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Purpose The majority of cytotoxic drugs for adults are dosed based on body surface area (BSA), aiming to reduce interpatient variability in drug exposure. We prospectively studied the usefulness of BSA-based dosing of cisplatin in patients at extremes of BSA values. Patients and Methods Patients were randomly assigned to receive a fixed dose of cisplatin in course 1, and a BSA-adjusted dose in course 2, or vice versa. The fixed dose was based on the average BSA for males and females, while extremes were set at BSA values exceeding the average ± 1 standard deviation. Subsequently, we retrospectively analyzed data from a normal population. Results In 25 patients assessable for both courses, the use of a fixed dose of cisplatin resulted in reduced exposure to unbound platinum in patients at the upper extremes of BSA (P = .003) and higher exposures in patients at the lower extremes (P = .009), as compared with exposures following the BSA-adjusted dose. Although clearance was related to BSA (R2 = 0.44; P < .001), only a small reduction in interpatient variability in clearance after correction for BSA was achieved (20.8% v 17.1%). In the retrospective analysis, compared with the average patient, the clearance of unbound platinum in patients with a BSA value ≤ 1.65 m2 was 16% slower (P < .001), while an 18% faster clearance (P < .001) was observed in patients with a BSA value ≥ 2.05 m2. Conclusion Unless better predictors for platinum clearance are identified, fixed-dose regimens per BSA cluster (≤ 1.65 m2; 1.66 m2 to 2.04 m2; ≥ 2.05 m2) are recommended.

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p

Male germ cell tumors (GCTs) are extremely sensitive to platinum-containing chemotherapy, with only 10% of patients showing therapy resistance. However, the biological basis of the high curability of disseminated GCTs by chemotherapy is still unknown. Recently, we demonstrated that the mammalian serine/arginine rich protein-specific kinase 1 (SRPK1) is a cisplatinsensitive gene, inactivation of which leads to cisplatin resistance. Because, in mammalians, the expression of SRPK1 is preferentially high in testicular tissues, cisplatin responsiveness of male GCTs might be associated with SRPKi levels. In the present study, we monitored SRPK1 protein expression in a unique series of nonseminomatous GCTs by immunohistochemistry. Randomly selected GCTs (n = 70) and tumors from patients responding to standard chemotherapy (n = 20) generally showed strong SRPKi staining. In contrast, expression in refractory GCTs (n = 20) as well as in GCTs from poor-prognosis patients responding to high-dose chemotherapy only (n = 11) was significantly lower (two-sided Wilcoxon rank sum test: P < .001). In conclusion, our data suggest that SRPK1 expression might be an important prognostic indicator for the chemoresponsiveness of nonseminomatous GCTs.

The objective of this study was to identify prognostic factors for survival in patients with advanced oesophageal cancer, who are treated with cisplatin-based combination chemotherapy. We analysed the baseline characteristics of 350 patients who were treated in six consecutive prospective trials with one of the following regimens: cisplatin/etoposide, cisplatin/etoposide/5-fluorouracil, cisplatin/paclitaxel (weekly) and cisplatin/paclitaxel (biweekly). Predictive factors in univariate analyses were further evaluated using multivariate analysis (Cox regression). The median survival of all patients was 9 months. The 1, 2 and 5-year survival rates were 33, 12 and 4%, respectively. The main prognostic factors were found to be WHO performance status (0 or 1 vs 2), lactate dehydrogenase (normal vs elevated), extent of disease (limited disease defined as locoregional irresectable disease or lymph node metastases confined to either the supraclavicular or celiac region vs extensively disseminated disease) in addition to the type of treatment (weekly or biweekly cisplatin/paclitaxel regimen vs 4-weekly cisplatin/etoposide with or without 5-fluorouracil). Although weight loss, liver metastases and alkaline phosphatase were significant prognostic factors in univariate analyses, these factors lost their significance in multivariate analyses. The median survival for patients without any risk factors was 12 months, compared to only 4 months in patients with WHO 2 plus elevated LDH and extensive disease. The performance status, extent of disease, LDH and the addition of paclitaxel to cisplatin are independent prognostic factors in patients with advanced oesophageal cancer, who are treated with cisplatin-based combination chemotherapy.

The therapeutic potential of the highly active anticancer agent cisplatin is severely limited by the occurrence of cellular resistance. A better understanding of the molecular pathways involved in cisplatin-induced cell death could potentially indicate ways to overcome cellular unresponsiveness to the drug and thus lead to better treatment results. We used the budding yeast Saccharomyces cerevisiae as a model organism to identify and characterize novel genes involved in cisplatin-induced cell kill, and found that SKY1 (SR-protein-specific kinase from budding yeast) is a cisplatin sensitivity gene whose disruption conferred cisplatin resistance. In cross-resistance studies, we observed resistance of yeast sky1 Delta cells (i.e., cells from which the SKY1 gene had been disrupted) to cisplatin, carboplatin (but not oxaliplatin), doxorubicin and daunorubicin, and hypersensitivity to cadmium chloride and 5-fluorouracil. Furthermore, these cells did not display reduced platinum accumulation, DNA platination or doxorubicin accumulation, indicating that the resistance is unrelated to decreased drug import or increased drug export. Based on the modification of the anticancer drug sensitivity profile and our finding that sky1 Delta cells display a mutator phenotype, we propose that Sky1p might play a significant role in specific repair and/or tolerance pathways. Disruption of the S. cerevisiae SKY1 gene would thus result in deregulation of such mechanisms and, consequently, lead to altered drug sensitivity.

PURPOSE: Most anticancer drugs are dosed based on body-surface area (BSA) to reduce interindividual variability of drug effects. We evaluated the relevance of this concept for cisplatin by analyzing cisplatin pharmacokinetics obtained in prospective studies in a large patient population. PATIENTS AND METHODS: Data were obtained from 268 adult patients (163 males/105 females; median age, 54 years [range, 21 to 74 years]) with advanced solid tumors treated in phase I/II trials with cisplatin monotherapy or combination chemotherapy with etoposide, irinotecan, topotecan, or docetaxel. Cisplatin was administered either weekly (n = 93) or once every 3 weeks (n = 175) at dose levels of 50 to 100 mg/m2 (3-hour infusion). Analysis of 485 complete courses was based on measurement of total and non–protein-bound cisplatin in plasma by atomic absorption spectrometry. RESULTS: No pharmacokinetic interaction was found between cisplatin and the anticancer drugs used in combination therapies. A linear correlation was observed between area under the curves of unbound and total cisplatin (r = 0.63). The mean plasma clearance of unbound cisplatin (CLfree) was 57.1 ± 14.7 L/h (range, 31.0 to 116 L/h), with an interpatient variability of 25.6%. BSA varied between 1.43 and 2.40 m2 (mean, 1.86 ± 0.19 m2), with an interpatient variability of 10.4%. When CLfree was corrected for BSA, interindividual variability remained in the same order (23.6 v 25.6%). Only a weak correlation was found between CLfree and BSA (r = 0.42). Intrapatient variability in CLfree, calculated from 90 patients was 12.1% ± 7.8% (range, 0.30% to 32.7%). CONCLUSION: In view of the high interpatient variability in CLfree relative to variation in observed BSA, no rationale for continuing BSA-based dosing was found. We recommend fixed-dosing regimens for cisplatin.

Interleukin 12 (IL-12) has potential efficacy in malignant, infectious and allergic diseases. Its side-effects include activation of coagulation and fibrinolysis, as documented in chimpanzees. We assessed the coagulative and fibrinolytic response in 18 patients with renal cell carcinoma after subcutaneous injection of 0.5 microg/kg recombinant human IL-12. IL-12 induced a fibrinolytic response in 17 patients (94%): plasmin-alpha2-anti-plasmin complexes (PAPc) increased from 11.8 +/- 6.6 nmol/l (mean +/- SD) to a maximum of 18.8 +/- 7.4 nmol/l at 72 h. Baseline levels of tissue plasminogen activator (tPA) and plasminogen-activator inhibitor-I (PAI) were elevated in eight and 14 patients respectively. tPA increased from 12.6 +/- 5.2 ng/ml to a maximum of 19.0 +/- 6.7 ng/ml at 72 h. PAI decreased from 111 +/- 69 ng/ml to a minimum of 65 +/- 53 ng/ml at 8 h, thereafter remaining below baseline. Elevation of PAPc correlated with elevation of tPA and reduction of PAI. A coagulative response occurred in nine patients (50%): thrombin-anti-thrombin III complexes increased from 29 +/- 53 ng/ml to a maximum of 460 +/- 322 ng/ml at 12 h. Patients with and without a coagulative response had similar levels of recombinant human IL-12, interferon-gamma or tumour necrosis factor-alpha. We conclude that IL-12 can activate both fibrinolysis and coagulation in a significant proportion of patients with cancer. The time-frame and sequence of these activation processes differ from those known for other cytokines.

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.

Lurtotecan (GI147211; LRT) is a semisynthetic and water-soluble analogue of the topoisomerase I inhibitor camptothecin. To determine whether the therapeutic efficacy of LRT in patients could be improved, the drug was encapsulated in liposomes (NX211; Gilead Sciences). In order to allow accurate description of the pharmacokinetic behavior of NX211 in cancer patients, we have developed sensitive RP-HPLC assays with fluorescence detection (lambdaex=378 nm; lambdaem=420 nm) for the determination of total LRT levels in human plasma and urine. Sample pretreatment involved deproteinization with 10% (w/v) aqueous perchloric acid-acetonitrile (2:1, v/v), and chromatographic separations were achieved on an Inertsil-ODS 80A analytical column. The lower limit of quantitation (LLQ) was established at 1.00 ng/ml in plasma (200-microl sample) and at 100 ng/ml in urine (200 microl of 40-fold diluted sample). The within-run and between-run precisions were7.5%. LRT concentrations in urine of100 ng/ml were determined by a modified procedure comprising a single solvent extraction with n-butanol-diethyl ether (3:4, v/v). In this assay, the fluorescence signal of LRT was increased 14-fold prior to detection by post-column exposure to UV light (254 nm) in a photochemical reaction unit. The LLQ of this assay was 0.500 ng/ml (150-microl sample) and the within-run and between-run precisions were10%.

A new HPLC method has been developed for the quantitative determination of methotrexate (MTX) and its 7-hydroxyl metabolite in human plasma. Samples were purified by protein precipitation with acetone and methanol, and a sample clean-up with a mixture of n-butanol and diethyl ether. The analytes were separated on an RP Inertsil ODS-80A column and eluted in a solvent system containing 5% (v/v) tetrahydrofuran in water (pH 2.0). UV absorption measurement was performed at 313 nm, and the detector response was linear in a concentration range of 10-10,000 ng/ml. The lower limit of quantitation of MTX was 10 ng/ml using 1 ml sample aliquots. Values for accuracy and (within-run and between-run) precision were between 95.5-111% and 3.69-11.0%, respectively, at four concentrations analyzed in quintuplicate on four separate occasions. The assay was applied to study the effects of docetaxel co-administration on the pharmacokinetics and metabolism of MTX in cancer patients.

The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C --T and C --S), and (3) TPT or SN-38 followed by cisplatin (T --C and S --C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C --T (or C --S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROV(CDDP) was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROV(CDDP) lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G2/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors.

We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3+, 4-, 8+, 16-, 56-. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3+, 4-, 8+) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov- 1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation.

A retrospective study on the incidence of catheter-related complications and catheter indwelling time (tCI) during treatment with continuous interleukin-2 (IL-2) infusion in patients with metastatic renal cell cancer, who were equipped with tunnelled central venous catheters (CVC). A group of 72 patients were treated with IL-2-based immunotherapy. Two induction treatment cycles of 35 days each were used. Treatment consisted of IL-2 as a continuous intravenous infusion (c.i.v.) with lymphokine-activated killer cells and interferon alpha intramuscularly. A tunnelled CVC was inserted at the start of treatment and was kept in place for the duration of the therapy or until the occurrence of complications. Out of 72 CVC, 30 (42%) functioned uneventfully for a median tCI of 64 days. In another 12 clinically uncomplicated cases (16%), catheter tips were positive in routine culture after a median tCI of 33 days. In 18 patients (25%), CVC-related infections were noted, including 8 (11%) local tunnel infections and 10 (14%) septic episodes. These complications occurred at a median tCI of 28 and 20 days respectively. In 15 (83%) of these 18 catheter infections, Staphylococcus aureus was isolated, whereas in the remaining 3 (17%) Staphylococcus epidermidis was found. Subclavian vein thrombosis was noted in 12 (17%) CVC at a median tCI of 31 days: 5 (36%) of these were diagnosed in the first 14 patients. This prompted us to administer prophylactic heparin 15,000 i.u. c.i.v. daily during IL-2 treatment. Thereafter the incidence of thrombosis dropped to 7 (12%) in the subsequent 58 CVC inserted (P = 0.03). In conclusion, in contrast to previous reports on the high incidence of CVC-related septicaemia and thrombosis, we observed a relatively low incidence of these complications, which we ascribe to the use of tunnelled catheters and prophylactic heparin.

Seventy-two patients with metastatic renal cell cancer were treated with the combination of high-dose interleukin-2 (IL2), interferon-alpha (IFN alpha), and lymphokine-activated killer cells (LAK). Seventeen patients were entered in a feasibility part of the study (protocol 1) and 55 in an efficacy part (protocol 2). Protocol 2 differed from protocol 1 in the addition of IFN alpha to the first 5 days of IL2 infusion. Each patient was planned to receive two induction cycles. IL2, 18 MIU/m2/day, was administered continuously i.v. on days 1-5, and IFN alpha, 5 MIU/m2/day (protocol 2), was administered i.m. on days 1-5, followed by three daily lymphaphereses on days 7-9. On day 12, treatment was resumed with IL2 and IFN alpha on days 12-15 and LAK reinfusions on days 12-14. In protocol 1, three complete (CR) and one partial (PR) responses were achieved (response rate 24%). The median duration of response and the median survival were 18.1 and 13.9 months, respectively. The 3-year survival was 35%. Of the 51 evaluable patients in protocol 2, 6 achieved a CR and 13 a PR (response rate 37%). The median duration of response was 11.1 months. The median survival was 16.9 months. The 3-year survival was 35%. There were three treatment-related deaths. Other severe toxicities included hypotension, cardiotoxicity, pulmonary edema, renal toxicity, and infectious complications. In the two induction cycles, only 54 and 42% of the planned doses could be administered. We conclude that the use of high-dose regimens of IL2 and IFN alpha is not warranted, unless we can define more accurately which patients may experience long-term survival as a result of treatment.

Imatinib mesylate (STI571), a potent tyrosine kinase inhibitor, is successfully used in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. However, the intended chronic oral administration of imatinib may lead to development of cellular resistance and subsequent treatment failure. Indeed, several molecular mechanisms leading to imatinib resistance have already been reported, including overexpression of the MDR1/ABCB1 drug pump. We examined whether imatinib is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump that is frequently overexpressed in human tumors. Using a panel of well-defined BCRP-overexpressing cell lines, we provide the first evidence that imatinib is a substrate for BCRP, that it competes with mitoxantrone for drug export, and that BCRP-mediated efflux can be reversed by the fumitremorgin C analog Ko-143. Since BCRP is highly expressed in the gastrointestinal tract, BCRP might not only play a role in cellular resistance of tumor cells but also influence the gastrointestinal absorption of imatinib.

Docetaxel is a new semi-synthetic taxoid, which acts as an antimicrotubule agent and is now clinically available. Clinical studies of docetaxel as a single agent and in combination with other cytotoxic drugs reveals favourable response rates in a number of solid tumor types. Docetaxel appears to have great potential in advanced breast cancer as first or secondline chemotherapy. The dose-limiting toxicity is an early and short-lasting neutropenia. Fluid retention is a cumbersome, sometimes disabling, side-effect. Other side-effects are usually mild and include alopecia, myalgia, mucostitis, neuropathy, hypersensitivity reaction, nail changes and cutaneous reactions.

The aims of the trial were to establish the response rate and determine the toxicity of combination chemotherapy with ifosphamide, vincristine and cisplatin (HOP regimen) in advanced metastatic seminoma and to study the role of post-chemotherapy consolidation treatment. Patients with bulky metastatic non-alpha-fetoprotein-producing seminomas were eligible for this phase II study [serum human chorionic gonadotropin or = 10 cm or had extra-gonadal seminoma or had relapsed after previous radiotherapy. The HOP regimen consisted of four 3-weekly cycles of the following drug combination: ifosphamide (days 1-5, 1.2 mg m-2 day-1), vincristine (day 1, 2 mg) and cisplatin (days 1-5, 20 mg m-2 day-1). Residual masses persisting 6 months after chemotherapy could be considered for consolidation surgery or radiotherapy. Maximal response to the HOP chemotherapy (evaluated at any time) was based on the WHO criteria. The median observation time was 2.5 years (range 1.8-5.5 years). Thirteen institutions treated 42 eligible patients within the study (testicular cancer stage > or = IID, 25; extragonadal, 5; relapse after previous radiotherapy, 12). Two patients were not evaluable for response owing to premature treatment discontinuation. Maximal response was as follows: complete remission (CR), 26 (65%); partial remission (PR) 11 (28%); no change (NC), 2 (5%); progressive disease (PD), 1 (3%). Four patients have died, three from their malignancy (two without previous irradiation and one with prior radiotherapy). The fourth patient died of treatment-related toxicity. The 3 year survival for all 42 eligible patients was 90%. Dose reduction and treatment postponement were necessary in 25 and 14 patients respectively. Ten patients experienced granulocytic fever. Previously irradiated patients tolerated chemotherapy as well as non-irradiated patients. Immediately after HOP chemotherapy a mass persisted in 16 of 17 patients with retroperitoneal masses of > or = 100 mm at presentation. Three of these residual lesions were resected within the following 6 months showing complete necrosis. Four lesions dissolved spontaneously during the first year of follow-up. Nine lesions persisted for > or = 1 year (one after consolidation radiotherapy) without leading to relapse. Four of seven patients with mediastinal lesions achieved CR and three a PR after HOP chemotherapy. The HOP chemotherapy regimen is highly effective in patients with advanced metastatic seminoma or those relapsing after previous radiotherapy, but is associated with a high risk of toxicity, in particular myelotoxicity.

Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis. Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2. Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis. Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo. Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar. Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis. In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation. Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar. No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity. In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment.

Univariate and multivariate analyses were performed in 632 patients treated with cisplatin combination chemotherapy for metastatic non-seminomatous testicular germ cell tumours. Multivariately, the most important poor prognosis factors for the prediction of survival were: HCG greater-than-or-equal-to 10,000 IU/l, lung metastases greater-than-or-equal-to 10, abdominal metastases with a horizontal diameter of greater-than-or-equal-to 5 cm and the presence of supraclavicular metastases. If patients had 2 or more of these factors the death rates increased from 30% for patients with 2 factors to 65% for patients with 3 or 4 factors. The patients studied here are not a random sample of the metastatic testicular cancer patients in general, as the population studied comprised a large proportion of low volume metastatic disease patients. Therefore, the final analysis will include patients from all the recently completed trials.

Twenty-five assessable patients with metastatic melanoma have been entered in a multicenter phase II study of two induction cycles of human recombinant interleukin-2(IL2), 18 x 10(6) IU/m2/d continuous intravenous (IV) infusion on days 1 to 5 and days 12 to 17. Dacarbazine (DTIC), 850 mg/m2 IV bolus was given on day 26. The cycle was repeated at 5 weeks. Maintenance therapy was scheduled 3 weeks after the completion of induction treatment, consisting of IL2, 18 x 10(6) IU/m2/d for 5 days alternating with DTIC, 850 mg/m2 IV every 3 weeks, for a total of 18 weeks. Six patients responded (24%); two complete and four partial. Stable disease was seen in five patients. None of the six patients with more than two sites of metastases responded. Maximum response was observed in the first 3 months of treatment. Progression-free periods of 6 months and longer were seen in the two complete responders (8 and 17+ months), in two of the four partial responders (7 and 12+ months), and in three of the five patients with stable disease (9+, 15, and 17+ months). Toxicity included fever, skin rash, fatigue, anorexia, and diarrhea in most patients. Two patients had a weight gain of more than 10%. Eight patients needed intensive care for the observation and treatment of a myocardial injury (one patient), ventricular tachycardia (one), hypotension and oliguria (four), and sepsis (two). Sequential treatment with IL2 and DTIC appears to be effective but not clearly better than could be expected of IL2 alone.

Three murine tumor lines, B16 melanoma, C38 colon cancer and M2661 breast cancer, were used to evaluate the therapeutic efficacy of the drug combination ACNU and ifosfamide. For each tumor type five treatment groups of 12 mice each were studied, which respectively received 16.5 mg/kg ACNU, 150 mg/kg ifosfamide, 33 mg/kg ACNU, 300 mg/kg ifosfamide or 16.5 mg/kg ACNU + 150 mg/kg ifosfamide. One group served as controls. Growth delay was measured as the endpoint. In the B16 and C38 tumor lines both drugs were active and showed additive antitumor effects. However, no synergism was observed. Neither ACNU or ifosfamide or the combination of both had any activity against the M2661 tumor line.

Summary We initiated a phase II study with combination chemotheraphy consisting of cisplatin, etoposide and bleomycin in a subset of patients with carcinomas of unknown primary site characterized by the presence of at least one of the following criteria: 1) age below 50 years; 2) clinical evidence of rapid tumor growth; 3) tumour located predominantly in a mid-line distribution; 4) good response to previous administered radiotheraphy. In 34 evaluable patients an objective response rate of 53% (95% confidence limits 35%–70%) was achieved. For patients with poorly differentiated adenocarcinomas the response rate was 35%, and, in most instances, of short duration. A response rate of 79% including complete responses and long-term survivals was achieved in patients with undifferentiated carcinomas. This difference in response rate was statistically significant (p = 0.02). No supplementary prognostic factors predicting response to chemotherapy could be identified. One patient with an initial diagnosis of undifferentiated carcinoma proved to have a malignant lymphoma after additional immunohistochemical investigation. Untila better characterization of this syndrome is possible patients with undifferentiated carcinomas of unknown primary site should be challenged with cisplatin-based chemotherapy.

Background: Interferon-α (IFN-α) treatment is often associated with psychiatric side effects and has been found to lower the amount of tryptophan (TRP) available to the brain. The alterations in tryptophan metabolism might underlie the psychiatric side effects during treatment with IFN-α. Methods: In this study, 43 oncology patients treated with IFN-α were included. In order to study de novo depressions, depressed patients at baseline were excluded. Psychiatric evaluation comprising clinical judgment combined with a structured psychiatric interview and observer-based and self-report rating scales was performed at baseline and at 4 weeks, 8 weeks and 6 months after the start of treatment with IFN-α, and in the case of emerging psychopathology. Blood samples were drawn at the same evaluation times and assessed for concentrations of TRP, large neutral amino acids, kynurenine, 5-hydroxyindole acetic acid, neopterin and biopterin. Results: During treatment with IFN-α, several alterations in laboratory parameters occurred that were consistent with an increased degradation of peripheral TRP. Psychometric ratings revealed hardly any psychiatric changes. No consistent associations were found between changes in the laboratory assessments determined and the diverse psychiatric measures. Conclusion: In this study, IFN-α was found to alter TRP metabolism without inducing psychiatric side effects. Therefore, a possible relationship between TRP metabolism and depression was not substantiated by this study.

The effectiveness of platinum drugs in the treatment of cancer is hindered by intrinsic and acquired resistance. The cause of clinical resistance to platinum compounds is still unknown. In an attempt to identify new cellular mechanisms of cisplatin resistance, a one-step cisplatin-selection procedure was used to generate resistant sublines of the platinum sensitive A2780 ovarian cancer cell line. In the present study we selected an A2780 subline, A2780-Pt, that has a significantly reduced ability to accumulate cisplatin (36% of the parent A2780 cell line) and consequently shows a clear cisplatin-resistant phenotype (resistance factor, i.e., RF: 8.6). The A2780-Pt cell line was specifically cross-resistant to carboplatin (RF: 12.0), tetraplatin (RF: 8.1) and oxaliplatin (RF: 6.1) which was associated with a reduced cellular platinum accumulation (50%, 54% and 58% of A2780, respectively). No cross-resistance was found for a variety of other anticancer agents. Further experiments to determine the cause of the platinum resistance of the A2780-Pt cell line revealed that: (1) impaired cellular platinum accumulation could not be attributed to aberrant expression of MRP2 (ABCC2), CTR1 (SLC31A1), ATP7A or ATP7B, (2) resistance was not associated with platinum inactivation by metallothionein and glutathione, (3) the platinum efflux rate was similar to that of A2780, (4) the defect in cellular accumulation and the resistance could be overcome by treatment with cisplatin nanocapsules, consistent with impaired influx, and (5) the defect in accumulation is specific for platinum compounds in the cis-configuration, since A2780-Pt cells did not show reduced accumulation of transplatin. This specificity suggests that not passive diffusion but an inward transporter is impaired in A2780-Pt. In conclusion, we generated an A2780 subline that showed a uniquely stable platinum resistance phenotype, which could theoretically be caused by an impaired inward transporter specific for cis-configurated platinum compounds.

The aim of this study was to evaluate the effect of pegylated interferon-alpha (PEG-IFN-alpha) on the plasma citrulline/arginine ratio, regarded as an index of nitric oxide (NO) synthesis, in patients with high-risk melanoma. Forty patients were randomly assigned to either PEG-IFN-alpha treatment (n = 22) or to observation only (control group, n = 18). The treatment group received 6 microg PEG-IFN-alpha/kg once a week during 8 weeks, followed by a maintenance dose of 3 microg/kg/wk. Blood was collected at different time points, plasma concentrations of citrulline and arginine were measured and the ratio of citrulline/arginine was calculated. Patients treated with PEG-IFN-alpha showed a significant decrease in the concentrations of citrulline and in the citrulline/arginine ratio during the whole study period, both compared to baseline values and to the control group. The data suggest that therapy with PEG-IFN-alpha results in a marked decrease in the synthesis of NO in melanoma patients.

The introduction of paclitaxel/platinum combination chemotherapy and (interval) debulking surgery has significantly improved the prognosis of patients with ovarian cancer. Yet, many patients die of drug-resistant disease. Second-line chemotherapy may result in prolonged secondary remissions with alleviation of symptoms and improvement of quality of life. The response to second-line chemotherapy is strongly related to platinum sensitivity. More than 60% of platinum-sensitive patients respond to a re-challenge with platinum-containing chemotherapy. In platinum-resistant patients, on the contrary, the response rate to a re-challenge with 3-weekly platinum or any nonplatinum chemotherapy is less than 20%. The response to dose-dense weekly platinum-based regimens ranged from 48% to 64% in platinum-resistant patients. Moreover, the majority of the patients responded within 8 weeks after the start of the treatment. The progression-free survival ranged from a median of 5 months in a study using cisplatin/etoposide, to 11 months in a study with paclitaxel/carboplatin. The median survival was 11-15 months. The outpatient weekly paclitaxel/carboplatin regimen, with paclitaxel at a dose of 90 mg/m(2) and carboplatin at area under the curve 4, seems similarly effective and is better tolerated. Dose-dense weekly paclitaxel/carboplatin is an effective and well-tolerated therapy for platinum-sensitive, as well as platinum-resistant tumors. Responses to therapy are observed within 8 weeks in the majority of the patients. Whether a weekly regimen indeed is more effective than 3-weekly paclitaxel/carboplatin needs to be answered in a randomized study.

The purpose of this study was to determine the mechanism of the pharmacodynamic interaction between docetaxel/paclitaxel and cisplatin. Cisplatin- induced DNA-adducts and cisplatin accumulation were quantitated in peripheral blood leukocytes (WBC). The WBC were obtained from patients treated with docetaxel or paclitaxel in phase I/II studies and were incubated in vitro with cisplatin. In addition, blank whole-blood samples were obtained from pa- tients and healthy subjects and incubated in vitro with cisplatin or docetaxel/paclitaxel and cisplatin. The cis- platin-induced DNA-adduct levels measured in WBC after treatment with docetaxel or paclitaxel were signif- icantly lower than those determined in non-pretreated WBC. Docetaxel and paclitaxel reduced the intracellu- lar accumulation of cisplatin in WBC by 46—47%. If the pharmacodynamic interaction between docetaxel/pac- litaxel and cisplatin also occurs in other normal tissues such as bone marrow, it may well contribute to the sequence dependent toxicity that has been observed in clinical studies.

A prospective study was performed to investigate the combination of the aromatase inhibitor aminoglutethimide and hydrocortisone in androgen-independent prostate cancer with changes in prostate-specific antigen (PSA) level as main determinant for response. Thirty-five patients were treated with aminoglutethimide 1000 mg daily and hydrocortisone acetate 40 mg daily. PSA measurements were performed every month. If evaluable lesions were present, objective tumor assessment was done by computed tomography scan and X-ray investigations. In 12 patients (37%) the PSA value showed a confirmed response with a decline in serum level of at least 50%. Median time to progression in responding and all patients was 10.5 and 4.5 months, respectively. Median duration of response in responding patients was 9 months. Median survival for these two groups was 23 and 14.5 months, respectively. Of seven patients with measurable disease, two showed a partial response and five a stable disease. Improvement in general condition, pain and feeling of well-being was noted in two-thirds of patients. Therapy was well tolerated with mainly grade I and II adverse events in 20% of patients. We conclude that aminoglutethimide is a valuable second-line therapy for patients with androgen-independent prostate cancer.

Our objective was to study the influence of pegylated interferon-alpha2b (PEG-IFN-alpha) on the metabolism of amino acids and pteridines. We used an exploratory study into plasma concentrations of large neutral amino acids, 5-hydroxyindolacetic acid (5-HIAA), total biopterin (BIOP) and neopterin (NEOP) in 40 high-risk melanoma patients. Patients were randomized to treatment with PEG-IFN-alpha once a week in a dose of 6 microg/kg/week s.c. during 8 weeks, followed by a maintenance treatment of 3 microg/kg/week s.c. or to observation only. We found that treatment with PEG-IFN-alpha decreases tryptophan (TRP) concentrations in the first 3 months of treatment to a maximum of 25.3% compared to controls [95% confidence interval (CI): 14.9 to 34.4]. The TRP:LNAA ratio, an index for the availability of TRP to the central nervous system (CNS), decreases during 6 months with 18.8% (95% CI: 11.9 to 25.2). Concentrations of NEOP rose; however, concentrations of BIOP, the sum of tetrahydrobiopterin [BH4] and its oxidative products, did not decrease. The ratio of phenylalanine to tyrosine was increased with 11.7% (95% CI: 1.0 to 23.5) during 6 months. We conclude that, like conventional IFN-alpha, PEG-IFN-alpha lowers TRP concentrations and decreases the availability of TRP to the CNS. PEG-IFN-alpha has a similar influence on pteridine metabolism as standard IFN-alpha. If a lowered availability of TRP and a consequent decrease of serotonergic neurotransmission are indeed a mechanism underlying neuropsychiatric side-effects of IFN-alpha, patients on PEG-IFN-alpha are not at a lower risk of developing neuropsychiatric side-effects as patients on conventional IFN-alpha.

The therapeutic potential of antitumor drugs is seriously limited by the manifestation of cellular drug resistance. We used the budding yeast Saccharomyces cerevisiae as a model system to identify novel mechanisms of resistance to one of the most active anticancer agents, cisplatin. We pinpointed NPR2 (nitrogen permease regulator 2) as a gene whose disruption conferred resistance to cisplatin. In addition, we observed a 4-fold cross-resistance of yeast npr2Delta cells (i.e., cells from which the NPR2 gene had been disrupted) to the anticancer drug doxorubicin, in combination with hypersensitivity to cadmium chloride. Furthermore, npr2Delta cells displayed unaltered cellular cisplatin and doxorubicin accumulation and showed an enhanced rate of spontaneous mutation compared with the isogenic parent. These data indicate that the npr2Delta phenotype overlaps that of the sky1Delta cells that we characterized previously (Mol Pharmacol 61:659-666, 2002). Therefore, we generated yeast npr2Delta sky1Delta double-knockout cells and performed clonogenic survival assays for cisplatin and doxorubicin, which revealed that NPR2 and SKY1 (SR-protein-specific kinase from budding yeast) are epistatic. The double-knockout strain was just as resistant to cisplatin and doxorubicin as the single-knockout strain that was most resistant to either drug. In conclusion, we identified NPR2 as a novel component involved in cell kill provoked by cisplatin and doxorubicin, and our data support the hypothesis that NPR2 and SKY1 may use mutual regulatory routes to mediate the cytotoxicity of these anticancer drugs.

Previously, a gender dependency of topotecan was found in the pharmacokinetics in the plasma compartment. Here, we prospectively studied the red blood cell (RBC) partitioning of topotecan and evaluated its consequences for overall drug disposition. Blood samples were obtained from 12 patients receiving cisplatin followed by i.v. topotecan. Topotecan pharmacokinetic analysis was performed in whole blood, plasma and RBCs. Significantly slower clearance was noted in females (n=7) compared to males (n=5) for lactone and total topotecan in plasma (p

Piritrexim is a lipid-soluble inhibitor of dihydrofolate reductase (DHFR) that enters tumour cells rapidly by passive diffusion, cannot be polyglutamated, and is as effective as methotrexate in inhibiting DHFR. Bioavailability after oral dosing is approximately 75%. We performed a phase II study with oral piritrexim in non-chemotherapy pretreated patients with metastatic urothelial cancer. Thirty-three patients were treated with 25 mg three times daily for 5 consecutive days, repeated weekly, with provision for dose escalation or reduction according to the toxicity observed. Of 29 evaluable patients, one patient achieved a complete response of 19+ weeks duration, and ten patients achieved a partial response with a median duration of 22 weeks (range 16-48), for a total response rate of 38%. Piritrexim was generally well tolerated, with myelosuppression as the major toxicity, that frequently required dose modification. We conclude that piritrexim appears to be an active agent in patients with metastatic urothelial cancer when administered as a 5-day, low-dose oral schedule. It would be attractive to investigate the combination of piritrexim and cisplatin.

The ether lipid miltefosine (hexadecylphosphocholine) was orally given to patients with various tumours in a dose-finding study. All patients initially received a daily total dose of 100 mg, which in the absence of side-effects was increased to 150 mg and further to 200 mg. A total of 54 patients were entered and were evaluable for gastrointestinal toxicity. Nausea and vomiting were found to be dose-limiting; 22% of patients ultimately tolerated a dose of 100 mg, 59% tolerated a dose of 150 mg and 19% tolerated a dose of 200 mg. In addition 30% of patients developed renal dysfunction, which was thought to be related to the drug. No other toxicities were observed. For further phase II studies it is recommended that one starts with a dose of 150 mg daily, divided over three administrations.

47 patients with advanced urothelial cancer and no prior chemotherapy were randomly assigned to therapy with either carboplatin or iproplatin. Both platinum analogues were administered intravenously every 28 days at doses of 400 mg/m 2 carboplatin and 300 mg/m 2 iproplatin. None of 14 evaluable patients treated with carboplatin responded. Therefore, this arm was closed and from then on all eligible patients were registered on the iproplatin arm. 5 of 29 evaluable patients treated with iproplatin achieved a partial response (17%) for a median duration of 27 weeks (range 22–37). Iproplatin did not induce renal function disturbance. Gastrointestinal toxicity was mild to moderate. Bone marrow toxicity predominantly consisted of thrombocytopenia and required platelet transfusions in 13% of patients. 2 patients developed hypersensitivity reactions. It is concluded that the bone marrow toxicity and the chance of hypersensitivity render iproplatin an unattractive alternative to cisplatin.

A high-performance liquid chromatographic assay with fluorescence detection has been developed for the determination of doxorubicin and its metabolite doxorubicinol in plasma of cancer patients. Quantitative extraction was achieved by a single protein-precipitation step of 1-ml samples with 500 microl of acetone in the presence of 100 microl of zinc sulfate [70% (w/v) in water]. Doxorubicin and doxorubicinol were separated isocratically on a column packed with Inertsil ODS-80A material and a mobile phase composed of water:acetonitrile:tetrahydrofuran (76:24:0.5, v/v/v). The related compound daunorubicin was used as internal standard. The column effluent was monitored fluorimetrically at an excitation wavelength of 480 nm and an emission wavelength of 560 nm, with a band width of 40 nm. The calibration graphs of doxorubicin and doxorubicinol were linear over a range of 1.0 to 100 and 0.50 to 50. 0 ng/mL, respectively, with lower limits of quantitation of 1.0 and 0.50 ng/ml. Results obtained from a 4-day validation study demonstrated excellent accuracy (91.0-106%) and precision (0.90-10. 2%) across the calibration ranges for both compounds. The developed method has been applied extensively to a clinical study to examine the pharmacokinetics and metabolism of doxorubicin in patients cotreated with a potent inhibitor of MDR1 P-glycoprotein activity, GF120918.

The induction of apoptosis by diverse apoptotic stimuli was studied in a panel of 6 testicular germ cell tumour(TGCT) cell lines with defined p53 status. Although the sensitivity to a particular stimulus varied considerably among the TGCT cell lines, the differences in response were not associated with the presence of functional p53. Mutant (mt) p53-expressing NCCIT and S2 (no p53 protein) were both readily triggered into apoptosis by cisplatin and doxorubicin, while wild-type(wt)-p53-transactivation-competent 2102 EP cells failed to undergo drug-induced apoptosis. Moreover, transactivation-deficient NCCIT cells and wtp53-expressing NT2 cells were equally sensitive to cisplatin, doxorubicin, gamma radiation, and cell-permeable C2-ceramide. Our p53 data suggest that, at least in this panel of non-isogeneic TGCT cell lines, hypersensitivity to therapeutic agents is not associated with p53 status. Next, we examined the impact of p53 inactivation on apoptosis induction in isogeneic NT2 sublines expressing human papillomavirus E6 protein. Evidently, abrogation of p53 function did not affect the hypersensitivity to apoptotic stimuli. We noted that drug-sensitive S2 cells were highly resistant to radiation-induced apoptosis, indicating distinct signalling pathways for chemotherapy and irradiation. The impaired radiation-induced apoptotic pathway in S2 and 2102 EP could not be restored by addition of cell-permeable C2-ceramide, suggesting that the blockade is downstream of ceramide generation. Ligation of Fas/APO-1/CD95 by anti-Fas effectively induced apoptosis in Fas-antigen expressing S2, 2102 EP and 833 KE. The efficient Fas-mediated activation of apoptosis in drug-, radiation-, and ceramide-resistant 2102 EP cells further suggests that diverse apoptosis-inducing factors may use distinct signalling pathways. In summary, we demonstrated the presence of distinct p53-independent apoptotic pathways in TGCT cells. Int. J. Cancer 81:620–628, 1999. © 1999 Wiley-Liss, Inc.

Testicular cancer is the most frequent malignant disease in men aged 15–40 years. Due to its sensitivity to chemotherapeutic drugs, most patients, including those with widespread metastatic disease, can now be cured. Bleomycin is an essential component of the most effective chemotherapy regimen for testicular cancer—ie, bleomycin, etoposide, and cisplatin [BEP].1 However, bleomycin is feared for its induction of bleomycin-induced pneumonitis (BIP), which is sometimes fatal.2 After reports in the 1980s of perioperative complications that were ascribed to bleomycin, high inspired-oxygen fractions during anaesthesia were avoided, as were high inspired-oxygen fractions under hyperbaric circumstances, such as scuba diving.

Docetaxel is a new antimicrotubule agent that induces a predominantly sensory neuropathy that is mild in most patients. This prospective study was performed to determine if corticosteroid co-medication reduces the incidence and severity of docetaxel-induced neuropathy. Two groups of patients treated with docetaxel in subsequent cohorts were prospectively analyzed for neurotoxicity. Group A consisted of 38 patients with a variety of solid tumors, who were treated in studies before corticosteroid co-medication was recommended, while 49 female patients in group B with metastatic breast cancer were treated after co-medication with corticosteroids was introduced as a routine. Neuropathy was evaluated by a clinical sum-score for symptoms and signs, and by measurement of the vibration perception threshold (VPT). The severity of neuropathy was graded according to NCI Common Toxicity Criteria. In 42% of patients of group A and in 65% of patients of group B a mainly mild neuropathy was documented. There was no statistically significant difference in neurotoxicity between group A and B. The cumulative dose of docetaxel showed a significant correlation with post-treatment scores of VPT, sensory sum-score, grade of paresthesias, and grade of neurosensory and neuromotor toxicity. Corticosteroid co-medication does not reduce the development of docetaxel-related neuropathy.

We investigated the efficacy and toxicity of induction chemotherapy with cisplatin and etoposide with either bleomycin or ifosfamide in patients with intermediate-prognosis testicular non-seminoma. A total of 84 eligible patients were randomized to receive four cycles of etoposide, ifosfamide, cisplatin (VIP), or four cycles of bleomycin, etoposide, cisplatin (BEP). Intermediate prognosis was defined as any of the following: lymph node metastases 5-10 cm in diameter, lung metastases more than four in number or > 3 cm, HCG 5000-50 000 IU l(-1), AFP > 1000 IU l(-1). The complete response (CR) rates to VIP and BEP were similar, 74% and 79% respectively (P = 0.62). Including the cases in whom viable cancer was completely resected with post-chemotherapy debulking surgery, the percentages of patients who achieved a no-evidence-of-disease status were 80% on VIP and 82% on BEP (P = 0.99). In addition, there were no differences in relapse rate, disease-free and overall survival after a median follow-up of 7.7 years. The 5-year progression-free survival was 85% (95% CI 74-96%) in the VIP arm and 83% (95% CI 71-96%) in the BEP arm, hazard ratio (VIP/BEP) 0.83 (95% CI 0.30-2.28). The VIP regimen was more toxic with regard to bone marrow function; the frequency of leucocytes below 2000 mu l(-1) throughout four cycles was 89% on VIP and 37% on BEP (P

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid-liquid extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water-methanol-tetrahydrofuran-ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10-500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6+/-8.52 and 93.7+/-5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.

DNA adducts formed by cisplatin [cis-diamminedichloroplatinum(II)] were measured in blood samples from 48 testicular cancer patients treated in four centers in Europe during four six cycles with cisplatin infusions on five successive days (total samples, 112). Total protein-bound platinum (Pt) in blood was also measured (total samples, 84). The mean on main DNA adduct, cis-Pt(NH3)(2) d(pGpG) (Pt-GG), was 0.75 fmol/mu g DNA [standard deviation (SD) = 0.66] on the first day of the first cycle, increased after the infusion at day 5 of the cycle (mean 1.74 fmol/mu g DNA, SD = 0.90) and decreased on the following day (mean 1.09 fmol/mu g DNA, SD = 0.62). In subsequent cycles, there was a tendency to an increase in the mean Pt-GG levels. The values of protein-bound Pt in blood showed little reduction between day 5 and 6 of each cycle, and a stable increase during the course of the therapy. Strong correlations were seen between day 1, 5 and 6 of the first cycle for both Pt-GG and protein-bound Pt in blood. A strong correlation (r = 0.62, p

We investigated the role of p53 and of the Bcl-2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin-induced apoptosis. In contrast to wild-type p53-containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with non-functional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells failed to undergo cisplatin-induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4-fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53-independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in drug-induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin-induced apoptotic pathway in TGCT cell lines might be p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat.

PURPOSE This prospective randomized trial was designed to compare the efficacy of etoposide plus cisplatin (EP) versus bleomycin, etoposide, and cisplatin (BEP) chemotherapy in patients with good-prognosis metastatic nonseminomatous testicular cancer. PATIENTS AND METHODS Four hundred nineteen patients with good-prognosis nonseminomatous testicular cancer were randomized to receive four cycles of cisplatin 20 mg/m2 on days 1 to 5 plus etoposide 120 mg/m2 on days 1, 3, and 5 with or without bleomycin 30 mg weekly. RESULTS Of 395 eligible patients, 169 of 195 patients allocated to EP (87%) and 189 of 200 patients allocated to BEP (95%) achieved a complete response with chemotherapy alone or after postchemotherapy surgery. These results are significantly different (P = .0075). After a median follow-up duration of 7.3 years, eight patients (4%) on each treatment arm relapsed. In view of the low number of unfavorable treatment outcomes (11%), no significant differences were detected in time to progression (P = .136) and survival (P = .262). Both the acute and late pulmonary toxicity and neurotoxicity were significantly greater in patients who received BEP, whereas Raynaud's phenomenon occurred exclusively in patients who received BEP (P < .001). Two patients treated with BEP died of bleomycin pulmonary toxicity. CONCLUSION BEP is the most effective combination regimen in the treatment of disseminated nonseminomatous germ cell cancer. In this particular BEP regimen with etoposide at a dose of 360 mg/m2 per cycle, even in good-prognosis patients, bleomycin cannot be deleted without compromising treatment efficacy, but its use is associated with more toxicity (particularly pulmonary) and efforts to reduce this merit further exploration.

We investigated the prognostic value of the serum half-life of human chorionic gonadotrophin (HCG) and alpha-fetoprotein (AFP) during induction chemotherapy and the relative prognostic importance of initial marker concentrations and marker half-life. Marker half-lives were calculated using two abnormal values observed between day 8 and day 22 of the first chemotherapy cycle, Moreover, analyses were carried out using day 43 as the second measurement point. Treatment failure at any time was chosen as the end point. The relative prognostic influence of marker half-lives and initial marker concentrations was tested in univariate and multivariate analyses. Half-lives were considered to be prolonged ii > 3 days for HCG and > 6 days for AFP. In addition, we separated patients into those with half-lives > 6 days for HCG and those with half-lives > 10 days for AFP to examine whether these long half-lives were associated with a poor prognosis. A group of 669 patients treated with cisplatin combination chemotherapy was studied. Forty-two per cent of the patients had normal HCG and 37% had normal AFP at the start of chemotherapy. At day 22, HCG was still elevated in 138 patients and AFP in 211, At day 43, the numbers of these patients were 35 and 80 respectively. Based on the measurements obtained on day 8 and day 22, a half-life of HCG > 3 days or > 6 days and/or a half-life AFP > 6 days or > 10 days did not accurately predict treatment failure (P=0.413 and P=0.851, respectively; values obtained using tests for trend). However, initial marker concentrations of HCG and/or AFP greater than or equal to 1000 IU l(-1) were highly significant prognosticators for treatment failure (P=0.001 and P

Sensitive high-performance liquid chromatographic assays have been developed to determine the levels of the lactone and lactone plus carboxylate (total) forms of the antitumor agent irinotecan (CPT-11) and its active metabolite SN-38, in human plasma. The related compound camptothecin was used as the internal standard. The selective sample pretreatment for the lactone forms involved a single solvent extraction with acetonitrile-n-butyl chloride (1:4, v/v), whereas the sample clean-up for the total forms was a simple protein precipitation with aqueous perchloric acid-methanol (1:1, v/v), which results in the conversion of the carboxylate to the lactone forms. Chromatography was carried out on a Hypersil ODS column, with detection performed fluorimetrically. The methods have been validated, and stability tests under various conditions have been performed. The lower limits of quantitation are 0.5 and 2.0 ng/ml for the lactone and total forms, respectively. The assays have been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.

Article abstract-Docetaxel, a new semisynthetic taxoid used as an antineoplastic agent, induced a predominantly sensory neuropathy in 20 of 41 patients. We assessed neurotoxicity in all patients participating in four phase II trials conducted in our institution. The neuropathy was evaluated by a clinical sum-score for symptoms and signs and by measurement of the Vibration Perception Threshold (VPT). The severity of neuropathy was graded according to the National Cancer Institute's Common Toxicity Criteria. Neuropathic symptoms were mild in most patients. However, at cumulative doses above 600 mg/m2, 3 of 15 patients developed a moderate and 1 of 15 patients a severe neuropathy. There was a significant correlation between the cumulative dose of docetaxel and the post-treatment sum-score (p = 0.002). We found no correlation between post-treatment VPT and clinical sum-score or between post-treatment VPT and the cumulative dose of docetaxel. We conclude that docetaxel produces a mild and predominantly sensory neuropathy in a high proportion of treated patients. This neurotoxicity appeared to be dose dependent and may be severe and disabling at higher dose levels. Determination of the VPT is not a reliable method to monitor docetaxel-induced neuropathy.NEUROLOGY 1996;46: 104-108

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.

We have previously reported the results of two multicenter European studies, investigating the efficacy and toxicity of continuous infusion Interleukin-2 (IL-2) alone (1) and IL-2 with LAK cells (2). The therapeutic outcome of these studies was in accordance with the results published in the world literature (3). This means that the response rates were between 15 and 25% and that the overall median survival of 8 months did not appear different from that observed in non-selected historical and IL-2 treated controls (4).

We have investigated whether an alternating induction chemotherapy regimen of PVB/BEP is superior to BEP in patients with poor-prognosis testicular non-seminoma. A total of 234 eligible patients were randomised to receive an alternating schedule of PVB/BEP for a total of four cycles or four cycles of BEP. Poor prognosis was defined as any of the following: lymph node metastases larger than 5 cm, lung metastases more than four in number or larger than 2 cm, haematogenic spread outside the lungs, such as in liver and bone, human chorionic gonadotrophin > 10,000 IU l-1 or alphafetoprotein > 1000 IU l-1. The complete response (CR) rates to PVB/BEP and BEP were similar, 76% and 72% respectively (P = 0.58). In addition, there was no significant difference in relapse rate, disease-free and overall survival at an average follow-up of 6 years. The 5-year progression-free and survival rates in both treatment groups were approximately 80%. The PVB/BEP regime was more toxic with regard to bone marrow function; the frequencies of leucocytes below 1000 microliters-1, leucocytopenic fever and platelets below 25,000 microliters-1, throughout four cycles were 28% vs 5% (P < 0.001), 16% vs 5% (P = 0.006), and 10% vs 1% (P = 0.001) respectively. Neuropathy also occurred more often in the PVB/BEP arm: 47% vs 25% (P = 0.001). This study shows that an alternating regimen of PVB/BEP is not superior to BEP and that it is more myelo- and neurotoxic.

The search for tumour specific immune reactions was triggered by the formulation of the immune surveillance hypothesis as early as 1909 [1] and by its later updates [2]. The central idea of immune surveillance is that the immune system has the capacity, not only to distinguish ‘self ’from ‘non-self’, but also to distinguish cancer cells in the body from their normal counterparts. As a result the immune system may arrest or eliminate the cancer cells from the tumour bearing host. The fascination with this concept has initiated and stimulated cancer immunologists to classify tumours on the basis of their antigenic ‘make-up’ and their reaction to cytokines. To this end a wide range of animal species were immunised with (human) tumour cells to raise antisera for the serological dissection of cancer. With the advent of monoclonal antibodies (mAb) an even more precise knowledge was gained about carbohydrate epitopes on the glycoprotein, glycolipid and ganglioside antigen structure of tumour cells [3]. All these efforts have generated serological reagents, that are now widely applied in the laboratory and clinic, ranging from application in fundamental tumour cell biology to the use for immunodiagnosis in the laboratory and in vivo and for immunotherapy.