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PubMed1997

Detection of neuroblastoma cells in CD34+ selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry.

A sensitive assay was developed for the detection of neuroblastoma cell contamination in CD34+ selected and unseparated peripheral blood stem cells (PBSC) used for autologous transplantation in stage 4 neuroblastoma patients. Specifically, we established a non-radioactive nested cDNA-PCR (nPCR) for detection of tyrosine hydroxylase (TH) gene expression combined with anti-disialoganglioside GD2 immunocytochemistry with the murine monoclonal antibody (MAb) 14G2a. Sensitivities of TH nPCR determined with a number of neuroblastoma cell lines and PBSCs correlated to cell line dependent basal TH gene expression levels and ranged from 1:10(4) to 1:10(6). The sensitivity obtained by immunocytochemistry was 1:10(5). We observed the highest PBSC contamination rate of 47% (18/38) among 38 PBSC specimens exclusively obtained from stage 4 neuroblastoma patients by using TH nPCR and GD2 immunocytochemistry in combination. Furthermore, a clinically applied purging method, CD34+ selection by immunoabsorption (CD34+ purity 42.4%), was used on 16 PBSCs. 10/16 (63%) preparations were contaminated prior to CD34+ selection and 56% (9/16) remained contaminated. A significant reduction of neuroblastoma cell contamination by CD34+ selection was not detectable, but the absolute amount of re-infused tumour cells was decreased due to 100-fold smaller cell counts of CD34+ selected grafts used for transplantation. 22 PBSC preparations were used for transplantation. A Kaplan-Meier analysis showed an event-free survival probability of 0.56 +/- 0.22 (n = 9) in the group with contaminated PBSCs versus 0.88 +/- 0.12 (n = 8) with no detectable neuroblastoma-cell contamination. Our data suggest that the combined use of TH nPCR and GD2 immunocytochemistry is optimal to detect contamination and monitor purging strategies.

Antigens, CD34↗
PubMed1990

Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate.

A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A. The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow. The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells. NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.

ADP Ribose Transferases↗
PubMed1988

Mafosfamide (ASTA-Z-7654) in vitro treatment of bone marrow does not eradicate Philadelphia-positive cells in chronic myeloid leukaemia.

Bone marrow from 9 patients with Ph1+CML was treated in vitro with varying concentrations of Mafosfamide in order to test the hypothesis that selective pharmacological killing of Ph1-positive cells in CML is feasible. A marked difference in sensitivity to Mafosfamide of CFU-GM was observed, but at all concentrations 100% persistence of Ph1+ cells was noticed. We conclude that, in classical cases of Ph1-positive CML, Mafosfamide purging of bone marrow will not be effective.

Adult↗
PubMed2006

Ex vivo expansion of mafosfamide-purged PBPC products.

BACKGROUND: Multiple studies have demonstrated that 'purging' of autografts with 4-hydroperoxycyclophosphamide (4HC) or the related compound mafosfamide (Mf), to eradicate residual leukemia, produces the best results associated with autologous blood and marrow transplantation for AML. However, 4HC purging results in prolonged aplasia. Therefore, we evaluated the potential of ex vivo expansion of Mf-treated CD34+ cells from mobilized PBPC. METHODS: CD34+ cells were isolated from PBPC products and treated with 30 microg/mL Mf. The Mf-treated CD34+ cells were washed and cultured for 14 days in StemLine II-defined media containing recombinant human (rh) SCF, G-CSF and thrombopoietin (Tpo). RESULTS: Treatment with Mf resulted in 90% killing of progenitor cells (GM-CFC) but maintenance of SCID-repopulating cells (SRC). Ex vivo culture of the Mf-treated CD34+ cells resulted in decreased cell numbers (10-20% of the starting cell dose) during the first week. Nevertheless, in the second week of culture the total cell numbers expanded to approximately 20-fold above starting cell numbers and progenitor cells returned to approximately pre-treatment levels. DISCUSSION: These studies demonstrate the potential of ex vivo culture to expand both total cell numbers and progenitor cells following treatment of PBPC CD34+ cells with Mf. Clinical studies are currently being initiated to evaluate the engraftment potential of these purged and expanded products.

Adjuvants, Immunologic↗
PubMed2003

Ex vivo purging with NK-92 prior to autografting for chronic myelogenous leukemia.

Although controversial, purging of the autograft may be necessary to optimize transplant outcome, especially if better treatments become available to eliminate or control residual disease that may be left after the conditioning regimen. The intent of this study was to show that immunological purging with the cytotoxic cell line NK-92 effectively reduces the number of clonogenic cells and that the method can be performed in compliance with GMP. Owing to the easy quantification of bcr-abl transcripts, chronic myelogenous leukemia (CML) was used as a model disease for proof of principle. A detection level of 10(-7) bcr-abl+ cells and purging efficiency of four logs were achievable for the bcr-abl+ cell line, K562. Leukapheresis products collected from CML patients after stem cell mobilization were then tested. For all patients tested, residual CML cells were highly sensitive to purging by NK-92 with a purging efficacy of several logs. No adverse effect on hematopoietic progenitor cell function was noted. These results demonstrate the efficacy of NK-92 as a purging agent to decrease or eliminate malignant contamination of autologous stem cell grafts and establish proof of principle for ex vivo purging of CML autografts using cytotoxic effector cells.

Antigens, CD34↗
PubMed2005

N-desmethyl clozapine as purging agent of leukemic cells in vitro.

Attempts have been made to improve the results of autologous transplant in leukemia by purging strategies to deplete malignant cells. Clozapine is an atypical antipsychotic agent, which has been associated with hematopoietic toxicity. It has been shown that N-desmethyl clozapine a metabolite of clozapine is toxic to the hematological precursors. There is also evidence that this toxicity is confined to precursors and does not affect hematopoietic stem cells. The metabolite of clozapine (N-desmethyl clozapine) has been found to be more toxic than clozapine itself. This suggests that perhaps this metabolite can be used for purging of leukemic cells. Here it is being hypothesized that N-desmethyl clozapine could be used as an in vitro purging agent for leukemia patients. This might improve the therapeutic efficacy of autologous transplantation in leukemic patients.

Bone Marrow Purging↗
PubMed1997

Treatment of non-Hodgkin's lymphoma with high-dose therapy and hematopoietic stem cell support.

Standard therapies for advanced low-grade lymphomas are not likely to provide a cure, prompting the use of more aggressive approaches. Patients with low-grade lymphoma who receive high-dose therapy with stem cell support appear to have a prolonged disease-free survival, although the benefit to overall survival remains unproven. Patients with chemotherapy-sensitive intermediate- or high-grade relapsed disease have improved survival with high-dose therapy, and those with high-risk disease may benefit from early consolidation while in first remission. Significant questions remain in terms of the proper timing of high-dose therapy, appropriate stratification by risk factors, the value of purging, the role of radiotherapy after transplantation, and the most appropriate source of stem cells for transplantation.

Antineoplastic Agents↗
PubMed1988

Leukaemia and lymphoma treatment with autologous bone marrow transplantation: preclinical studies.

Following the limited success of T-cell depletion in patients undergoing allogeneic bone marrow transplantation (BMT), recent interest is focused on autologous BMT. The selection of sensitive methods for detecting residual lymphoid malignancy, the choice of very efficient complement-fixing lytic monoclonal antibodies against residual disease, and the "tailoring" of these antibodies (or their cocktails) to individual patients lead to a very high remission rate in acute lymphoid leukaemia (ALL) of bad prognosis and second remission. The future of this extension of chemotherapy toward BMT seems to be promising, but controlled trials are required to show that the purging of bone marrow contributes to this good result. Very efficient antibodies for elimination of B lymphoma cells are also available.

Antibodies, Monoclonal↗
PubMed2000

Immunologic methods of purging in autologous stem cell transplantation.

Malignant cells in the stem cell product have been shown to contribute to disease recurrence in patients who relapse after autologous transplantation. Immunologic methods of purging tumor cells from stem cell products focus on either removal of specific target cells or positive selection of HPC. mAb are the key component of many purging strategies and are employed both in vitro and in vivo. Cytotoxic cellular therapies are an emerging method of tumor cell eradication.

Antibodies, Monoclonal↗
PubMedDate not supplied

Heat sensitivity, thermotolerance and protein synthesis of granulocyte and macrophage progenitors from mice and from long-term bone marrow cultures.

Ex-vivo purging of leukaemic cells by single or fractionated heating during bone marrow transplantation for acute myeloid or lymphoblastic leukaemias may be possible since many leukaemic cells may be more sensitive to killing by heat than normal bone marrow colony-forming unit-granulocyte and macrophage (CFU-GM). In these studies we have compared heat response, thermotolerance response and heat shock protein (HSP) synthesis of bone marrow progenitors from intact mice or bone marrow obtained from 37 degrees C or 33 degrees C long-term bone marrow cultures (LTBMCs). Such studies were done to examine whether CFU-GM responses to heat are influenced by in vitro growth conditions. In terms of heat response CFU-GM progenitors from LTBMCs showed increased heat sensitivity when compared to CFU-GM from mice, with CFU-GM from 33 degrees C cultures being more heat-sensitive than those from 37 degrees C cultures. The kinetics of thermotolerance development and decay in CFU-GM were similar from all three systems. Thermotolerance was maximum at 3-9 h, began to decay by 24 h and was absent by 48 h from cells obtained from mice and LTBMCs grown at 37 degrees C. However, CFU-GM from 33 degrees C LTBMCs showed a delay in decay of thermotolerance. The kinetics of the development and decay of thermotolerance remained as above and was independent of incubation conditions whether bone marrow was left in situ or incubated in vitro in medium containing various colony-stimulating factors (CSF) or medium containing no CSF. The synthesis of HSPs was measured by one- and two-dimensional gel electrophoresis. Synthesis of HSP-70 kd was detected following a triggering dose of heat at 0-3 h for bone marrow heated in vivo and 0-6 h for bone marrow from LTBMCs. These studies indicate that CFU-GM heat and thermotolerance response was influenced by the temperature at which the cultures were maintained but not by other differences present in their microenvironment.

Animals↗
PubMed1998

A single step density gradient separation for large scale enrichment of mobilized peripheral blood progenitor cells collected for autotransplantation.

Peripheral blood leukocytes are becoming the preferred source of hematopoietic progenitor/stem cells for autologous transplantation. However, in vitro purging procedures are complex and expensive when applied to peripheral blood progenitor cells harvests. This is mainly due to the large quantities of nucleated cells present in leukapheresis collections. Aiming to reduce total cellularity without significant loss of CD34+ cells, we developed an in vitro cell separation procedure based on ficoll/metrizoate gradient used at a final density of 1.067 g/ml. To obtain this density, standard Lympho-prep (1.077 g/ml) was diluted with normal saline solution (NaCl 9 g/l). Twenty-six leukapheresis collections (median cellularity 21.1 x 10(9), range 2.8-60) from 14 patients with non-Hodgkin's lymphoma, multiple myeloma or plasma cell leukemia were processed (median two leukaphereses per patient). Mean (+/- s.d.) recovery of total nucleated cells, CD34+ cells and CFU-GM was 20.9 +/- 10%, 74.7 +/- 22% and 70.5 +/- 19%, respectively. Cumulative per patient progenitor cell recovery was always above 50%, and as high as 80% in 10/14 patients, while total cellularity was reduced to a median 21.5% (10-33%) of pre-separation values. Contaminating neoplastic cells, identified by immunofluorescence in five collections, were reduced by 1-2 logs. The results indicate that our density gradient separation is an effective method to reduce total cellularity prior to immunological purging, without significant loss of progenitor cells.

Antigens, CD34↗
PubMed1999

Targeting myeloid leukemia with a DT(390)-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice.

The IL-3 receptor was expressed on a high frequency of myeloid leukemia cells and also on hematopoietic and vascular cells. We previously showed that a recombinant IL-3 fusion immunotoxin (DT(390)IL-3) expressed by splicing the murine IL-3 gene to a truncated diphtheria toxin (DT(390)) gene selectively killed IL-3R(+) expressing cells and was not uniformly toxic to uncommitted BM progenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J. and Vallera,D.A., 1996, Blood, 88, 1445-1456). Thus, we explored the feasibility of using DT(390)IL-3 as an anti-leukemia agent. DT(390)IL-3 was toxic when administered to mice at doses as low as 0.1 microg/day. The dose limiting toxicity appeared to be related to platelet and bleeding effects of the fusion toxin. Because of these effects, DT(390)IL-3 was studied ex vivo as a means of purging contaminating leukemia cells from BM grafts in a murine autologous BM transplantation. In this setting, as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid 32Dp210 leukemia cells were lethal. An optimal purging interval of 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BM mixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneic mice. Mice given treated grafts containing BM and a lethal dose of 32Dp210 cells survived over 100 days while mice given untreated grafts did not survive (P < 0.00001). DT(390)IL-3 may prove highly useful for ex vivo purging of lethal malignant leukemia cells from autologous BM grafts.

Animals↗
PubMed1987

Preferential differentiation of murine CFU-S toward granulopoiesis and megakaryocytopoiesis after in vitro incubation of bone marrow with ASTA-Z 7557.

We investigated the in vitro effect of ASTA-Z 7557 on the qualitative aspects of murine CFU-S differentiation, as assessed by the histological nature of day-9 colonies generated in the spleen of irradiated mice by bone marrow exposed to the drug at concentrations ranging from 0 to 150 micrograms/ml. The proportion of erythrocytic colonies declined linearly with the logarithm of the dose (a 22% decrease per log), whereas the granulocytic and megakaryocytic colony proportions increased linearly (a 10% increase per log for both cell lineages). This suggests a preferential channeling of CFU-S differentiation toward granulopoietic and megakaryocytic cell lineages as a consequence of the in vitro chemotherapy, and supports the hypothesis that some alteration of the qualitative potential of CFU-S to differentiate after in vitro purging of bone marrow with ASTA-Z 7557 takes place prior to autologous bone marrow transplantation.

Animals↗
PubMed1994

CD34 selection for purging in multiple myeloma and analysis of CD34+ B cell precursors.

Selection of CD34+ hematopoietic progenitor cells from autografts may be performed in multiple myeloma (MM) to minimize contamination with tumor cells. This approach is based on the assumption that the malignant cells do not express the CD34 antigen. Therefore, we first compared the CD34+/CD10+ and CD34+/CD19+ subpopulations from bone marrow (BM) and peripheral blood (PB) of fourteen MM patients and five normal controls. No difference between the respective early B cell subsets of both groups could be observed. Using tricolor flow cytometry, the CD19 expression on CD34+/CD10+ cells in BM was found to increase continuously from CD19- to CD19dim. In contrast, circulating CD34+/CD10+ cells did not coexpress the CD19 antigen. This population may contain myeloid progenitor cells or bipotential progenitor cells of the myeloid and lymphoid lineage as suggested by data obtained with fetal liver cells. Further functional studies are required. Enrichment of CD34+ cells with immunomagnetic beads was performed from BM of three MM patients and four normal donors. The CD34+ cells were selected with the HPCA-1 antibody and detached from the beads with chymopapain. Compared with the starting cell preparation, a 3.97 +/- 0.48 log (mean +/- SE) reduction of plasma cells could be achieved after CD34 selection. On morphological examination, 84% +/- 4% of the cells in the CD34+ fraction (MM) were immature blasts. The plating efficiency for hematopoietic colony forming cells was 9.7% +/- 2.8% in the CD34 selected fraction of the MM group, reflecting a 51-fold increase as compared with the starting population.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1998

Autologous stem-cell transplantation for non-Hodgkin's lymphomas: the role of graft purging and radiotherapy posttransplantation--results of a retrospective analysis on 120 patients autografted in a single institution.

PURPOSE: To analyze retrospectively survival and prognostic factors of patients with non-Hodgkin's lymphoma (NHL) autografted from 1979 to 1995 in a single institution. PATIENTS AND METHODS: A total of 120 patients, 64 with aggressive and 56 with low-grade NHL, were autografted. The carmustine (BCNU), etoposide, cytarabine, and melphalan (BEAM) regimen was used in 104. The autograft was marrow in 101 patients. Marrow was purged in vitro by mafosfamide for 63 patients (adjusted dose [AD] in 32; unique dose [UD] in 31); 27 patients received a CD34+-selected graft. Following intensification, 45 patients received additional radiotherapy on previous sites of involvement. RESULTS: Outcome at 5 years for patients transplanted with low-grade NHL in first complete remission (CR1), in first partial remission (PR1), and in second complete remission (CR2) or beyond showed an event-free survival (EFS) of 75% +/- 12%, 46% +/- 18%, and 57% +/- 24%, a relapse incidence (RI) of 21% +/- 12%, 49% +/- 19%, and 43% +/- 25%, and a transplant-related mortality (TRM) of 5% +/- 5%, 10% +/- 7%, and 0%, respectively. For patients with aggressive NHL transplanted in CR1, in PR1, in CR2 or beyond, and in resistant relapse or in primary refractory disease, the EFS was of 73% +/- 9%, 58% +/- 19%, 29% +/- 16%, and 10% +/- 9%, the RI 22% +/- 9%, 14% +/- 9%, 77% +/- 18%, and 66% +/- 20%, and the TRM 6% +/- 6%, 32% +/- 21%, 11% +/- 10%, and 71% +/- 22%, respectively. In patients autografted upfront in first remission, additional radiotherapy was associated with a higher EFS, in univariate (P = .03) and multivariate analysis (P = .02, relative risk [RR] = .021). The role of graft purging with mafosfamide on the outcome reflected by the dose of colony-forming unit-granulocyte-macrophage (CFU-GM) per kilogram infused postpurging was assessed by univariate analysis: patients in first remission who received lower doses of CFU-GM had a lower RI and a higher EFS. CONCLUSION: This retrospective analysis suggests that marrow purging and posttransplant radiotherapy improve the outcome of patients with NHL autografted in first remission.

Adult↗
PubMed2001

Ex vivo expansion of normal progenitor cells from acute myeloid leukemia cell-contaminated CD34+ peripheral blood progenitor cells after mafosfamide purging.

The rationale for purging of autologous acute myeloid leukemia (AML) grafts is to eradicate contaminating leukemic cells that might contribute to relapse. However, in vitro purging generally delays post-transplant hematopoietic recovery, thus increasing treatment-related complication rates. Theoretically, this prolonged aplasia might be shortened by the additional transplantation of ex vivo-generated progenitor cells. Therefore, we investigated whether nonleukemic progenitors could be expanded ex vivo from AML cell-contaminated CD34(+) peripheral blood progenitor cell (PBPC) preparations. Nonleukemic CD34(+)-selected PBPC and AML cells (Kasumi-1, KG-1, primary AML blasts) were cultured in cytokine-supplemented liquid culture for up to 19 days. Cells were used either unmanipulated or following in vitro purging with mafosfamide (30, 50, 75 microg/ml). Ex vivo-generated cells were assessed by flow cytometry, progenitor cell assays, and polymerase chain reaction. Without prior purging, ex vivo culture markedly amplified AML cells as well as nonleukemic CD34(+) PBPC (day 12: Kasumi-1, 18.5 +/- 0.6-fold; KG-1, 52.2 +/- 2.6-fold; CD34(+), 74.1 +/- 5.6-fold). Co-culture with leukemic cells did not affect CD34(+) cell growth and vice versa. Following in vitro purging, CD34(+) PBPC were expanded even at the highest mafosfamide dose (day 19: 25 +/- 15-fold), whereas leukemic cells were markedly depleted (approx. 1.5 log). Furthermore, normal colony-forming units (CFU) could be effectively recovered (day 19: 10 +/- 3.1% of prepurging input CFU), whereas CFU-L were depleted to undetectable levels in six of seven experiments. Finally, leukemic cells were undetectable following ex vivo co-culture of purged cells (CD34(+) PBPC plus 10% Kasumi-1 cells or primary blasts), but were clearly detectable without purging. Taken together, these data demonstrated that ex vivo expansion of normal progenitors from mafosfamide-purged AML cell-contaminated grafts might be feasible.

Acute Disease↗