to microbeads linked with either type of antibody, as revealed by secondary binding with 125I rabbit antimouse Ig, was identical, suggesting that similar numbers of antibody binding sites were available. In cell depletion studies, it was found that if IgG1 isotype monoclonal antibodies were employed as binding intermediaries between the target cells and the microbeads, the efficiency of target cell depletion was superior with the anti-mouse-IgG1(Fc)-coated beads, even if the amount of MoAb coating the target cells was suboptimal. However, if the intermediary antibodies were of the IgG2 isotype, the efficiency of target cell depletion with these beads was inferior. These findings indicate that the efficiency of immunomagnetic bone marrow purging is dependent upon matching of the targeting MoAb and the secondary antibodies that link to the surface to the microbeads.

Bone Marrow Transplantation↗
PubMed1994

Autologous bone marrow transplantation for myeloma patients using PNA- and CD19-purged marrow rescue.

A new method of in vitro bone marrow purging using a lectin and monoclonal antibody in combination has been used for the first time in vivo. Two patients with advanced myeloma were treated with high-dose melphalan and total body irradiation and then rescued with autologous bone marrow which had been purged in vitro to remove malignant cells by using a combination of a plasma cell-binding lectin (peanut agglutinin, PNA) and the anti-B lymphocyte monoclonal antibody anti-CD19, bound to magnetised microspheres. Both patients showed rapid engraftment of the purged bone marrow and remain well 36 and 46 months later with normal bone marrow morphology, although one patient still has a low level of circulating paraprotein. This is a promising form of therapy for what has been an invariably fatal condition.

Adult↗
PubMed2002

Oncogenic interaction between BCR-ABL and NUP98-HOXA9 demonstrated by the use of an in vitro purging culture system.

Chronic myelogenous leukemia (CML) is a clonal stem cell disease caused by the BCR-ABL oncoprotein and is characterized, in its early phase, by excessive accumulation of mature myeloid cells, which eventually leads to acute leukemia. The genetic events involved in CML's progression to acute leukemia remain largely unknown. Recent studies have detected the presence of the NUP98-HOXA9 fusion oncogene in acute leukemia derived from CML patients, which suggests that these 2 oncoproteins may interact and influence CML disease progression. Using in vitro purging of BCR-ABL-transduced mouse bone marrow cells, we can now report that recipients of bone marrow cells engineered to coexpress BCR-ABL with NUP98-HOXA9 develop acute leukemia within 7 to 10 days after transplantation. However, no disease is detected for more than 2 months in mice receiving bone marrow cells expressing either BCR-ABL or NUP98-HOXA9. We also provide evidence of high levels of HOXA9 expressed in leukemic blasts from acute-phase CML patients and that it interacts significantly on a genetic level with BCR-ABL in our in vivo CML model. Together, these studies support a causative, as opposed to a consequential, role for NUP98-HOXA9 (and possibly HOXA9) in CML disease progression.

Animals↗
PubMed1991

Monoclonal antibody-purged autologous bone marrow transplantation therapy for multiple myeloma.

Eleven patients with plasma cell dyscrasias underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-treated autologous bone marrow transplantation (ABMT). The majority of patients had advanced Durie-Salmon stage myeloma at diagnosis, all were pretreated with chemotherapy, and six had received prior radiotherapy. At the time of ABMT, all patients demonstrated good performance status with Karnofsky score of 80% or greater and had less than 10% marrow tumor cells. Eight patients had residual monoclonal marrow plasma cells and 10 patients had paraprotein. Following high-dose melphalan and total body irradiation (TBI) there were seven complete responses, three partial responses, and one toxic death. Granulocytes greater than 500/mm3 were noted at a median of 21 (range 12 to 46) days posttransplant (PT) and untransfused platelets greater than 20,000/mm3 were noted at a median of 23 (12 to 53) days PT in 10 of the 11 patients. Natural killer cells and cytotoxic/suppressor T cells predominated early PT, with return of B cells at 3 months PT and normalization of T4:T8 ratio at 1 year PT. Less than 5% polyclonal marrow plasma cells were noted in all patients after transplant. Three of the seven complete responders have had return of paraprotein, two with myeloma, and have subsequently responded to alpha 2 interferon therapy. Eight patients are alive at 18.9 (8.9 to 43.1) months PT and four remain disease-free at 12.3, 17.5, 18.9, and 29 months PT. This preliminary study confirms that high-dose melphalan and TBI can achieve high response rates without unexpected toxicity in patients who have sensitive disease, and that MoAb-based purging techniques do not inhibit engraftment. Although the follow-up is short- and long-term outcome to be determined, relapses post-ABMT in these heavily pretreated patients suggest that ABMT or alternative treatment strategies should be evaluated earlier in the disease course.

Adult↗
PubMed1991

4-Hydroperoxycyclophosphamide purging of breast cancer from the mononuclear cell fraction of bone marrow in patients receiving high-dose chemotherapy and autologous marrow support: a phase I trial.

We designed an ex vivo bone marrow treatment for breast cancer patients receiving high-dose chemotherapy and autologous bone marrow support (ABMS), using 4-hydroperoxycyclophosphamide (4-HC), an active derivative of cyclophosphamide with known activity against breast cancer. This phase I bone marrow purging trial used ficoll-separated mononuclear cells (MNC) (devoid of granulocytes and RBCs), as opposed to the buffy coat. Twenty-five patients with metastatic breast cancer were studied. Patients received three cycles of the Adriamycin (doxorubicin; Adria Laboratories, Columbus, OH), fluorouracil, and methotrexate (Duke AFM) regimen, followed by marrow harvest. An MNC fraction of marrow was prepared and treated with 4-HC in concentrations of 20 micrograms/mL (four patients), 40 micrograms/mL (four patients), 60 micrograms/mL (nine patients), or 80 micrograms/mL (eight patients) and cryopreserved. Patients then received high-dose systemic cyclophosphamide, cisplatin, and carmustine, followed by infusion of the purged marrow. The study end point was marrow engraftment, defined as WBC count greater than 1,000 cells per microliter. At the first three dose levels (20, 40, and 60 micrograms/mL 4-HC), there was no significant delay in time to engraftment (19, 20, and 23 days, respectively) compared with the unpurged historical controls (17 days). At 80 micrograms/mL, engraftment was significantly delayed compared with the lower concentrations (P = .027), and further escalation of 4-HC was not attempted. A significant correlation was observed between the time of leukocyte engraftment and the 4-HC concentration (P = .017). With a methylcellulose-based tissue culture assay, we demonstrated a statistically significant correlation between the colony-forming unit-granulocyte-macrophage (CFU-GM) content in the purged marrow and the days to engraftment. Ninety-five percent of patients responded clinically to the entire program, 55% of them completely. Longer follow-up is required to assess the ultimate benefit of intensive therapy on long-term survival.

Adult↗