Amifostine cytotoxicity and induction of apoptosis in a human myelodysplastic cell line.

Amifostine (AMF), a phosphorylated aminothiol, has been used to treat myelodysplastic syndrome (MDS), where it produces a stimulatory effect on hematopoiesis in bone marrow. To determine if AMF also produced a direct effect on human MDS cells, we planned a study to evaluate the effect of a continuous exposure to AMF on a human MDS cell line. AMF was shown to have a growth-inhibitory effect on MDS cells, with an IC(50) of 14 microM after a 5 day exposure. Cell cycle analysis revealed that a 5 day exposure to 20 microM AMF increased the percentage of cells in G0/G1 and this was accompanied by a decrease in the percentage of cells in S phase. Cytoflorometric and agarose-gel electrophoretic analysis revealed that this effect correlated with cell membrane alterations and DNA fragmentation consistent with an induction of apoptosis without affecting the expression of p53 protein or inducing any lymphoid or myeloid differentiation in the MDS cell line. We conclude that the continuous exposure of a human MDS cell line to AMF is cytotoxic and associated with an induction of apoptosis independent of alterations in p53 expression.

Taurolidine: preclinical evaluation of a novel, highly selective, agent for bone marrow purging.

Taurolidine has been shown to have remarkable cytotoxic activity against selected human tumor cells at concentrations that spare normal cells. In this study we have extended this observation and assessed the ability of Taurolidine to purge tumor cells from chimeric mixtures of bone marrow (BM) and neoplastic cells. Normal murine BM and human leukemic (HL-60) or ovarian (PA-1) tumor cell lines were used as models. Exposure of tumor cells to 2.5 mM Taurolidine for 1 h resulted in the complete elimination of viable cells. In contrast, exposure of BM to 5 mMTaurolidine for 1 h reduced CFU-GM, BFU-E and CFU-GEEM colony formation by only 23.0%, 19.6% and 25.2%, respectively. Inhibition of long-term BM culture (LTBMC) growth following a 1 h exposure to 5 mM Taurolidine also was approximately 20% compared to untreated LTBMC. Finally, chimeric cultures were generated from BM and HL-60GR or PA-1GR cells (tumor cells transfected with the geneticin resistance gene). Exposure of these chimeric cultures to 5 mM Taurolidine for 1 h totally eliminated viable cancer cells while minimally reducing viable BM cells. This finding was confirmed by subsequent positive selection for surviving tumor cells with geneticin. These findings reveal that Taurolidine holds promise for use in BM purging.

High-dose cyclophosphamide followed by autografting can improve the outcome of relapsed or resistant non-Hodgkin's lymphomas with involved or hypoplastic bone marrow.

We report our experience of high-dose cyclophosphamide (HDCY) followed by high-dose therapy (HDT) and peripheral blood progenitor cell (PBPC) autografting in patients with diffuse, intermediate and high-grade non-Hodgkin's lymphomas who have failed conventional treatment. From 1991 to 1996, 54 consecutive patients pre-treated with a median of two chemotherapy lines entered the study. Eighteen patients (33%) were still responders to conventional chemotherapy (sensitive relapse), and 20 patients (37%) were in partial response (PR) after chemotherapy (CT). Sixteen patients (30%) were resistant to conventional CT either at presentation (non responder) or in relapse (resistant relapse). Thirty-nine patients had bone marrow involved by disease and fifteen had an hypoplastic marrow following conventional treatment. Patients received HDCY (7gr/m2) and G-CSF or GM-CSF in order to collect PBPC. Median collected CD34+ cells was 12.3 x 10(6)/Kg (range 0.7-197). After HDT (BEAM or Melphalan + TBI) 50 patients underwent PBPC autografting. According to intention to treat, 44 (81%) of 54 patients achieved complete remission (CR) (50% after HDCY and 31% after HDT). Procedure related death occurred in 6 patients (11%), one after HDCY and 5 after autografting. Twenty-nine (66%) of 44 patients are still in CR, 7 to 63 months (median 27 months) after the procedure. Three-year probability of survival, disease-free survival and progression-free survival are 63%, 64% and 52% respectively. In conclusion, HDCY is an effective procedure not only in mobilizing PBPC, but also in reducing tumour burden. HDT with PBPC support may further improve the outcome in this category of high-risk non-Hodgkin's lymphomas.

Analysis of the proliferative and phenotypic properties of tumor infiltrating lymphocytes expanded in vitro in the course of the clinical trial of adoptive immunotherapy of metastatic melanoma.

Adoptive immunotherapy with in vitro expanded tumor infiltrating lymphocytes (TIL) and recombinant interleukin-2 (rIL-2) is a recent option in the treatment of advanced melanoma resistant to conventional chemotherapy. In the course of a protocol of treatment of advanced melanoma with in vitro expanded TIL and rIL-2, we obtained 38 samples from 27 different patients. Lymphocytes derived were cultured in the presence of rIL-2 in vitro for a 4-6 week period and 23 resulted in proliferative cultures. Eighteen samples were infused in the course of the clinical trial. The median number of lymphocytes obtained was 18x10(9) (range 1-43x10(9)) cells. Phenotypic analysis showed that all samples contained a pure population of T cells. These data confirm that lymphocytes can be expanded from advanced metastatic sites, have peculiar characteristics and are suitable to be infused in vivo.

Adoptive immunotherapy with tumor-infiltrating lymphocytes and subcutaneous recombinant interleukin-2 plus interferon alfa-2a for melanoma patients with nonresectable distant disease: a phase I/II pilot trial. Melanoma Istituto Scientifico Tumori Group.

BACKGROUND: On the basis of our previous experience, we designed this study to determine the activity and toxicity of outpatient treatment with autologous tumor-infiltrating lymphocytes (TIL) together with intermediate-dose recombinant interleukin-2 (rIL-2) and low-dose recombinant interferon alfa-2a (rIFN-alpha2a), for patients with metastatic melanoma. METHODS: Between April 1992 and October 1994, we processed 38 melanoma samples derived from 36 patients with metastases. Proliferative cultures of expanded lymphocytes (TIL) were infused only once into patients with metastatic melanoma. rIL-2 was administered subcutaneously for 1 month, starting on the day of TIL infusion, at an escalating dose of 6-18 x 10(6) IU/m2/day for the first week and at the maximum-tolerated dose for the subsequent 3 weeks and then, after a 15-day interval, for 1 week/month for 3 months. rIFN-alpha2a was administered subcutaneously at 3 X 10(6) IU three times each week until progression. RESULTS: Of 38 melanoma samples, 19 (50%) resulted in proliferative cultures and were infused. The median number of expanded lymphocytes was 18 x 10(9) (range, 1-43 x 10(9)), and the median period of culture was 52 days (range, 45-60). rIL-2 was administered at doses ranging between 6 and 18 x 10(6) IU/m2/day. Toxicity was mild or moderate, and no life-threatening side effects were encountered. Two of 19 treated patients experienced complete responses of their metastatic sites (soft tissue), 10 had stable disease, and 7 showed progressive disease. The response rate was 11% (95% confidence interval, 2-35%). CONCLUSIONS: Outpatient treatment with TIL plus rIL-2 and rIFN-alpha2a is feasible, although, within the context of the small sample size, the activity of the combination was no different from the reported activity of any of the components used alone.