Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa.
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Thiotepa is a bifunctional alkylating anticancer drug that is a rodent carcinogen and a suspected human carcinogen. In order to determine the sensitivity of mutant induction in the Hprt lymphocyte assay for detecting tumorigenic doses of thiotepa, Fischer 344 rats were treated for 4 weeks with thiotepa using a procedure adapted from a carcinogenesis protocol. At various times after beginning the treatment regimen, rats were killed and the lymphocyte Hprt assay was performed on splenic lymphocytes isolated from the animals. The 6-thioguanine-resistant T lymphocyte mutant frequency increased with time during the period of thiotepa exposure and declined slightly thereafter. Significant dose-dependent increases in mutant frequency were found using concentrations of thiotepa that eventually result in lymphoproliferative tumors. Hprt mRNA from mutant lymphocytes was reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. This analysis indicated that the major type of point mutation was G:C-->T:A transversion and that 33% of the mutants contained simple or complex frameshifts. Also, a multiplex PCR performed on DNA from mutant clones that were expanded in vitro indicated that 34% of the clones had deletions in the Hprt gene. These results indicate that the induction of lymphocyte Hprt mutants is a sensitive biomarker for the carcinogenicity of thiotepa and that the types of mutations found in the lymphocyte Hprt gene reflect the kinds of DNA damage produced by thiotepa. (+info)
Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers.
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Previous studies have demonstrated a synergistic interaction between rhuMAb HER2 and the cytotoxic drug cisplatin in human breast and ovarian cancer cells. To define the nature of the interaction between rhuMAb HER2 and other classes of cytotoxic drugs, we applied multiple drug effect/combination index (CI) isobologram analysis to a variety of chemotherapeutic drug/rhuMAb HER2 combinations in vitro. Synergistic interactions at clinically relevant drug concentrations were observed for rhuMAb HER2 in combination with cisplatin (CI=0.48, P=0.003), thiotepa (CI=0.67, P=0.0008), and etoposide (CI=0.54, P=0.0003). Additive cytotoxic effects were observed with rhuMAb HER2 plus doxorubicin (CI=1.16, P=0.13), paclitaxel (CI=0.91, P=0.21), methotrexate (CI=1.15, P=0.28), and vinblastine (CI=1.09, P=0.26). One drug, 5-fluorouracil, was found to be antagonistic with rhuMAb HER2 in vitro (CI=2.87, P=0.0001). In vivo drug/rhuMAb HER2 studies were conducted with HER-2/neu-transfected, MCF7 human breast cancer xenografts in athymic mice. Combinations of rhuMAb HER2 plus cyclophosphamide, doxorubicin, paclitaxel, methotrexate, etoposide, and vinblastine in vivo resulted in a significant reduction in xenograft volume compared to chemotherapy alone (P<0.05). Xenografts treated with rhuMAb HER2 plus 5-fluorouracil were not significantly different from 5-fluorouracil alone controls consistent with the subadditive effects observed with this combination in vitro. The synergistic interaction of rhuMAb HER2 with alkylating agents, platinum analogs and topoisomerase II inhibitors, as well as the additive interaction with taxanes, anthracyclines and some antimetabolites in HER-2/neu-overexpressing breast cancer cells demonstrates that these are rational combinations to test in human clinical trials. (+info)
The role of thiotepa in allogeneic bone marrow transplantation for genetic diseases.
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Graft-versus-host disease (GVHD), graft rejection, disease recurrence and long-term toxicity remain significant obstacles to successful allogeneic bone marrow transplantation (BMT) in children with genetic diseases. In an attempt to improve results, we used a preparative regimen consisting of three alkylating agents, busulfan (BU), thiotepa (TTP) and cyclophosphamide (CY), for T cell-depleted allogeneic bone marrow transplantation instead of the conventional BU-CY protocol. The effect of this intensified regimen was investigated in 26 consecutive children with genetic diseases who underwent T cell-depleted BMT from HLA-identical siblings. Sixteen patients were males and 10 females, of median age 5 (0.2-14) years. The diseases included beta-thalassemia major, osteopetrosis, severe combined immunodeficiency, Wiskott-Aldrich syndrome, familial agranulocytosis, congenital idiopathic hemolytic anemia (CIHA), Gaucher's disease, Niemann-Pick disease, Hurler's syndrome, and adrenoleukodystrophy. The conditioning regimen consisted of BU 4 mg/kg x 4 days (-8 to -5), TTP 5 mg/kg x 2 days (-4 and -3), and CY 60 mg/kg x 2 days (-2 and -1). Engraftment was as expected, with WBC >1.0 x 10(9)/l at day +19 (10-33), ANC >0.5 x 10(9)/l at day +22 (10-56) and platelets >25 x 10(9)/l at day +32 (18-131). Transplant-related mortality was 19%. Overall survival and disease-free survival (DFS) at 60 months follow-up were both 77%. Our results with the BU-TTP-CY regimen followed by T cell-depleted BMT in genetic diseases may provide a basis for prospective comparison with the standard conditioning regimen of BU-CY in the management of children suffering from these conditions. (+info)
Efficacy and safety of simultaneous immunomagnetic CD34+ cell selection and breast cancer cell purging in peripheral blood progenitor cell samples used for hematopoietic rescue after high-dose therapy.
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We have established a new simultaneous positive/negative selection procedure using the Baxter Isolex 300i system. We tested its tumor cell (TC) purging efficacy by tumor contamination tests ex vivo and its safety in a group of 17 breast cancer (BC) patients by measuring hematopoietic recovery after high-dose (HD) therapy and autologous stem cell rescue with the selected cells. Tumor contamination tests resulted in a TC depletion of 4.1-6.0 log steps. The CD34+ cell yield in this experimental setting was 38.9-91.5%, and the CD34+ cell purity was 86.0-96.0%. In a group of 17 BC patients (5 high-risk adjuvant, > or = 10 lymph nodes positive, and 12 metastatic), we processed leukapheresis products (LPs) by simultaneous positive/negative selection. In these clinical samples, the mean CD34+ cell yield was 56.2% (range, 14.0-80.1%), and the CD34+ cell purity was 94.5% (range, 69.0-99.8%). Additionally, we screened samples of the patients' LPs before and after the purging procedure for contaminating TC by immunocytochemistry. In 15 of 17 tested cases, TCs were detectable prior to the purging procedure. After the procedure, we could not detect residual TCs in 16 of 17 cases. In one case, we found a highly reduced number of TCs. Furthermore, we evaluated the times for hematopoietic reconstitution in a group of five BC patients in the high-risk adjuvant situation who underwent HD chemotherapy and hematopoietic rescue with positive/negative selected stem cells and compared it with our own data from 10 BC patients who, after identical HD therapy, received only positively selected CD34+ cells and 14 patients who, after identical HD therapy, received autografts purged by incubation with toxic ether lipids (ET-18-OCH3). In all groups, a leukocyte count of >2000 cells/microl was reached at day +10. A platelet count of > 50,000 cells/microl was reached at day +12 in the ET-18-OCH3 group and at day +14 in the other two groups. Furthermore, 12 patients with metastatic disease rescued with positive/negative selected stem cells after HD therapy also showed fast and comparable hematopoietic recovery. The new simultaneous immunomagnetic positive/negative selection using a closed system is effective and safe. Processing LPs leads to a similar CD34+ cell yield, a higher TC depletion compared to standard CD34+ cell selection, and no delay in hematopoietic recovery. (+info)
Repetitive high-dose therapy with ifosfamide, thiotepa and paclitaxel with peripheral blood progenitor cell and filgrastim support for metastatic and locally advanced breast cancer: results of a phase I study.
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BACKGROUND: This phase I study was designed to determine the optimal dosages of a novel repetitive high-dose therapy regimen for patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: The planned treatment was three cycles of high-dose ifosfamide, thiotepa and conventional-dose paclitaxel delivered every 28 days with progressive dose-escalation in successive cohorts. Each cycle was supported by peripheral blood progenitor cells (PBPC) and filgrastim. RESULTS: Twenty-three patients were entered into this trial. Of the planned 69 treatment cycles, 59 were delivered and fifteen patients completed all three cycles. The dose-limiting toxicities were renal tubular acidosis, encephalopathy, mucositis and enterocolitis. There was one treatment-related hemorrhagic death. CONCLUSIONS: The recommended doses for phase II or III studies are ifosfamide (10 g/m2), thiotepa (350 mg/m2) and paclitaxel (175 mg/m2). (+info)
Thiotepa, busulfan and cyclophosphamide as a preparative regimen for allogeneic transplantation for advanced chronic myelogenous leukemia.
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Thirty-six adults with chronic myelogenous leukemia (CML) in second or greater chronic phase, accelerated phase, or blast crisis underwent marrow or blood stem cell transplantation from an HLA-matched sibling using high-dose thiotepa, busulfan and cyclophosphamide (TBC) as the preparative regimen. All evaluable patients engrafted and had complete donor chimerism. One patient failed to clear meningeal leukemia, and one patient had one of 30 metaphases positive for the Philadelphia chromosome at 2 months post transplant. The remainder of the patients studied had eradication of CML documented by cytogenetics and/or Southern blot for BCR gene rearrangement, and 13 of 15 patients studied became negative for the BCR gene rearrangement by polymerase chain reaction. Three-year relapse rate is 42% (95% CI, 19-64%). The relapse rate was significantly lower for patients transplanted without blast crisis (9% vs 100%, P < 0.001). Eight (22%, 95% CI, 10-39%) patients had severe or fatal veno-occlusive disease (VOD). Elevated liver enzymes within 1 month prior to transplantation and transplantation using marrow were significantly associated with the occurrence of VOD. Three-year survival is 28% (95% CI, 13-43%). Survival was significantly higher for patients transplanted without blast crisis (45% vs 0%, P = 0.01). TBC is an effective preparative regimen for CML in accelerated phase but not refractory blast crisis, and it should be used with caution in patients with prior hepatopathy who have an increased risk of severe VOD. (+info)
LDH elevation after autologous stem cell transplantation.
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We have anecdotally noted that serum LDH values rise after the infusion of autologous peripheral blood progenitor cells (PBPCs), presumably the result of cellular lysis. To further study this issue, we retrospectively reviewed 203 consecutive patients undergoing autologous PBPC transplant from August 1996 to December 1997. 194 patients were evaluable, having documented LDH values on day -1 to day +5 post-transplant. All patients received cytokine mobilization for PBPC collection, and 99% received a chemotherapy-only preparative regimen. 99% of patients had a rise in serum LDH after PBPC infusion. 76% had a normal LDH prior to PBPC infusion that became abnormally high after infusion; 22% began with an abnormally high value that became higher with the infusion of PBPCs. The LDH rose at least 50% in 91% of patients. 92% of the patients had their peak LDH value occur on day +1 post-transplant. The total dose of CD34+ cells infused negatively correlated with peak percentage change of LDH (P = 0.0007). This result was initially perplexing, as we believed that a higher number of infused cells would correlate with a higher LDH value as a result of in vivo cellular lysis. However, the total number of CD34+ cells also negatively correlated with the number of phereses required to collect our threshold of 2.0 x 10(6) CD34+ cells/kg (P = 0.0001). After adjusting for CD34+ cell dose, we found that the number of phereses correlated highly with a peak percentage of change of LDH (P = 0.001). We conclude that the serum LDH increases universally after autologous PBPC infusion, peaking on day +1. We believe that this rise in serum LDH is a result of cell lysis that occurs ex vivo, and correlates with the number of pheresis procedures required to collect an adequate number of CD34+ cells. This may be a result of increasing red blood cell contamination with more phereses, and resultant RBC hemolysis. (+info)
Randomized trial of peripheral blood progenitor cell vs bone marrow as hematopoietic support for high-dose chemotherapy in patients with non-Hodgkin's lymphoma and Hodgkin's disease: a clinical and molecular analysis.
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Filgrastim (r-metHuG-CSF)-mobilized peripheral blood progenitor cells (PBPC) and unstimulated bone marrow (BM) were evaluated and compared for reconstitution after high-dose chemotherapy in patients with relapsed Hodgkin's disease (HD) or non-Hodgkin's lymphoma (NHL) with respect to engraftment, overall and relapse-free survival, and contamination by lymphoma cells using molecular analysis of immunoglobulin gene rearrangements. Forty-four patients with either NHL or HD underwent autologous transplantation after high-dose chemotherapy. Patients were randomized to receive either Filgrastim-mobilized PBPC (n = 15) or unstimulated BM (n = 14). An additional 15 patients received PBPC without randomization because of a recent history of marrow involvement by lymphoma. Use of PBPC was associated with faster neutrophil engraftment than BM (11 vs 14 days to an absolute neutrophil count >0.5 x 10(9)/l, P = 0.04), but without any difference in platelet engraftment, infectious complications, or overall or event-free survival. Both BM (65%) and PBPC (73%) were frequently contaminated by tumor cells as assessed by CDR3 analysis. Patients with negative polymerase chain reaction analysis of a BM sample during the study had a trend towards an improved survival; however, BM involvement by disease had no impact on the ability to mobilize or collect PBPC. We conclude that PBPC are as effective as BM in reconstituting hematopoiesis after high-dose chemotherapy and that both products are frequently contaminated by sequences marking the malignant clone. (+info)