Phase I/II trial of doxorubicin and fixed dose-rate infusion gemcitabine in advanced soft tissue sarcomas: a GEIS study.

The aim of the study was to determine the dose-limiting toxicity and maximum tolerated dose of a first-line combination of doxorubicin and gemcitabine in adult patients with advanced soft tissue sarcomas and to explore its activity and toxicity, and the presence of possible interactions between these agents. Patients with measurable disease were initially treated with doxorubicin 60 mg m(-2) by i.v. bolus on day 1 followed by gemcitabine at 800 mg m(-2) over 80 min on days 1 and 8, every 21 days. Concentrations of gemcitabine and 2',2'-difluorodeoxyuridine in plasma, and gemcitabine triphosphate levels in peripheral blood mononuclear cells were determined during 8 h after the start of gemcitabine infusion. Myelosuppression and stomatitis were limiting toxicities, and the initial dose level was applied for the Phase II trial, where grade 3-4 granulocytopenia occurred in 70% of patients, grade 3 stomatitis in 46% and febrile neutropenia in 20%. Objective activity in 36 patients was 22% (95% CI: 9-35%), and a 50% remission rate was noted in leiomyosarcomas. Administration of doxorubicin preceding gemcitabine significantly reduced the synthesis of gemcitabine triphosphate. Clinical activity, similar to that of single-agent doxorubicin, and the toxicity encountered do not justify further studies with this schedule of administration.

Simultaneous determination of gemcitabine di- and triphosphate in human blood mononuclear and cancer cells by RP-HPLC and UV detection.

A reverse-phase HPLC method based on ion-pair formation with UV detection was set up for the simultaneous determination of gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) in human cells. The separation was achieved on a Tracer Excel ODSA column (100 mm x 4.6mm i.d., 3 microm particle size) at room temperature. Nine nucleotides were separated by isocratic elution in 26 min. Accuracy, linearity, sensitivity and precision studies for dFdCDP, dFdCTP, adenosine diphosphate (ADP) and triphosphate (ATP) validated this method. This assay was used to provide data from gemcitabine treated patients and in vitro grown human cancer cells.

Phase II Trial of Doxorubicin Plus Escalated High-Dose Ifosfamide in Patients With Advanced Soft Tissue Sarcomas of the Adult: A Study of the Spanish Group for Research on Sarcomas (GEIS).

Background. To explore the tolerance and the activity of high-dose ifosfamide (IFOS) combined with doxorubicin (DXR) at 50 mg/m(2) every 4 weeks in patients with soft tissue sarcomas. Methods. DXR was given IV bolus and IFOS by continuous infusion at 2 g/m(2)/day. Initial IFOS dose (12 g/m(2)) was adjusted to 10, 13, or 14 g/m(2) according to toxicity. Results. Seventy patients received 277 cycles (median 3 cycles, range 1-10), 34% with IFOS dose increased, 30% decreased, and 48% delivered at 12 g/m(2). Toxicity grade 4 occurred on granulocytes (67% of patients) or platelets (19%), 54% had febrile neutropenia, 31% grade 3/4 asthenia, and 26% abandoned the study due to toxicity. Three toxic deaths occurred. In 57 non-GIST patients objective activity was 45.6% (95% CI, 32 to 58%). Conclusion. At least 4 cycles were tolerated by 71% of patients, most receiving DXR 50 mg/m(2) plus IFOS 10-12 g/m(2), with substantial toxicity.

Determination of thiamine and its phosphorylated forms in human plasma, erythrocytes and urine by HPLC and fluorescence detection: a preliminary study on cancer patients.

In man, neurotoxicity associated to ifosfamide treatment can be reversed by intravenous thiamine administration. Trying to explain this clinical finding, we decided to study possible changes in thiamine availability and activation in patients exposed to ifosfamide. Free thiamine and its phosphate esters levels were measured in plasma, erythrocytes and urine by an ion-pair HPLC method with pre-column derivatization, which allowed separation of the fluorescent compounds in less than 10 min. The method was validated by linearity, sensitivity and reproducibility studies, whose values met the demands for bioanalytical assays. This method was applied to assess thiamine status in cancer patients exposed to ifosfamide therapy for advanced disease.

Phase II Clinical Trial With Pegylated Liposomal Doxorubicin (CAELYX(R)/Doxil(R)) and Quality of Life Evaluation (EORTC QLQ-C30) in Adult Patients With Advanced Soft Tissue Sarcomas: A study of the Spanish Group for Research in Sarcomas (GEIS).

BACKGROUND: Pegylated liposomal doxorubicin (PLD), a formulation with pharmacokinetic differences with respect to doxorubicin (DXR), might benefit patients with advanced soft tissue sarcoma (STS) pretreated with DXR. PATIENTS AND METHODS: Patients with measurable and progressive STS received PLD at 35 mg/(2) every 3 weeks. Quality of life before and during treatment was assessed with EORTC QLQ-C30. RESULTS: Twenty-eight patients, 22 DXR-pretreated, were given 140 cycles (median 3, range 1-18). Activity in 27 patients (5 GIST): one complete and one partial remission (both non-GIST and without prior DXR), 12 stabilizations and 13 progressions (response rate 7.4%, 95% CI: 0-17%). Grade 3 toxicity: palmar-plantar erythrodysesthesia (19% of patients), stomatitis (4%) or cutaneous (4%). Neutropenia grade>/=3 was detected in 16% of patients. Median relative dose intensity was 95%. Progression-free rate at 3 and 6 months was, respectively, 48 and 22%, median progression-free survival 5.8 months and median overall survival 8.7 months. QLQ-C30 at baseline and at weeks 6-11 in 23 and 13 patients, respectively, showed good reliability and validity. Quality of life did not seem to worsen during therapy. CONCLUSIONS: PLD did not induce objective remissions in 22 STS patients pretreated with DXR, but progression-free rate figures support the use of this agent in patients who have not progressed under a DXR-containing regimen. The toxicity observed was comparable to that of other PLD schedules.

Down-regulation of the beta-chemokine receptor CCR6 in dendritic cells mediated by TNF-alpha and IL-4.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha (TNF-alpha), and stem cell factor. Using an anti-CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this beta-chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein-3alpha (MIP-3alpha), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF-alphaq treatments. Interleukin-4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP-3alpha/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens.

Temozolomide in adult patients with advanced soft tissue sarcoma: a phase II study of the EORTC Soft Tissue and Bone Sarcoma Group.

Temozolomide, an oral imidazotetrazine derivative, was given to 31 patients with advanced soft tissue sarcoma. The dose of 750 mg/m2 was divided over 5 consecutive days, and escalated to 1000 mg/m2 over 5 days at cycle 2 if myelosuppression no worse than common toxicity criteria grade 2 was noted in the first 28-day cycle. A total of 99 treatment cycles were given to 31 patients. The drug was well tolerated, with nausea and vomiting as the most common side-effects. Only one partial tumour response was documented, giving a response rate of 3.33%, 95% confidence interval, (CI) 0.1-17.2%. The median time to progression was 8 weeks and the median survival was 27 weeks. These results indicate that temozolomide in this schedule is not active as second-line treatment in advanced soft tissue sarcoma.

Phase III trial of cyclophosphamide, epirubicin, fluorouracil (CEF) versus cyclophosphamide, mitoxantrone, fluorouracil (CNF) in women with metastatic breast cancer.

BACKGROUND: The mitoxantrone combination CNF and the epirubicin combination CEF have shown similar activity and less toxicity than the standard CAF combination in metastatic breast cancer (MBC). A prospective randomised study was started to compare safety and activity between CEF and CNF administered using a classical chemotherapeutic schedule in MBC. PATIENTS AND METHODS: From December 1987 to June 1993, 151 patients were randomised to receive cyclophosphamide (C) 100 mg m(-2) p.o. days 1-14, fluorouracil (F) 500 mg m(-2) i.v. days 1 and 8, and epirubicin (E) 30 mg m(-2) i.v. days 1 and 8, or mitoxantrone (N) 6 mg m(-2) i.v. days 1 and 8, every 4 weeks. Seventy-three patients were eligible for CEF and 72 for CNF. RESULTS: Objective responses were observed in 61.6% of the CEF group and 44.4% in CNF group (p = 0.004). The median duration of response was 64 weeks in CEF and 50 weeks in CNF group (p = 0.02) and median time to progression was 51 and 33 weeks, respectively (p = 0.0004). At the time of analysis, all except six patients (one in CNF and five in CEF) had died and the median survival time in the CEF group was longer than in CNF (74.4 weeks vs 51.4 weeks; log-rank chi2 test p = 0.015). CNF produced more hematologic toxicity than CEF (WHO scale; grades 2-4); leucopenia 84% vs 68% (p = 0.03) and thrombocytopenia 17% vs 4.5% (p = 0.01); CEF caused more grade 2 and 3 alopecia: 93% vs 70% (p = 0.001). CONCLUSION: The combination CEF using this schedule and dosage in metastatic breast cancer is more effective with less toxicity than CNF, except for alopecia, and was associated with longer survival.

Drosophila grim induces apoptosis in mammalian cells.

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.

Phase II trial of first-line high-dose ifosfamide in advanced soft tissue sarcomas of the adult: a study of the Spanish Group for Research on Sarcomas (GEIS)

BACKGROUND: The agent Ifosfamide (IFOS) is active against soft tissue sarcomas (STS), and patients who progress to IFOS at doses < or = 10 g/m2 show remissions when exposed to high-dose ifosfamide (HDI) (i.e., doses > 10 g/m2), which supports a dose-response relationship for this drug. Because of a lack of first-line studies in adult STS patients, we decided to test the activity and toxicity of HDI in a phase II trial. PATIENTS AND METHODS: Forty-eight patients were enrolled in the study. IFOS was administered at a dose of 14 g/m2 by continuous infusion over six days every four weeks. Granulocyte-macrophage colony-stimulating factor (GM-CSF) at 5 micrograms/kg/day for 10 consecutive days was systematically administered after an episode of neutropenic fever or a delay in hematologic recovery. Patients were treated until progression or the occurrence of severe toxicity, and surgical rescue was attempted when possible. RESULTS: Six pathology-established complete remissions and 11 partial remissions were observed in 45 assessable patients with a response rate of 37.7% (95% CI: 25.5%-50%). Grade 3-4 toxicity (% of cycles) was noted by hemoglobin (17%), leukocyte (75%), granulocyte (75%) and platelet (13%) counts in 158 evaluable cycles. GM-CSF was administered to 28 patients, and 25 suffered one or more episodes of neutropenic fever. Renal toxicity was mild and reversible with some degree of tubular and glomerular dysfunction detected in up to 60% of patients. Grade 3 CNS toxicity was observed in 32% of patients but only one required interruption of therapy. Sixty-four per cent of the patients had asthenia grade 2-3 and 20% were excluded from the study due to excessive toxicity. There was one treatment-related death. CONCLUSIONS: HDI is an active drug in first-line therapy against adult STS. Different administration schedules should be evaluated in an attempt to improve its therapeutic index.

Molecular cloning and expression of stromalin protein from Drosophila melanogaster: homologous to mammalian stromalin family of nuclear proteins.

We report the cloning of a new cDNA from Drosophila melanogaster that encodes an open reading frame of 1116 amino acid residues. It is the insect homolog of the previously reported stromalin (SA) family of nuclear proteins in mammals (Carramolino et al. [1997]. Gene 195, 151-159). Taking into account the identical domain present in all the SA family members characterized to date, we have carried out polymerase chain reaction (PCR) using degenerate oligonucleotides from the 5' and 3' ends of one of those regions of the molecule and cDNA from D. melanogaster embryos. We isolated the homologous domain of the putative Drosophila SA molecule (DSA). This cDNA fragment was used as a radiolabeled probe for screening a cDNA library from Drosophila embryos, and we have cloned a full-length cDNA for the SA homolog from an insect. The protein shows a good degree of identity with the mammalian stromalins SA-1 and SA-2, with the N and C ends being the most divergent regions of the molecule. The mRNA coding for this protein shows a molecular size of about 3.7 kb by Northern blot analysis and is essentially expressed in embryonic stage. The in situ hybridization experiments indicate that the DSA messenger is expressed mainly in neurogenic territories in the embryonic development of Drosophila. The DSA protein has been cloned and expressed in a baculovirus system, and polyclonal antibodies were generated against the recombinant molecule. Western blot analysis using these antibodies detected a main band corresponding to about 120 kDa, principally in embryos.

Expression and purification of human stromelysin 1 and 3 from baculovirus-infected insect cells.

Stromelysin 1 (ST1) is a member of the matrix metalloproteinase (MMP) family probably involved in extracellular matrix degradation. Stromelysin 3 (ST3), considered by sequence homology to be a member of the MMP family of proteases, is specifically expressed in the stroma adjacent to the invasive tumoral cells, but its role in cancer progression remains to be elucidated. Genes encoding ST1 and ST3 were expressed in lepidopteran insect cells using the baculovirus expression vector system. Recombinant baculoviruses were obtained after cloning the full-length cDNA of ST1 and ST3 in plasmids pBacPAK1 and pBacPAK9, respectively. Sf9 insect cells infected with the recombinant baculovirus overexpressed the zymogen proST1 (60 kDa) in an insoluble form, a peak of expression being reached from 24 h postinfection. After solubilization in 8 M urea, and further refolding, activation, and purification, 0.3 mg of mature ST1 (30 kDa), purified to 90% homogeneity, was obtained per 5 x 10(8) infected cells. Recombinant ST1 exhibited proteolytic activity on alpha2-macroglobulin, casein, fibronectin, alpha1-antitrypsin, and laminin. The recombinant zymogen proST3 (55 kDa) was expressed as a soluble form in insect cells, maximal expression occurring at 72 h postinfection. After purification to 95% homogeneity, 2.5 mg of proST3 was obtained per 5 x 10(8) infected cells. A number of proteases including plasmin, urokinase, and ST1 were shown to be able to cleave proST3 giving rise to defined bands of 50-30 kDa. The ST3 mature form of 45 kDa (mST3) was also expressed in the baculovirus system and the obtained protein, 2. 5 mg per 5 x 10(8) infected cells purified to 80% homogeneity, was shown to be active on both casein degradation and alpha2-macroglobulin entrapment assays. Our results suggest that the baculovirus system offers a convenient and efficient means to produce ST1 and ST3 in order to carry out further biochemical studies.

Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3.

To elucidate the physiological role of human stromelysin-3 (hST-3) in tumor progression and/or wound healing, insulin-like growth factor-binding protein-1 (IGFBP-1) was analyzed as a potential physiological substrate. hST-3 proteolysis generates two fragments of 16 and 9 kDa that react with IGFBP-1 monoclonal antibody, although they do not bind insulin-like growth factor-I (IGF-I) in ligand blot. N-terminal sequencing shows that hST-3 cleaves IGFBP-1 at the His140-Val141 bond located in the IGFBP-1 midregion. We show that IGFBP-1 inhibits IGF-I-induced survival and proliferation of BAF/3 cells, as well as IGF-I-mediated activation of phosphatidylinositol 3-kinase (PI 3-K). Co-incubation of the IGF-I. IGFBP-1 complex with hST-3 restores IGF-I-induced proliferation and PI 3-K kinase activity in these cells. BAF/3 proliferation is significantly increased with the hST-3-treated IGF-I.IGFBP-1 complex compared with that obtained using IGF-I alone. To produce this enhanced proliferation, IGF-I must bind to IGFBP-1 before hST-3 proteolysis, demonstrated using an IGF-I variant that does not bind IGFBP. IGFBP-1 also inhibits IGF-I-induced proliferation of the MCF-7 breast adenocarcinoma, and this inhibition was not seen in hST-3-transfected MCF-7 cells. Such proteolysis may thus play a role in in vivo tumor progression. These results indicate that hST-3 may regulate IGF-I bioavailability by proteolyzing IGFBP, thus favoring cell survival and proliferation.

Surgical treatment of lung metastases: The European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group study of 255 patients.

BACKGROUND: Several reports have shown a prolonged survival after surgical treatment of pulmonary metastases from soft tissue sarcomas. However, it is still unclear which prognostic factors predict a favorable outcome. Series are not comparable and the data are conflicting. Therefore, a multi-institutional study was undertaken to analyze prognostic factors in selecting patients for resection of pulmonary metastases from soft tissue sarcomas. METHODS: This report is a retrospective study of the European Organization for Research and Treatment of Cancer-Soft Tissue and Bone Sarcoma Group. Two hundred fifty-five patients underwent complete resection of lung metastases from soft tissue sarcomas. Cases with chondrosarcoma and small round cell sarcomas like Ewing sarcoma were excluded. RESULTS: The 3 year and 5 year overall postmetastasectomy survival rates were 54% and 38%, respectively. The disease free postmetastasectomy survival rates were 42% and 35%, respectively. Analysis of prognostic factors for a more favorable outcome revealed disease free intervals of 2.5 years or more, following a resection with microscopically free margins, age less than 40 years, and Grade I and II tumors. These prognostic factors have an independent influence on overall survival, using a multivariate Cox regression model. CONCLUSIONS: Surgical excision of lung metastases from soft tissue sarcomas is well accepted and should be considered as a first line of treatment if preoperative evaluation indicates that complete clearance of the metastases is possible. Further investigation is needed before chemotherapy can be recommended as additional therapy.

Combination chemotherapy with cisplatin and 5-fluorouracil 5-day infusion in the therapy of advanced gastric cancer: a phase II trial.

Fifty-six patients with measurable or evaluable advanced gastric cancer were treated with cisplatin, 100 mg/m2 in continuous infusion of 24 hours, and 5-fluorouracil, 1000 mg/m2/day (by continuous 5-day infusion) every 4 weeks. Three patients were found ineligible for the study. A response rate of 41% (22/53) was obtained (95% confidence interval: 28%-54%), with a median duration of remission of 10.2 months and an overall median survival time of 10.6 months. Leukopenia and thrombocytopenia were mild. Nausea and vomiting were common, and 23.5% of the patients had grade 3 stomatitis. Peripheral neuropathy and renal insufficiency increased with the number of cycles, representing the cumulative dose-limiting toxicity. This study indicates that the combination of cisplatin plus 5-fluorouracil is synergistic or at least has additive antitumor activity. We think that this association of 2 drugs should be considered for further phase III clinical trials.