Amifostine and 2-CdA: a novel purging strategy in CML autografting?
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Adult↗
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Controversies on the role of bone marrow transplantation for patients with hematopoietic malignancies.
Over the past decade, bone marrow transplantation has evolved as a treatment modality for patients with hematopoietic malignancies and for patients with responsive solid tumors. In this paper, I review (a) the indications for marrow transplantation, with emphasis on patients with hematopoietic malignancies, including acute and chronic leukemias and lymphomas; (b) the role of the donor, including the rapid expansion of compatible volunteer donors; (c) the role and design of preparative regimens; (d) the procurement of bone marrow, including marrow purging and T-cell depletion, and (e) the care of the transplant patient, including a discussion of the more commonly occurring complications.
Anemia, Aplastic↗
In vivo administration of recombinant human granulocyte/macrophage colony-stimulating factor in acute lymphoblastic leukemia patients receiving purged autografts.
Based on the recent reports that recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) accelerates the rate of engraftment in a variety of autologous bone marrow transplantation settings, we have investigated its effects on hematopoietic recovery of patients with acute lymphoblastic leukemia (ALL) undergoing autologous bone marrow transplantation. Our studies, which involved 25 autologous ALL recipients who received rhGM-CSF and 27 controls similar for disease status (remission or relapse) and disease type (B- or T-lineage) differed from previous studies in one important aspect: the bone marrows were purged with 4-hydroperoxcyclophosphamide (4HC) and anti-T or anti-B-cell lineage-specific antibodies before transplantation. Such treatments frequently lead to a reduction in the CFU-GM content of the transplanted marrow. Eighteen of 25 patients completed the entire course of rhGM-CSF. Of the 16 patients who received greater than or equal to 64 micrograms/M2/d for at least eight days, there were five patients who had an apparent rhGM-CSF response and 11 patients who did not respond. Of the parameters analyzed, only the number of CFU-GM progenitor cells infused per kilogram was significantly associated with an rhGM-CSF response. All patients receiving greater than or equal to 1.2 x 10(4) CFU-GM progenitors per kilogram achieved an absolute neutrophil count (ANC) greater than or equal to 1,000/microL by day 21 and had a greater than 50% decrement in ANC within 48 to 72 hours of discontinuing rhGM-CSF, as contrasted to none of the patients receiving less than or equal to 7.2 x 10(3) CFU-GM progenitors per kilogram.(ABSTRACT TRUNCATED AT 250 WORDS)
Bone Marrow Transplantation↗
The cytotoxicity of alkyl-lysophospholipid on clonogenic leukemia cells and on normal bone marrow progenitor cells is highly, but differentially, increased by cryopreservation.
We studied the cytotoxic effect of alkyl-lysophospholipid (ALP) in combination with cryopreservation on human clonogenic leukemia cells from 10 patients with acute leukemia and on normal bone marrow progenitors (committed stem cells) from 10 donors, in order to assess the applicability of ALP as a purging agent in autologous bone marrow transplantation (ABMT). The tumoricidal effect of ALP was greatly increased by the cryopreservation procedure, both on leukemic progenitors and to a lesser extent on normal bone marrow progenitors. The cytotoxic effects of ALP and of cryopreservation were synergistic. As a consequence, the ALP dose for ex vivo purging has to be adjusted. Furthermore, the cryopreservation procedure itself is more cytotoxic for leukemic progenitors than for normal marrow progenitors, indicating that cryopreservation has a purging effect in AMBT.
Bone Marrow Purging↗
Immunomodulation of autoimmunity in MRL/lpr mice with syngeneic bone marrow transplantation (SBMT).
MRL-lpr/lpr mice spontaneously develop a severe autoimmune syndrome, characterized by massive generalized lymphadenopathy, arthritis, arteritis, dermatitis and immune complex-mediated glomerulonephritis. Bone marrow transplantation (BMT) from MHC-matched systemic lupus erythematosus (SLE)-resistant donors to susceptible recipients has proved effective in correcting autoimmune manifestations in autoimmune-prone mice. We investigated the effect of syngeneic BMT from MRL/lpr (donor) to immunocompromised MRL/lpr (recipient), after purging the bone marrow inoculum with MoAbs against mature T cells (anti-Thy 1.2). All the untreated mice developed lymphadenopathy and by the age of 36 weeks five of the eight were dead; in contrast, all the mice which underwent syngeneic BMT following acute immunosuppression with total body irradiation (900 cGy) (TBI) remained disease-free. In an additional experiment, it was found that conditioning with cyclophosphamide (CY) before BMT was more effective than TBI in inhibiting delayed-onset autoimmune manifestations (mean survival 350 days in the CY group and 305 days in the TBI group, versus 197 days in untreated controls). Under both immunosuppressive regimens T cell-depleted bone marrow grafts produced far better results than did unmanipulated BMT. Following syngeneic BMT the incidence of proteinuria and the level of serum anti-DNA (dd) antibodies were significantly reduced, compared with that of the age-matched untreated controls. CY was more effective than TBI in reducing the anti-DNA titres. Likewise, T depletion of bone marrow inocula before BMT induced a more drastic drop in autoantibodies, following both CY and TBI conditioning protocols. After syngeneic BMT (either CY or TBI) no signs of lymphadenopathy were observed even at an advanced age. Upon histopathological examination, the BMT-treated mice displayed normal glomeruli with occasional minimal signs of glomerulonephritis. Syngeneic T cell-depleted BMT following acute cytoreduction of anti-self immune lymphocytes may represent a new therapeutic approach for drug-resistant autoimmune diseases.
Animals↗
When, how, what, and where to purge are not the questions either.
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Bone Marrow Purging↗
Adenoviral vectors in hematology: purging, stem cell gene transfer, or both.
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Adenoviridae↗
Long-term bone marrow cultures: their use in autologous marrow transplantation.
When cultured together, hemopoietic cells and marrow stromal cells can support hemopoiesis in vitro in the absence of added growth factors. The continued production of stem cells, progenitor cells, and mature cells within these "long-term bone marrow cultures" (LTBMC) requires their physical association with the stromal cells. For reasons that are not well understood, leukemic cells often fail to survive in such cultures. We and others have undertaken studies to examine whether LTBMC can be exploited as a way of purging the bone marrow of leukemia patients prior to autologous marrow transplantation.
Bone Marrow Cells↗
Prediction of graft-versus-host disease by phenotypic analysis of early immune reconstitution after CD6-depleted allogeneic bone marrow transplantation.
Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic bone marrow transplantation (BMT). Because GVHD is frequently refractory to treatment, the early identification of high-risk patients could have significant clinical value. To identify such patients, we examined early immunologic recovery in 136 patients with hematologic malignancies who received anti-T12 (CD6)-purged allogeneic bone marrow over a 9-year period. The majority of patients received marrow from HLA-matched sibling donors after ablation with cyclophosphamide and total body irradiation. No patients received any immune suppressive medications for GVHD prophylaxis. The fraction and absolute numbers of peripheral blood lymphocytes (PBL) expressing the CD3, CD4, CD8, and CD56 surface antigens were determined weekly by immunofluorescence analysis in patients beginning 8 to 14 days (week 2) after marrow infusion. Results in patients who did or did not subsequently develop GVHD post-BMT were compared. Within 2 weeks of marrow infusion, patients who developed grades 2-4 GVHD had significantly higher percentages and absolute numbers of CD8+ T cells and a lower fraction of CD56+ natural killer (NK) cells than individuals who remained free of GVHD. Thirty-five percent of patients whose PBL were greater than 25% CD8+ in the second posttransplant week developed GVHD, compared with only 3% of patients who had < or = 25% CD8+ cells (odds ratio 37.8; 95% confidence interval [CI] 4.1 to 397). A subgroup of patients at very high risk for GVHD could be identified based on the combined frequency of CD8+ T cells and NK cells in blood. Seventy-five percent of patients with greater than 25% CD8+ cells and < or = 45% CD56+ cells during week 2 post-BMT developed GVHD, compared with only 11% of the remaining patients (odds ratio 24.9; 95% CI, 5.3 to 117.0). None of the 23 patients with both less than 25% CD8+ cells and greater than 45% CD56+ cells in the second posttransplant week developed grades 2-4 GVHD. Our findings indicate that CD8+ T cells play an important role in the pathogenesis of GVHD in humans. Analysis of immune reconstitution early after BMT is useful in predicting the onset of GVHD and can help direct the implementation of treatment strategies before the appearance of clinical manifestations. Such interventions may decrease the morbidity and mortality associated with allogeneic BMT and ultimately improve overall survival.
Adult↗
Variable cytotoxicity of diphtheria toxin 388-granulocyte-macrophage colony-stimulating factor fusion protein for acute myelogenous leukemia stem cells.
In this study, the utility of DT388-granulocyte-macrophage colony-stimulating factor (GM-CSF) for the ex vivo purging and direct administration to patients with acute myeloid leukemia (AML) is tested using clonogenic assays, long-term cultures (LTC), and NOD/SCID mice as assays for leukemic progenitors. We compare the ability of 24-hour exposure to 0.3 microg/mL (4 nM) DT388-GM-CSF to kill AML colony forming cells (CFC) and the more primitive AML progenitors detected after 6 weeks in stromal cocultures (AML LTC-initiating cells or AML LTC-IC) and after 8 weeks in NOD/SCID mice.AML samples (n = 10), expressing a mean of 35 to 1466 GM-CSF receptors/blast, showed mean (range) percent kills of AML CFC and LTC-IC of 61 (17-98) and 46 (0-94) respectively with a direct correlation (r = 0.69) between the % kills detected in the in vitro assays. Among 5 evaluable samples the percent reduction in AML cell engraftment in NOD/SCID marrow following ex vivo DT388-GM-CSF treatment varied from 38% to 100%. 40% to 56% of normal bone marrow CFC and 31% to 48% of normal LTC-IC survived the same ex vivo treatment (n = 3). In subsequent experiments, NOD/SCID mice received AML blast cell injections intravenously followed in 24 hours by 1.5 microg DT388-GM-CSF daily intraperitoneally for 5 days. A reduction of marrow blast cells was seen with 7 of 9 samples tested 4 to 12 weeks post one course of toxin. Repeating the 5-day course of toxin 2 or 3 times at 4-week intervals did not improve the response, while delaying administration until 4 to 8 weeks post AML cell injection reduced the toxin's effectiveness (n = 5).This fusion toxin may prove useful for in vitro purging of stem cell harvests from selected AML patients and for direct administration to such patients.
Animals↗
Continuous infusion of complement by an automated cell processor enhances cytotoxicity of monoclonal antibody sensitized leukemia cells.
We report the use of the Haemonetics cell processor for monoclonal antibody (MoAb) and complement (C')-mediated lysis of leukemia cells. Using the HL-60 promyelocytic leukemia cell line, we can achieve a six-log depletion of HL-60 colony-forming cells in cell mixtures containing 1% HL-60 cells and 99% normal peripheral blood or bone marrow leukocytes at 10(7)/ml after a single 60-min treatment with an anti-myeloid MoAb, PM-81, plus C'. In this procedure, we continuously infuse a solution of fresh C' while removing medium containing spent C'. We have utilized this procedure to purge remission bone marrow from patients with acute myelogenous leukemia in preparation for autologous bone marrow transplantation. There were no adverse effects to normal progenitor cells of the granulocyte, monocyte and erythrocyte lineages as measured in colony-forming assays. Other potential benefits of using the cell processor method of cytotoxicity include reduction in treatment time and the amounts of costly reagents.
Antibodies, Monoclonal↗
Rituximab: enhancing outcome of autologous stem cell transplantation in non-Hodgkin's lymphoma.
High-dose chemotherapy and autologous stem cell transplantation (ASCT) is a potentially curative therapy for younger patients with relapsed aggressive non-Hodgkin's lymphoma, and is under investigation as first-line treatment and as therapy for indolent and mantle cell non-Hodgkin's lymphoma. However, between 40% and 70% of all patients relapse after ASCT because of contamination of the stem cell product or persistence of residual tumor cells. Evidence is emerging that the administration of rituximab as an in vivo purging agent before ASCT is effective in eliminating lymphoma cell contamination, as measured by clearance of bcl-2-positive cells from stem cell harvests. Furthermore, in vivo purging with rituximab does not adversely affect the stem cell yield or function. Maintenance therapy with rituximab post-transplantation has also been explored as a means of eliminating residual tumor cells. Results suggest that rituximab may eradicate minimal residual disease post-transplant and help prevent relapse. The efficacy of both in vivo purging and maintenance therapy with rituximab is currently being investigated in a large, multicenter, randomized trial by the European Group for Blood and Bone Marrow Transplantation in patients with follicular non-Hodgkin's lymphoma. Results from this and other ongoing trials will confirm the full potential of rituximab in ASCT.
Antibodies, Monoclonal↗
Cytokine treatment of human bone marrow activates anti-leukaemia effector cells: monitoring of purging by polymerase chain reaction and DNA analysis.
The aims of this study were to investigate the role of cytokines (tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-2 (IL-2) in augmenting graft-versus-leukaemia (GVL). We have investigated the effector cells involved in GVL, by studying the role of these cells in purging of the cell line K562 in short-term bone marrow cultures. The effect of the addition in vitro of rGCSF was also studied. Monitoring of purging was achieved by cytotoxicity assays, DNA analysis and the use of the polymerase chain reaction for the detection of bcr/abl transcripts in the Philadelphia positive (Ph+) K562 cell line. Supernatants from IL-2-treated and non-treated bone marrow were tested for cytokine production (TNF alpha and IFN gamma). The results have shown that the main cytotoxic effector cells in the bone marrow generated by IL-2 have the CD56+ CD8+ phenotype. Overnight incubation of bone marrow was sufficient to generate cytotoxic cells as measured by Chromium51 (Cr51) release assays. Measurable levels of TNF alpha but not IFN gamma were also detected in supernatants. Addition of TNF alpha and IFN gamma to the IL-2 in the bone marrow cultures augmented the cytotoxicity but tended to inhibit progenitor cell growth as measured by granulocyte-macrophage colony-forming unit (GM-CFU) and erythroid blast-forming unit (BFU-e) assays. An estimate of the purging of the marrow could also be achieved by DNA analysis of K562 DNA in bone marrow. The bcr/abl transcript could still be detected by PCR analysis in marrow containing 1% K562 and treated with IL-2 for 24 h, but by 6 days of incubation the bcr/abl transcript was weak or undectable. The results suggest that although reduction in the proportion of leukaemia in contaminated marrow can be detected after incubation with IL-2 for 24 h, complete elimination of minimal residual disease requires longer incubation times.
Biomarkers, Tumor↗
Transduction of bone marrow cells by the AdZ.F(pK7) modified adenovirus demonstrates preferential gene transfer in myeloma cells.
Adenoviral vectors can efficiently infect myeloma cell lines, but transduction of fresh myeloma cells performed at low multiplicity of infections (MOIs) showed only partial efficacy. The modified adenoviral vector AdZ.F(pK7), through binding of polylysines to heparan sulfate-containing receptors, could increase virus adsorption and gene transfer efficiency in myeloma cells, which express heparan sulfate-containing receptors. Thus, we investigated the ability of AdZ.F(pK7) vector to achieve efficient gene transfer in primary cultured fresh myeloma cells. Transduction of 16 primary cultured myeloma samples showed that gene transfer was much more efficient with AdZ.F(pK7) than with control AdZ.F. Both addition of soluble heparin and cell treatment with heparinase I dramatically inhibited gene transfer in myeloma cells by AdZ.F(pK7) but had no effect with AdZ.F, while addition of recombinant fiber protein inhibited AdZ.F but not AdZ.F(pK7), confirming that AdZ.F(pK7) gene transfer in myeloma cells is mediated by the targeting of heparan sulfates. AdZ.F(pK7) transduction of bone marrow cells showed that myeloma cells and hematopoietic progenitor AC133-, CD34-, and CD33-positive cells were efficiently transduced at an MOI of 100, but that only myeloma cells were significantly transduced at an MOI of 12. Thus, AdZ.F(pK7) vector seems to be well suited for immunological approaches of gene therapy or bone marrow-purging applications in multiple myeloma.
Adenoviridae↗
Growth inhibition of chronic myelogenous leukemia cells by ODN-1, an aptameric inhibitor of p210bcr-abl tyrosine kinase activity.
p210bcr-abl-Related tyrosine kinase activity has been shown to cause chronic myelogenous leukemia (CML), a disease of bone marrow stem cells. Having previously demonstrated that the aptameric oligonucleotide, ODN-1, could inhibit p210bcr-abl kinase activity, the current study sought to determine if ODN-1 could selectively inhibit the growth of CML cells relative to that of normal bone marrow. ODN-1, when introduced by electroporation into peripheral blood mononuclear cells (PBMC) from patients with CML, decreased the number of committed progenitors (CML CFU-GM) by an average of 67%+/-19% (mean+/-SEM, range 28-98%). Treatment of CML PBMC with ODN-1 was also shown to decrease the number of more primitive cobblestone area-forming cells (CAFC) by 35%-87%. In contrast, there was little suppressive effect by the combination of electroporation and ODN-1 on either CFU-GM or CAFC numbers from normal donor bone marrow. These studies suggest that inhibition of p210bcr-abl protein-tyrosine kinase (PTK) activity by ODN-1 is associated with some degree of selective growth inhibition of p210bcr-abl-transformed cells. p210bcr-abl kinase inhibitory agents may be useful for the ex vivo purging of bone marrow or peripheral blood progenitor/stem cells in the setting of autologous transplantation for CML.
Animals↗
[CD34+ cells: biological aspects].
The CD34 antigen is a glycosilated transmembrane protein with a molecular weight of 105-120 kDs, whose molecular function is still unknown. At present different epitopes of this antigen are recognized by more than 20 monoclonal antibodies. By flow cytometry is quite simple to identify and enumerate the CD34+ cells, present in physiological conditions on 1-3% of normal bone marrow, 0,1-0,5% of cord blood and 0,001-0,01% of peripheral blood cells. The concomitant expression of other monoclonal antibodies allows the identification of different subsets, lineage negative or already lineage "committed", so that CD34+ cells represent an heterogeneous population with only a small number of undifferentiated progenitors. The number of circulating progenitors has highly increased in peripheral blood for few hours during the fast hematopoietic recovery after high dose chemotherapy. Growth factors are able to mobilize CD34+ cells if used alone in a short treatment schedule and the effect is amplified by combining growth factors with chemotherapy. With this treatment CD34+ cells can increase in peripheral blood up to 100-1000 fold the baseline concentration. Collection of large scale of peripheral stem cells is now possible using different models of continuous-flow blood cell separators. The vast majority of the cells harvested by apheresis are constituted by "committed" elements, myeloid peroxidase positive cells (40%), T lymphocytes (30%), monocytes (20%), B lymphocytes (1-2%), the CD34+ representing not more than 3-4% of the total cells collected. The main biological characteristic of the CD34+ cells is the capacity to reconstitute the myelo and lymphopoietic system after a myeloablative treatment. For this reason in the last few years there has been an increasing interest in using these particular stem cells in many clinical settings. Peripheral blood autografting is widely used in a large number of trials for the treatment of chemosensitive tumors. At present peripheral blood allogeneic transplants have been done in a number of patients sufficient to conclude that it is safe and able to give rise to a sustained marrow engraftment. Moreover, due to the fact that circulating stem cells are a mixture of indifferentiated progenitors and "committed" cells, the hematopoietic recovery is significantly faster both in autologous and allogeneic transplant setting. The increasing use of peripheral blood stem cells for autografting has raised the problem of tumoral contamination. The role of reinfused tumoral cells in promoting the relapse had been proved in the past. Attempts to "purge" the bone marrow of patients affected by low-grade non-Hodgkin lymphoma were done several years ago at Dana Farber Institute, strongly suggesting the importance of tumor cells left in the inoculum in modifying the prognosis. In certain tumors, such as myeloma for example, using a PCR based method, the contamination was found in all the aphereses tested. Similar data were found in samples derived from advanced breast cancer or small-cell lung cancer patients. These findings have brought to the development of different systems of stem cell "purging" or CD34+ positive selection. At present at least two or three different methods are available on the market for small and large scale bone marrow or peripheral blood stem cell processing. The ongoing trials will clarify the clinical utility. In the end the availability of large amount of enriched CD34+ cells have suggested to several investigators a possible target for gene therapy. The first data seem to suggest this is a good way to pursue, even if a clinical application remains still far from being satisfying.
Animals↗
Minimal disease detection in patients with alveolar rhabdomyosarcoma using a reverse transcriptase-polymerase chain reaction method.
BACKGROUND: Polymerase chain reaction (PCR) assays that detect fusion genes resulting from consistent chromosomal translocations have been used to detect minimal residual disease, primarily in hematologic malignancies. Molecular assays have been developed recently that detect the PAX3-FKHR or PAX7-FKHR fusion transcript resulting from the t(2; 13) or t(1; 13) translocation consistently observed in alveolar rhabdomyosarcoma. Because of the tumor's propensity to disseminate widely, our aim was to determine whether or not reverse transcriptase-PCR assays could detect submicroscopic disease in bone marrow or peripheral blood specimens. METHODS: We analyzed 19 bone marrow samples from 11 patients with a known gene fusion in their primary tumor. Specimens were collected at diagnosis, remission, and relapse. Fourteen peripheral blood samples were obtained serially from 5 of these patients. A reverse transcriptase-PCR assay was used to detect the presence of the PAX3-FKHR or PAX7-FKHR fusion transcript. These results were compared with the results of microscopic examination of the bone marrow. Medical records of all 11 patients were reviewed. RESULTS: Adequate amplifiable RNA was obtained in 17 of 19 marrow samples. The PCR assay detected fusion products in all 4 specimens obtained from 3 patients with histologic evidence of bone marrow involvement. In addition, PAX3-FKHR fusion products were detected in bone marrows from 2 patients for whom there was no histologic evidence of disease. The fusion transcripts were not detected in any of the peripheral blood samples. CONCLUSIONS: The detection of submicroscopic disease is possible in alveolar rhabdomyosarcoma patients using PCR. These assays may have a role in staging, monitoring therapy, purging protocols for autologous bone marrow transplantation, and post therapy follow-up. Larger prospective studies are needed to address the clinical relevance of these results.
Bone Marrow↗
Elimination of malignant tumor cells from human bone marrow using monoclonal antibodies and immunomagnetic beads.
Conditions for depletion of malignant tumor cells from human bone marrow using monoclonal antibodies and immunomagnetic beads were studied. Human hepatoma HA22T/VGH cells labeled with the supravital DNA binding dye Hoechst 33342 were seeded into normal human bone marrow at a ratio of tumor cells to marrow cells of 1:9. The immunobeads used were M-450 magnetic microspheres coated with sheep anti-mouse immunoglobulin. The marrow-tumor cell mixtures were incubated with a panel of antihuman hepatoma cell monoclonal antibodies (9B2, E10A1, and C2E6) at 4 degrees C for 40 min, and then reacted with immunomagnetic beads for 60 min longer. After incubation, the bead-coated tumor cells were removed from the marrow with a magnetic particle concentrator. Tumor residual was measured in a limiting dilution test, and the recovery of marrow progenitor cells was assessed by CFU-GM assay. The efficiency of tumor depletion was closely related to the amount of monoclonal antibody bound to tumor cells and the immunobead/tumor cell ratio. Monoclonal antibodies used in combination were more effective than their individual use alone. Two cycles of purging with both monoclonal antibodies and immunomagnetic beads resulted in an apparent improvement in tumor depletion as compared with one cycle. Using a cocktail of three monoclonal antibodies and purging the marrow for two cycles, 4 to 5 logs of tumor cells could be removed with a sufficient recovery (70%) of CFU-GM. However, multiple purging treatments (> or = 3 cycles) would be apparently damaging to hematopoietic progenitor cells.
Animals↗