Assessment of the efficacy of purging by using gene marked autologous marrow transplantation for children with AML in first complete remission.
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Acute Disease↗
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Evaluation of monoclonal antibody DF3 conjugated with ricin as a specific immunotoxin for in vitro purging of human bone marrow.
DF3 is an IgG1 monoclonal antibody (MAb) generated against a Mr 350,000-400,000 glycoprotein expressed by approximately 80% of human breast cancers. We have coupled MAb DF3 to ricin. Purification of the immunotoxin (DF3-IT) was obtained by affinity and size exclusion chromatography. DF3 antigen-positive breast cancer cell lines (ZR-75-1, BT-20, and MCF-7) and DF3 antigen-negative lung cancer cell lines (A549 and CALU6) were tested for cytotoxicity with metabolic labeling and clonogenic assays. The cells were exposed for 3 h to different concentrations of DF3-IT, MAb DF3, ricin, and a combination of unconjugated MAb DF3 and ricin. In the presence of 100 mM lactose, DF3-IT specifically inhibited protein synthesis of lines expressing DF3 antigen on their cell surface. Moreover, clonogenic survival experiments demonstrated that DF3-IT at a concentration of 1 x 10(-9) M specifically kills 2.6-2.8 log of ZR-75-1 and BT-20 cells and 1.6 log of MCF-7 cells. At the same concentration, nonspecific toxicity of DF3-IT resulted in a 30% reduction of bone marrow granulocyte and macrophage colony formation. In bone marrow-purging experiments, tumor cells were mixed with an excess of bone marrow cells and treated with DF3-IT or ricin. Tumor cell clonogenic survival assays demonstrated that the presence of bone marrow cells had no detectable effect on activity or specificity of the DF3-IT. These results thus indicate that MAb DF3 is an effective vehicle to specifically deliver toxins to cancer cells which express DF3 antigen on their surface and that DF3-IT may be useful for in vitro purging of bone marrow.
Antibodies, Anti-Idiotypic↗
Autologous bone marrow transplantation for leukemia.
Autologous bone marrow transplantation for leukemia was developed to extend the apparent curative potential of myeloablative therapy with allogeneic bone marrow transplantation to leukemia patients without histocompatible marrow donors. The conceptual problem with this approach is obvious: if the need for the transplant is based on overt or occult contamination of the marrow by leukemia, the use of autologous marrow seems destined to failure because of reinfusion of leukemia cells along with the harvested marrow. For this reason, ex vivo antileukemic treatment ("purging") of remission marrow was developed to justify autologous transplants for leukemia. Clinical trials involving thousands of patients worldwide have demonstrated curative potential of autologous bone marrow transplants, using purged or untreated remission marrow, for selected patients with acute myelogenous leukemia and acute lymphocytic leukemia. Purging appears to contribute to increased leukemia-free survival, at least in a subset of patients who are at very high risk of relapse, but this has not been tested in a prospective randomized trial and remains controversial. In acute myelogenous leukemia, in which the greatest experience exists, procedure-related mortality is much less for autologous than for allogeneic transplants; however, since leukemia relapse is much more frequent for autologous than for allogeneic transplants, the long-term disease-free survival is similar. In general, autologous transplants are preferred for older individuals and those without matched related donors, whereas allogeneic transplants are preferred for younger patients with matched related donors. Leukemia relapse has greatly limited the success of autologous transplants for acute lymphocytic leukemia. Autologous transplants for the chronic leukemias are in a much earlier stage of investigation. Autologous transplantation for leukemia is a fertile area for research. Important topics include conditioning regimens with improved antileukemic efficacy, the value of purging and the best method(s) for leukemia stem cell purging or normal stem cell selection, the possibility of inducing an autologous graft versus leukemia reaction, the use of immunomodulatory cytokines for postgrafting immune system manipulation, and the use of hematopoietic growth factors for ex vivo stem cell expansion and postgrafting support of marrow recovery.
Animals↗
High-dose chemotherapy with reinfusion of purged autologous bone marrow following dose-intense induction as initial therapy for metastatic breast cancer.
We assessed the toxicity and efficacy of high-dose chemotherapy consolidation with reinfusion of purged autologous bone marrow in women with metastatic breast cancer responding to a dose-intense outpatient regimen. Thirty women with hormone-unresponsive metastatic breast cancer, previously untreated with adjuvant doxorubicin or with any chemotherapy for metastatic disease, were treated with cyclophosphamide, methotrexate, doxorubicin, fluorouracil, vincristine, and leucovorin for 16 weeks. Twenty-four patients responded to therapy; 8 showed a complete response, and 16 showed a partial response. These patients proceeded to the next phase of the protocol, ie, marrow harvest and treatment with 6000 mg/m2 cyclophosphamide and 800 mg/m2 thiotepa given over 4 days. Harvested marrow was purged with 100 micrograms/mL 4-hydroperoxycyclophosphamide, and all patients engrafted satisfactorily. The predominant side effects were myelosuppressive and gastrointestinal, and there were no deaths from toxic effects. Three of the 16 patients who showed a partial response after the outpatient phase of treatment achieved a complete response after high-dose therapy. The partial response seen in two more patients converted to a complete response at all sites except bone. The median time to disease progression for all patients in this study was 13 months, and the median survival was 22 months. Four of the original 30 patients remained without disease progression a median of 27 months from entry into the study. This study indicates that this dose-intense regimen can be safely administered, even with the use of purged marrow, with an acceptable toxicity profile. This approach results in a high response rate in women with metastatic breast cancer and could form the basis for a regimen to be tested in the high-risk adjuvant setting.
Adult↗
[The role of autologous hematopoietic cell transplantation in adult acute myelogenous leukemia].
High dose chemotherapy with autologous hemopoietic cell transplantation (AHCT) is a common method of treatment of acute myelogenous leukemia (AML). AHCT is a treatment of choice for patients who have no matched family donor. AHCT is particularly recommended for older patients, excluded from allogeneic transplantation procedures. Prospective randomised trials have shown better efficacy of AHCT comparing with conventional chemotherapy in postremission treatment of AML. Both in vitro and in vivo bone marrow purging allow to achieve better transplantation results. Since two years peripheral blood instead of bone marrow is increasingly used as a source of transplant material. It allows more rapid hemopoiesis regrowth. Various methods of immunotherapy such as interleukin-2, Linomid and mixed hemopoietic cell transplantation (delayed donor lymphocytes transfusion) are used to evoke an autologous graft versus leukemia (GvL) phenomenon and to reduce AML relapse rate. Analysis of prognostic factors allows to identify a group of AML patients for whom AHCT is strongly recommended.
Adult↗
Major histocompatibility complex class II expression is required for posttransplant immunological but not hemopoietic reconstitution in mice.
We had previously shown in a canine model that the administration of anti-MHC class II monoclonal antibody (MAB) immediately after autologous marrow transplantation prevented hemopoietic reconstitution. Since MHC class II expression in mice differs from that in dogs we were interested in determining the effect of MHC class II manipulation on posttransplant hemopoietic and immunological recovery in mice. Three murine models including MHC class II knock-out mice were studied. BALB/c mice (I-E+, I-A+) given anti-MHC class II MAB H81.9 (anti-I-E; 1 mg/kg/day, days 0-4) after TBI and infusion of syngeneic marrow or infused with anti-I-E purged marrow both showed normal hemopoietic reconstitution. Similarly, C57B1/6 mice (I-E-, I-A+) transplanted with M5/114 (anti-I-A) purged marrow recovered normal hemopoiesis. MHC class II knock-out (C2D) mice, which lack class II completely, also recovered normal hemopoiesis after TBI and transplantation with either normal or class II-deficient (C2D) marrow, although the kinetics of platelet recovery as determined by megakaryocytopoiesis and platelet counts on day 14 were slightly delayed. C57B1/6 mice transplanted with C2D marrow recovered normally. Immunologic recovery, however, was abnormal both in C2D recipients and in normal mice transplanted with C2D marrow: While CD8+ T lymphocytes recovered normally, no (or only very few) CD4+ T cells were identified posttransplant. Treatment of normal mice with anti-MHC class II MAB in vivo or transplantation of MHC class II-purged marrow, however, did not interfere with complete immunological recovery, although T cell maturation was slightly delayed. Thus, complete immunological reconstitution requires the expression of MHC class II on marrow-derived precursor cells, while the expression of MHC class II antigens is not a requirement for hemopoietic reconstitution in mice.
Animals↗
Transfer of marker genes into hemopoietic progenitor cells.
Ex vivo gene marking of normal and malignant hemopoietic cells allows the cells to be subsequently tracked in vivo. Marking has shown that even when marrow is in remission, it may contain malignant cells that contribute to relapse. These studies have also shown that it is possible to obtain long-term gene expression in human long-lived hemopoietic progenitor cells and T lymphocytes in vivo. Current marker studies use two distinguishable vectors to track two distinctively treated cell populations in a single individual. This modification greatly increases the power of the technique. It is now possible to study the effects of purging on residual malignant cells in marrow, to determine the action of growth-promoting agents (such as cytokines and stroma) on short- and long-term repopulation by transduced marrow, and to discover which phenotypic subsets of hemopoietic progenitor cells have long-term repopulating potential. The information gained will be invaluable for improving therapeutic gene transfer protocols in which marrow-derived cells are the targets.
Bone Marrow Cells↗
Incorporating monoclonal antibodies in blood and marrow transplantation.
The development of therapeutic monoclonal antibodies had been a significant advance in the treatment of B-cell non-Hodgkin's lymphoma (NHL). In the setting of blood and marrow transplantation (BMT), initial research was focused on in vitro graft purging. More recently, investigators have designed in vivo purging protocols using rituximab with the goal of obtaining tumor-free grafts. Exciting developments stem from the use of iodine 131 ((131)I)-tositumomab as well as yttrium 90 ((90)Y)-ibritumomab in myeloablative doses in blood and marrow transplantation protocols for high-risk patients. Finally, vigilance towards complications in particular unusual infections is advised given the profound immunosupressive state caused by these antibodies.
Antibodies, Monoclonal↗
A novel gene therapy strategy for elimination of prostate carcinoma cells from human bone marrow.
We report a novel means to purge bone marrow of a specific subset of prostate carcinoma cells based on transductional and genetic selectivity. Using both adenovirus-polylysine-DNA complexes and E1A/B-deleted replication-deficient adenoviruses, we have demonstrated a transductional preference of these vectors for the prostate carcinoma cell lines DU 145, LNCaP, and PC-3 over primary human bone marrow cells and the leukemia cell line KG-1. We have also shown a genetic selectivity of an anti-erbB-2 intracellular single-chain antibody (sFv) encoding adenovirus, Ad21, for the erbB-2-positive prostate carcinoma cell lines DU 145 and LNCaP. Delivery of Ad21 resulted in cytotoxicity to the DU 145 and LNCaP, but not PC-3, cell lines and reduced the clonogenic capacity of DU 145 cells cultured alone or mixed with various ratios of irradiated human bone marrow. Finally, quantitative, competitive reverse transcription polymerase chain reaction (QC-RT-PCR) analysis demonstrated that Ad21 could effectively reduce DU 145 and erbB-2-positive primary prostate tumor contamination in bone marrow cultures. Delivery of Ad21 had no effect on the ability of progenitor cells to form colonies. These results suggest that an anti-erbB-2 sFv-encoding adenoviral vector is efficacious for removal of erbB-2-positive prostate carcinoma cells from human bone marrow, and demonstrates a novel method for ex vivo genetic purge of malignant cells from bone marrow for autologous bone marrow transplantation (ABMT) therapy.
Adenoviridae↗
Role of autotransplantation in neuroblastoma.
Neuroblastoma is the most enigmatic disease treated by pediatric oncologists. The same pattern of apparent metastatic spread is associated with a high rate of spontaneous regression in children under 1 year of age (stage IVS) but an invariably fatal course in older children. For these children with truly advanced neuroblastoma, recent studies have demonstrated that the possibility of complete remission and disease-free survival is increased with more intensive conventional combination chemotherapy. Supportive care with autologous marrow has enabled further significant dose escalation of cytotoxic therapy active against neuroblastoma, resulting in disease-free survival in between 25% and 50% of patients at 2 years, an improvement over historical experience with conventional chemotherapy. The development of more effective cytotoxic combinations active against neuroblastoma, improvements in ex vivo techniques to purge marrow of neuroblastoma cells, and the application of cytokines to hasten marrow recovery should further enhance the effectiveness of autotransplantation in the treatment of advanced neuroblastoma and further improve the chances for complete remission and longer remission duration. Whether this improved remission induction and duration will translate into an increased cure rate remains to be determined. Parallel advances in therapies not involving autologous marrow support will undoubtedly modify the role of autotransplantation in neuroblastoma. Currently, however, autotransplantation offers one potential solution to a major challenge in the treatment of advanced neuroblastoma--obtaining and maintaining complete remissions--and a promising basis for future therapeutic studies.
Antineoplastic Combined Chemotherapy Protocols↗
Polymerase chain reaction: a method for monitoring tumor cell purge by long-term culture in BCR/ABL positive acute lymphoblastic leukemia.
We report the successful purging of leukemia cells bearing the Philadelphia chromosome and BCR/ABL transcripts by long-term marrow culture (LTC), and subsequent grafting of the purged marrow in a case of refractory acute lymphoblastic leukemia. The efficiency of the purge was evaluated by polymerase chain reaction (PCR) for BCR/ABL transcripts. In two LTCs initiated in the blastic stage, we demonstrated the selective effect of three culture media (serum dependent, serum-free (SF) supplemented or not with IL3 and GM-CSF) on the proliferative potential of normal hematopoietic (CFU-GM/BFU-E) and leukemic progenitors (CFU-ALL). BCR/ABL positive cells disappeared after 3 to 4 weeks of culture. The addition of IL3 and GM-CSF to the SF medium enhanced the growth of CFU-GM/BFU-E and shortened the purging period. We therefore carried out a LTC in the presence of IL3 and GM-CSF with marrow harvested in morphological remission. BCR/ABL positivity was detected at the outset, although no leukemia cells could be identified. The BCR/ABL was no longer found by PCR in the 7 and 14 day LTCs. The patient, consolidated by high dose polychemotherapy and total body irradiation, was infused with the 14 day LTC. This study indicates that PCR is a useful and sensitive technique for monitoring tumor cell reduction after LTC prior to autografting.
Bone Marrow Purging↗
Culture purging in leukemia: past, present, and future.
Over the past 5 years we have been evaluating the feasibility of using cultured marrow autografts to allow patients with chronic myeloid leukemia (CML) to receive intensive, potentially curative therapy. The rationale for this approach is based on two important findings. The first is that leukemic stem cells (operationally defined as Ph-positive long-term culture-initiating cells, or LTC-IC) are present in the marrow of many CML patients at relatively low levels by comparison to co-existing normal stem cells (i.e., Ph-negative LTC-IC). The second finding is that leukemic LTC-IC are selectively "purged" following their incubation in vitro for 10 days under LTC conditions. As a result, cultured CML marrow preparations show, on average, a 300-fold selection in favour of normal LTC-IC. However, there is considerable variation in the initial normal and leukemic LTC-IC content of marrow samples from different CML patients. Thus in only approximately one third of cases does the number of leukemic LTC-IC in the marrow decrease to undetectable levels within the first 10 days of culture with the number of co-existing normal LTC-IC remaining at or above 1/50th of the average value for normal marrow. We have now transplanted 22 such CML patients with 10 day cultured marrow autografts following their treatment with myeloablative therapy. Fifteen of these patients were in first chronic phase and 7 had more advanced disease.(ABSTRACT TRUNCATED AT 250 WORDS)
Bone Marrow↗
Monoclonal antibody-based therapy for neuroblastoma.
Dose-intensive combination chemotherapy can improve the clinical response of many pediatric solid tumors. However, cure remains elusive. Stage 4 neuroblastoma stands out as an exception. Part of this success is a result of antibody-based strategies, which include immunomagnetic purging of autologous marrow prior to autologous marrow transplantation and immunotherapy directed at minimal residual disease. It is striking that treatment with monoclonal antibodies, even when targeted at a single antigen, namely, ganglioside G(D2), can affect long-term progression-free survival among these patients. The potential role of the idiotype network in tumor control can be exploited clinically. The genetic engineering of these antibodies into novel forms holds great promise for more specific and effective targeting possibilities, including the delivery of cytokines and cells. Preclinical results are also promising. It is expected that the availability of novel antibodies directed at a broader spectrum of pediatric solid tumors will facilitate the successful application of this approach to more patients. Experience with metastatic neuroblastoma has provided proof of this principle. It is likely that other tumors will fall.
Antibodies, Monoclonal↗
Busulfan plus etoposide as a preparative regimen for autologous bone marrow transplantation for acute myelogenous leukemia: an update.
We have used a new preparative regimen prior to autologous bone marrow transplantation in patients in remission of acute myelogenous leukemia (AML). Sixty-two patients were treated with preparative chemotherapy consisting of 1 mg/kg of busulfan every 6 hours for 4 days (total dose 16 mg/kg) on days -7 to -4 followed by an intravenous infusion of 60 mg/kg of etoposide on day -3. Autologous bone marrow that had been purged with 4-hydroperoxycyclophosphamide was infused on day 0. Thirty-five of the treated patients were in first remission, 21 were in second or third remission, and six had primary refractory AML and required aggressive salvage regimens to achieve remission. Among patients in first remission, one treatment-related death and five relapses have occurred. With a median follow-up of 25 months, the actuarial relapse rate is 19% +/- 8% and the disease-free survival rate is 79% +/- 8% at 3 years. The 16 patients with favorable FAB subtypes (M3 or M4E0) showed an excellent response to this treatment protocol, with no relapses after median follow-up of 35 months. Among patients in second or third remission, there were five treatment-related deaths and five relapses. With a median follow-up of 41 months, the actuarial relapse rate is 30% +/- 11% and the disease-free survival rate is 52% +/- 11% at 3 years. Four of six primary refractory patients died during treatment and one remains in remission with short follow-up. These preliminary data indicate that aggressive preparative regimens followed by the infusion of purged autologous bone marrow may be a promising approach to improving the clinical outcome of adult patients with AML.
Adolescent↗
Alpha-interferon broadens the difference between surviving fractions of normal and leukemic progenitor cells in vitro by heat: its application to marrow purging.
Alpha-interferon (IFN) may inhibit the proliferation of human leukemic progenitor cells (L-CFU) in vitro and enhance the anti-tumor effects by heat. In this study, the combined effects of IFN and hyperthermia on the growth of L-CFU and human granulocyte-macrophage progenitors (CFU-GM) were examined to determine if this combination resulted in a greater selective killing of L-CFU than that obtained by heat treatment alone. The survival of normal CFU-GM without IFN decreased at elevated temperatures (42-44 degrees C). However, IFN added during heating (42 and 43 degrees C) appeared significantly to protect against the hyperthermic killing of CFU-GM in vitro leaving over 50% of CFU-GM surviving. The optimal dose to protect CFU-GM in vitro dropped to a rather low dose (100 U/ml). On the other hand, the addition of IFN to leukemic cell suspensions enhanced the hyperthermic killing of myeloid leukemic cell lines (HEL and KG-1) as well as a T lymphoblastic cell line (CEM) in a dose-related manner. In addition, similar results were observed in the study of L-CFU from patients with acute myelogenous leukemia. These results suggest that IFN can be used to broaden the difference between surviving fractions of CFU-GM and L-CFU by heat. Thus, this combination could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous remission marrow graft before autologous bone marrow transplantation.
Bone Marrow Purging↗
[Studies on purging leukemic cells by photosensitizer PSD-007 laser photoradiation in vitro].
The sensitivity to photosensitization mediated by the hematoporphyrin photosensitizer PSD-007 of acute promyelocytic leukemic cell line (HL-60) was compared with normal human hemopoietic progenitor cells. The results showed that the leukemic cells were more sensitive. After being treated with 10 micrograms.ml-1 PSD-007 followed by 2J.cm-2 copper vapor laser light irradiation, the clonogenic leukemic (HL-60) cells were reduced 98%, but the survival rate of human granulocyte-macrophage colony-forming units (CFU-GM) was 40 +/- 8%. Mixing of normal human marrow cells with leukemic (HL-60) cells (ratio 100:1) did not interfere with elimination of tumor cells. The ultrastructure changes of HL-60 cells treated by laser photoradiation was observed under the trans-electronic microscope. The mitochondria, endoplasmic reticulum and cell membrane were involved. This means that the cell biomembrane is the main target to be attacked. It is considered that PSD-007-laser photoradiation therapy is efficient for killing leukemic cells.
Adult↗
Ex vivo purging of allogeneic marrow with L-Leucyl-L-leucine methyl ester. A phase I study.
L-Leucyl-L-leucine methyl ester (LLME) is a lysosomatropic compound that is converted by dipeptidyl peptidase I to metabolites that are membranolytic for cytotoxic T cells, NK cells, and LAK cells. Ex vivo treatment of murine marrow with LLME ameliorates acute graft-versus-host-disease (GVHD), which led to consideration of a clinical study. A phase I study design was initiated to evaluate the effects of ex vivo purging of allogeneic marrow on engraftment, since LLME also suppresses human progenitor cells. All patients received a preparative regimen of cyclophosphamide plus total body irradiation. GVHD prophylaxis consisted of cyclosporine +/- corticosteroids. This study included 19 patients with high risk disease undergoing allogeneic transplantation from an HLA-identical sibling (n = 12) or a partially HLA-matched family donor (n = 7). Marrow mononuclear cells were treated ex vivo in a dosage escalation study with LLME concentrations of 0.25 mM, 0.375 mM, and 0.5 mM. Marrow NK and LAK activities were essentially eliminated at concentrations > or = 0.375 mM LLME. CD8+ cells were also reduced. Granulocyte macrophage colony-forming unit recovery was 3% at 0.5 mM LLME. The median time to an absolute neutrophil count of 500/microliters was 17 days after transplantation (95% confidence interval = 14-18 days). One patient that received marrow treated with 0.5 mM LLME died of secondary graft failure. Complete donor chimerism was documented in each evaluable case. NK recovery was delayed at LLME concentrations > or = 0.375 mM LLME. Grade II/IV GVHD occurred in 4/18 evaluable patients. Ex vivo treatment of human marrow with LLME diminishes NK activity, LAK activity, CD8+ cells, and granulocyte macrophage colony-forming units, but does not totally prevent acute GVHD.
Acute Disease↗
Fractionation of chronic myelogenous leukemia marrow cells by stroma adherence: implications for marrow purging.
Chronic myelogenous leukemia (CML) progenitor cells have been shown to be defective in their ability to adhere to marrow stroma. It was the aim of the present study to investigate at the cytogenetic level marrow-derived CML clonogenic cells fractionated on the basis of their ability to adhere to preformed, allogeneic, normal marrow-derived stromal layers. Mononuclear marrow cells from CML patients (n = 15) were incubated with mafosfamide (100 micrograms/ml) or control medium, seeded onto marrow stromal layers and allowed to adhere (3 hrs, 37 degrees C). Following a short-term liquid culture, the different cell fractions were harvested and incorporated in methylcellulose cultures. CFU-GM grown from these cultures were analyzed by single colony karyotyping. On direct cytogenetic analysis, the overall mean (+/- SD) percentage of Ph-negative metaphases was 7 +/- 20%. Following stroma adherence and shortterm suspension culture, the mean (+/- SD) percentages of Ph-negative clones were as follows: 33 +/- 25% for adherent CFU-GM, 59 +/- 40% for adherent, mafosfamide-treated CFU-GM, 12 +/- 16% for non-adherent CFU-GM, and 32 +/- 26% for non-adherent-mafosfamide-treated CFU-GM. If only the patients showing a percentage of Ph-negative clones > or = 20% were included in this analysis, the mean (+/- SD) percentages of Ph-negative clones were 47 +/- 19% for adherent CFU-GM, and 81 +/- 21% for adherent-Mafosfamide-treated CFU-GM. In contrast, the majority of pH-positive CFU-GM were detected within the stroma non-adherent cell fraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Bone Marrow Cells↗