Phase I/II trial of autologous stem cell transplantation in systemic sclerosis: procedure related mortality and impact on skin disease.
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BACKGROUND: Systemic sclerosis (SSc, scleroderma) in either its diffuse or limited skin forms has a high mortality when vital organs are affected. No treatment has been shown to influence the outcome or significantly affect the skin score, though many forms of immunosuppression have been tried. Recent developments in haemopoietic stem cell transplantation (HSCT) have allowed the application of profound immunosuppression followed by HSCT, or rescue, to autoimmune diseases such as SSc. METHODS: Results for 41 patients included in continuing multicentre open phase I/II studies using HSCT in the treatment of poor prognosis SSc are reported. Thirty seven patients had a predominantly diffuse skin form of the disease and four the limited form, with some clinical overlap. Median age was 41 years with a 5:1 female to male ratio. The skin score was >50% of maximum in 20/33 (61%) patients, with some lung disease attributable to SSc in 28/37 (76%), the forced vital capacity being <70% of the predicted value in 18/36 (50%). Pulmonary hypertension was described in 7/37 (19%) patients and renal disease in 5/37 (14%). The Scl-70 antibody was positive in 18/32 (56%) and the anticentromere antibody in 10% of evaluable patients. Peripheral blood stem cell mobilisation was performed with cyclophosphamide or granulocyte colony stimulating factor, alone or in combination. Thirty eight patients had ex vivo CD34 stem cell selection, with additional T cell depletion in seven. Seven conditioning regimens were used, but six of these used haemoimmunoablative doses of cyclophosphamide +/- anti-thymocyte globulin +/- total body irradiation. The median duration of follow up was 12 months (3-55). RESULTS: An improvement in skin score of >25% after transplantation occurred in 20/29 (69%) evaluable patients, and deterioration in 2/29 (7%). Lung function did not change significantly after transplantation. One of five renal cases deteriorated but with no new occurrences of renal disease after HSCT, and the pulmonary hypertension did not progress in the evaluable cases. Disease progression was seen in 7/37 (19%) patients after HSCT with a median period of 67 (range 49-255) days. Eleven (27%) patients had died at census and seven (17%) deaths were considered to be related to the procedure (direct organ toxicity in four, haemorrhage in two, and infection/neutropenic fever in one). The cumulative probability of survival at one year was 73% (95% CI 58 to 88) by Kaplan-Meier analysis. CONCLUSION: Despite a higher procedure related mortality rate from HSCT in SSc compared with patients with breast cancer and non-Hodgkin's lymphoma, the marked impact on skin score, a surrogate marker of mortality, the trend towards stabilisation of lung involvement, and lack of other treatment alternatives justify further carefully designed studies. If future trials incorporate inclusion and exclusion criteria based on this preliminary experience, the predicted procedure related mortality should be around 10%. (+info)
Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia.
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Successful autologous hematopoietic stem cell (HSC) transplantation in childhood acute lymphoblastic leukemia (ALL) requires the ability to either selectively kill the leukemia cells or separate normal from leukemic HSC. Based on previous studies showing that more than 95% of childhood B-lineage ALL express CD38, this study evaluated whether normal CD34(+)CD38(-) progenitors from children with B-lineage ALL could be isolated by flow cytometry. CD34(+) cells from bone marrow samples from 10 children with B-lineage ALL were isolated at day 28 of treatment, when clinical remission had been attained. The CD34(+) progenitor cells were flow cytometrically sorted into CD34(+)CD38(+) and CD34(+)CD38(-) populations. The absolute numbers of CD34(+)CD38(-) cells that could be isolated ranged from 401 to 6245. The cells were then analyzed for the presence of clonotypic rearrangements of the T-cell receptor (TCR) Vdelta2-Ddelta3 locus. Only patients whose diagnostic marrow had an informative TCR Vdelta2-Ddelta3 rearrangement were included in this study. Detection thresholds were typically 10(-4) to 10(-5) leukemic cells in normal marrow. In 6 of 10 samples analyzed, the sorted CD34(+)CD38(-) cells had no detectable Vdelta2-Ddelta3 rearrangements. In 4 cases, the clonotypic leukemic Vdelta2-Ddelta3 rearrangement was detected in the CD34(+)CD38(-) population, indicating that the putative normal HSC population also contained leukemic cells. The data indicate that although most childhood ALL cells express CD34 and CD38, leukemic cells are also frequently present in the CD34(+)CD38(-) population. Therefore, strategies to isolate and transplant normal HSC from children with ALL will require a more stringent definition of the normal HSC than the CD34(+)CD38(-) phenotype. (Blood. 2001;97:3925-3930) (+info)
A virus-directed enzyme prodrug therapy approach to purging neuroblastoma cells from hematopoietic cells using adenovirus encoding rabbit carboxylesterase and CPT-11.
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Tumor cells that contaminate hematopoietic cell preparations contribute to the relapse of neuroblastoma patients who receive autologous stem cell rescue as a component of therapy. Therefore, effective purging methods are needed. This study details in vitro experiments to develop a viral-directed enzyme prodrug purging method that specifically targets neuroblastoma cells. The approach uses an adenovirus to deliver the cDNA encoding a rabbit liver carboxylesterase that efficiently activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11). The data show that an adenoviral multiplicity of infection of 50 transduces 100% of cultured neuroblastoma cells and primary tumor cells, irrespective of the level of tumor cell line contamination. Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34(+) cells at a ratio of 10:90 to replication-deficient AdRSVrCE for 24 h and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three neuroblastoma cell lines (SJNB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential. Also, after "purging," RNA for neuroblastoma cell markers (tyrosine hydroxylase, synaptophysin, and N-MYC) was undetectable by reverse transcription-PCR. In contrast, the purging protocol did not affect the number or type of colonies formed by CD34(+) cells in an in vitro progenitor cell assay. No bystander effect on CD34(+) cells was observed. The method described is being investigated for its potential clinical utility, particularly its efficacy for use with patients having relatively high tumor burdens, because no published methods have been shown to be efficacious when the tumor burden exceeds 1%. (+info)
Double reinforcement with fludarabine/high-dose cytarabine enhances the impact of autologous stem cell transplantation in acute myeloid leukemia patients.
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Reinforced chemotherapy based on a double high-dose consolidation regimen could be a different way to enhance in vivo purging prior to autologous stem cell transplantation (auto-SCT) in acute myeloid leukemia (AML). We investigated the impact on outcome of auto-SCT after two different strategies of early intensification performed after an identical induction regimen in adult patients with AML. Between January 1993 and December 1998, 140 consecutive AML patients were enrolled in a program consisting of an identical anthracycline-based induction (ICE) and two different consolidation regimens: one cycle, cytarabine-based (single-NOVIA: 91 patients); two cycles, fludarabine-based (double-FLAN: 49 patients). Seventy out of 91 patients received single-NOVIA consolidation: 60 underwent a transplantation procedure (allogeneic bone marrow transplantation (allo-BMT):16 patients; auto-SCT: 44). Thirty-five out of 49 patients received double-FLAN consolidation: 31 underwent a transplantation procedure (allo-BMT: 10; auto-SCT: 21). The double consolidation regimen was well-tolerated with only minor side-effects. Median follow-up observation time for surviving patients was 38 months (range, 17-71) for the double-FLAN consolidation group and 70 months (range: 48-93) for the single-NOVIA consolidation group. Among the patients who received auto-SCT, the double consolidation strategy produced a superior disease-free survival curve at 36 months (78.6% (95%CI: 59.4-97.8) vs 47.7% (95%CI: 33-62.4)) compared with the single-NOVIA group. This difference was confirmed when the patients were analyzed for intention to treat (P = 0.04). In addition, the double-FLAN consolidation group showed a superior overall survival and lower relapse rate (P = 0.02). We conclude that the double-FLAN reinforcement strategy is safe and enhances the clinical impact of auto-SCT for AML patients in first complete remission. It may provide specific clinical benefit for patients undergoing auto-SCT. (+info)
Ganglioside GM2/GD2 synthetase mRNA is a marker for detection of infrequent neuroblastoma cells in bone marrow.
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GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-Cer (GM2)/GalNAcbeta1-4(NeuAcalpha2-8NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer (GD2) synthetase [beta-1,4-N-acetyl-galactosaminyl transferase (GalNAc-T)] mRNA, which encodes a key glycosyltransferase for ganglioside GD2 synthesis, was assessed as a molecular marker for detecting metastatic neuroblastoma cells in bone marrow (BM). GalNAc-T mRNA expression by neuroblastoma cell lines (n = 15), primary untreated neuroblastoma tumors (n = 29), morphologically normal BM (n = 22), peripheral blood stem cells (n = 10) from patients with cancers other than neuroblastoma, and blood mononuclear cells from normal donors (n = 17) was assessed by using reverse transcriptase-polymerase chain reaction (RT-PCR) and electrochemiluminescence detection assay (RT-PCR/ECL). BM harvested from 15 neuroblastoma patients was tested before and after ex vivo immunomagnetic bead purging, and results were compared to immunocytological analysis of the same specimens. All neuroblastoma cell lines (mean, 653 x 10(3) ECL units) and primary tumors (mean, 683 x 10(3) ECL units) were positive for significant expression of GalNAc-T mRNA compared to normal blood and BM cells. The RT-PCR/ECL assay could detect GalNAc-T mRNA in 100 pg of total RNA, and in a mixture of one neuroblastoma cell among 10(7) normal BM or blood cells. Eight of 15 autologous BM cells harvested from patients with neuroblastoma had tumor cells detectable by immunocytology, and all 15 were positive for GalNAc-T mRNA. After ex vivo purging, none of the BM cells was immunocytology-positive, but six remained positive by the RT-PCR/ECL assay. GalNAc-T mRNA provides a specific and sensitive molecular marker for RT-PCR/ECL detection of infrequent neuroblastoma cells in BM. (+info)
Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.
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PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens. PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays. RESULTS: MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR. CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease. (+info)
High-dose melphalan with autologous hematopoietic stem cell transplantation for acute myeloid leukemia: results of a retrospective analysis of the Italian Pediatric Group for Bone Marrow Transplantation.
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This retrospective study from the Italian Association of Pediatric Hematology Oncology-Bone Marrow Transplant Group (AIEOP-TMO) reports the results of consolidation with high-dose melphalan and autologous hematopoietic stem cell transplantation (auto-HSCT) in patients with acute myeloid leukemia (AML) in first complete remission (CR1). From October 1994 to July 1999, 20 patients (median age 9.9 years, range 0.11-16.2) were treated in six centers. Eighteen had de novo AML and two had secondary AML. According to BFM criteria, 10 were classified as standard- and 10 as high-risk patients, respectively. The median time from diagnosis to CR1 and from diagnosis to Auto-HSCT were 1.1 months (range 0.8-1.6) and 4.3 months (range 3.1-6.2), respectively. Purging with either mafosfamide (three) or in vivo interleukin-2 (four) was performed in seven of 20 patients. Melphalan was administered at a dosage of 150-220 mg/m(2) (median 180). Median total number of nucleated cells infused was 2.5 x 10(8)/kg (range 1.1-8.9). The myeloablative regimen was well tolerated with no toxic death, veno-occlusive disease or life-threatening complications. All patients had hematopoietic recovery in a median time of 27 days for neutrophils and 44 days for platelets. Eight of 20 patients relapsed after a median time of 7.2 months from transplant (range 5.7-15.9). Six of them died (five of progression of disease and one of sepsis) while the remaining two patients are alive in CR2. The 3-year cumulative probability of survival and event-free-survival (EFS) is 62% and 56%, respectively. This study showed that in pediatric patients with AML consolidation of CR1 with high-dose melphalan allows survival and EFS to be obtained comparable to other auto-HSCT or chemotherapy published series with a potential sparing effect both on duration of treatment (with respect to chemotherapy) and on long-term side-effects (with respect to auto-HSCT with TBI or busulfan containing regimens). (+info)
Purging of autologous peripheral-blood stem cells using CD34 selection does not improve overall or progression-free survival after high-dose chemotherapy for multiple myeloma: results of a multicenter randomized controlled trial.
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PURPOSE: Although high-dose chemotherapy supported by autologous peripheral-blood progenitor-cell (PBPC) transplantation improves response rates and survival for patients with multiple myeloma, all patients eventually develop progressive disease after transplantation. It has been hypothesized that depletion of malignant plasma cells from autografts may improve outcome by reducing infused cells contributing to relapse. PATIENTS AND METHODS: A randomized phase III study using the CEPRATE SC System (Cellpro, Bothell, WA) to enrich CD34(+) autograft cells and passively purge malignant plasma cells was completed in 190 myeloma patients randomized to receive an autograft of CD34-selected or unselected PBPCs. RESULTS: After CD34 selection, tumor burden was reduced by 1.6 to 6.0 logs (median, 3.1), with 54% of CD34-enriched products having no detectable tumor. Median time to count recovery, number of transfusions, transplantation-related mortality, and days in hospital were equivalent between the two transplantation arms. With a median follow-up of 37 months, 33 patients (36%) in the selected and 34 patients (35%) in the unselected arm had died (P =.784). Median overall survival in the selected arm was reached at 50 months and is not reached at this time in the unselected arm (P =.78). Median disease-free survival was 100 versus 104 weeks (P =.82), with 67% of patients in the selected arm and 66% of patients in the unselected arm relapsing. CONCLUSION: This phase III trial demonstrates that although CD34 selection significantly reduces myeloma cell contamination in PBPC collections, no improvement in disease-free or overall survival was achieved. (+info)