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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.

Purpose: To determine the maximum-tolerated dose, toxicity profile, and pharmacokinetics of a fixed dose of paclitaxel followed by increasing doses of carboplatin, given weekly to patients with advanced esophageal or gastric junction cancer. Experimental Design: Paclitaxel was administered on day 1 as a 1-h infusion at a fixed dose of 100 mg/m2 followed by a 1-h infusion of carboplatin targeting an area under the curve (AUC) of 2–5 mg × min/ml, with cycles repeated on days 8, 15, 29, 36, and 43. Results: Forty patients [36 males; median (range) age, 57 (40–74) years] were enrolled. Dose-limiting toxicity was observed at a carboplatin AUC of 5 mg × min/ml and consisted of treatment delay attributable to myelosuppression. No grade 3/4 treatment-related nonhematological toxicity was observed. The highest dose intensity (>95% of the planned dose over time) was achieved with a carboplatin AUC of 4 mg × min/ml. The mean (±SD) AUCs of unbound (Cu) and total paclitaxel were 0.662 ± 0.186 and 7.37 ± 1.33 μm × h, respectively. Clearance of Cu was 188 ± 44.6 liter/h/m2, which is not significantly different from historical data (P = 0.52). Cremophor EL clearance was 123 ± 23 ml/h/m2, similar to previous findings. Of 37 patients evaluable for response, 1 had complete response, 19 had partial response, and 10 had stable disease, accounting for an overall response rate of 54%. Conclusions: This regimen is very tolerable and effective, and the recommended doses for additional studies are paclitaxel (100 mg/m2), with carboplatin targeting an AUC of 4 mg × min/ml.

The clinical development of interleukin 12 (IL-12) as a single agent for systemic cancer therapy has been hindered by its significant toxicity and disappointing anti-tumor effects. The lack of efficacy was accompanied by, and probably related to, the declining biological effects of IL-12 in the course of repeated administrations at doses approaching the maximum tolerated dose (MTD). Nevertheless, IL-12 remains a very promising immunotherapeutic agent because recent cancer vaccination studies in animal models and humans have demonstrated its powerful adjuvant properties. Therefore, IL-12 may re-enter the arena of cancer therapy. Here, we review the immune modulating characteristics of IL-12 considered responsible for the adjuvant effects, as well as the results of animal and human cancer vaccination studies with IL-12 applied as an adjuvant. In addition, we discuss how studies with systemic IL-12 in cancer patients, and several other lines of evidence, indicate that IL-12 may exert optimal adjuvant effects only at low dose levels. Therefore, the MTD may not constitute the maximum effective dose of IL-12 for adjuvant application.

Purpose: Despite dose calculation using body-surface area (BSA), pharmacokinetics of most anticancer drugs show wide interindividual variability. In this study, we evaluated the role of BSA in paclitaxel disposition. Patients and Methods: Paclitaxel pharmacokinetics were prospectively studied in 12 patients that were treated in a randomized cross-over design with paclitaxel (3-hour infusion at a 3-week interval) at 175 mg/m2 in cycle 1 (A) and a flat-fixed dose of 300 mg in cycle 2 (B), or vice versa. Blood samples were collected up to 24 hours after dosing and analyzed for total and unbound paclitaxel. Results: The area under the curves (AUC) of unbound paclitaxel were similar in both dosing groups, with mean values ± SD (A v B) of 1.34 ± 0.158 versus 1.30 ± 0.329 μM•h, indicating that BSA-based dosing reduced the coefficient of variation by 53.3%. Unbound and total paclitaxel clearance was also significantly related to various body-size measures, including BSA (R ≥ 0.617; P ≤ .033), weight (R ≥ 0.621; P ≤ .031), and lean-body mass (r ≥ 0.630; P ≤ .028). We hypothesize that this is caused by the association of paclitaxel in the circulation with Cremophor EL, the distribution of which is linked to total blood volume, and thus to BSA. Conclusion: This study indicates that paclitaxel disposition is significantly related to BSA. This provides a pharmacokinetic rationale for BSA-based dosing of this drug.

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.

Previous studies have shown that STI571, a selective tyrosine kinase inhibitor of c-KIT, is highly effective in c-KIT/CD117-positive gastrointestinal stromal tumours (GIST), especially those that have activating mutations in the c-kit exon 11 that encodes the juxtamembrane (JM) domain of the c-KIT oncoprotein. We examined the prevalence of activating exon 11 c-kit mutations in 26 small-cell lung cancer (SCLC) cases in order to explore whether this disease is also a potential target for treatment with STI571. Expression of c-KIT, estimated by immunohistochemistry, was demonstrated in 14 out of 22 SCLC samples (64%); nine samples showed moderate to strong staining (41%), five samples were weakly positive (23%), whereas eight samples (36%) were negative for CD117. Next, we examined the mutational status of exon 11 of the c-kit gene, by single-stranded conformational polymorphism (SSCP) and sequencing in all of the cKIT/CD117-positive tumours. However, no activating mutations in the c-kit exon 11 were found by either technique. Apparently, c-KIT oncoprotein expression in SCLC was not correlated with activating mutations in c-kit exon 11. In analogy to GISTs, our results could imply that SCLC patients would not benefit from treatment with STI571.

New solubilizers, including Sorporol 230, Sorporol 120Ex, Aceporol 345-T, Aceporol 460 and Riciporol 335, as potential new delivery vehicles for paclitaxel were investigated, since recent studies have shown that the paclitaxel delivery vehicle Cremophor EL significantly alters the pharmacokinetics of paclitaxel. Cremophor EL and Tween 80 were used as a reference. As in the case of Cremophor EL, alteration of blood distribution of paclitaxel occurred in the presence of all tested vehicles. Also, no differences in the affinity of paclitaxel for the tested solubilizers was found during equilibrium dialysis experiments. The different vehicles could be distinguished by a different rate of esterase-mediated breakdown, which was correlated with the fatty acid content of the solubilizers. The activation of the complement cascade was less pronounced for all solubilizers, except Riciporol 335, compared to Cremophor EL. The strategies presented here provide the possibility to rapidly screen future candidate delivery vehicles with optimal characteristics for use as a solubilizer in clinical formulations of paclitaxel or other poorly water-soluble drugs.

In a phase I study we demonstrated the feasibility of a bi-weekly combination of paclitaxel 180 mg m−2 with cisplatin 60 mg m−2. In this study we further assessed toxicity and efficacy of this schedule in the treatment of advanced cancer of the oesophagus or the gastro-oesophageal junction. Patients received paclitaxel 180 mg m−2 administered over 3 h followed by a 3-h infusion of cisplatin 60 mg m−2. Patients were retreated every 2 weeks unless granulocytes were

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.

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.

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.

Purpose: This study was performed to deter mine the correlation of CA15.3 and TPS with disease course in patients with metastatic breast cancer.Methods: Levels of CA15.3 and tissue polypeptide antigen using the M3 monoclonal antibody (TPS) were determined in the serum of 60 patients with metastatic breast cancer. CA15.3 and TPS were measured at two assay times.Results: A change of more than 25% in the serum level of CA15.3 or TPS was highly correlated with tumor response. The association between response and change in marker levels was stronger for CA15.3 (P=0.0001) than for TPS (P=0.0005). Distinct mismatches between marker changes and the tumor response were observed for both CA15.3 and TPS.Conclusion: CA15.3 and TPS are useful in the determination of response to treatment. Because of observed disagreement, marker changes can only be regarded as indicative of disease course.

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.

We treated 72 patients with metastatic renal-cell cancer according to 2 protocols consisting of two 5-week induction cycles of continuous i.v. high-dose interleukin-2 (IL-2), i.m. interferon-alpha (IFN alpha) and ex vivo IL-2-activated lymphocytes, followed for patients with stable disease (SD), partial response (PR) or complete response (CR) by four 4-week maintenance cycles of IL-2 and IFN alpha. Protocol 2 (55 patients) differed from protocol 1 (17 patients) in (i) the addition of IFN alpha to the first IL-2 infusions in both induction cycles; (ii) the use of Teceleukin IL-2, reconstituted with carrier protein, instead of Proleukin IL-2 without carrier protein. We classified 23 patients with CR and PR as responders (4 in protocol 1 and 19 in protocol 2) and 45 patients with SD and progressive disease as non-responders. Prior to immunotherapy, patients entered into protocol 2 already had higher IFN gamma serum concentrations, higher peripheral blood CD56-,3+ and CD8-,4+ lymphocyte numbers and lower NKK562 activity than those entered into protocol 1. These differences persisted during and after immunotherapy. In line with these observations, ex vivo IL-2-activated lymphocytes had larger proportions of CD56-,3+ and CD8-,4+ lymphocytes and lower NKK562 activity in protocol 2 than in protocol 1. Higher IL-2 serum concentrations were reached during the IL-2 infusion in protocol 2 than in protocol 1. In addition, the immunomodulation in protocol 2 was stronger than in protocol 1 as indicated by higher TNF alpha serum concentrations and a more pronounced eosinophilia. Differences between responders and non-responders treated according to the 2 protocols were not significant, except for the total number of lymphocytes obtained by apheresis, which was higher in responders than in non-responders.

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.

Background. In a group of patients with metastatic melanoma treated with high dose immunotherapy, there was an unexpectedly high incidence of severe cardiac adverse effects. Methods. Sixteen patients with metastatic melanoma were treated with high dose interleukin-2 (IL-2) and alpha-interferon (α-IFN). Each treatment cycle consisted of IL-2 at a dose of 12 MIU/m2 and α-IFN at a dose of 3 MIU/m2, given as intravenous bolus injections every s hours on Days 1-5, every 3 weeks for a total of three cycles. Before treatment, careful cardiologic screening was performed, including electrocardiogram (ECG), stress test, cardiac multiple uptake-gated acquisition (MUGA) scan, and echocardiography. During therapy, patients were monitored with daily ECG and creatine phospokinase measurements. Once cardiac damage was suspected, IL-2 and α-IFN were discontinued, and echocardiography, stress test and MUGA-scan were repeated. If indicated, cardiac catheterization with endomyocardial biopsies was performed. Results. Despite pretreatment cardiac screening, seven patients (44%) exhibited myocardial injury. Acute myocardial infarction occurred in one patient, cardiomyopathy developed in four, asymptomatic ECG changes appeared in one, and 1 patient died of acute cardiac arrest. Echocardiography showed hypokinesis and decreased left ventricular ejection fraction. These abnormalities disappeared within 6 months. Cardiac catheterization in four affected patients revealed normal coronary arteries, but endomyocardial biopsies showed interstitial edema, vacuolation, and degeneration of myocytes. Electron-microscopic examination showed fragmentation of myofibrils, swelling of mitochondria and loss of mitochondrial cristae. Conclusions. This intensive treatment schedule of IL-2 and α-IFN is prohibited by severe and life-threatening cardiac toxicity.

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.

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.

Following a previous EORTC GU-Group study, in which linomide showed some activity in poor prognosis patients, this study was initiated to determine the effect of linomide in more favourable patients. 35 patients with metastatic renal cell carcinoma with good prognostic factors, i.e. good performance status, prior nephrectomy, no prior systemic therapy, and no liver, bone or brain metastases, were treated with linomide, a quinoline derivative with immunomodulating properties, at a dose of 10 mg daily, after an initial dose escalation during the first 4 weeks of treatment. In 29 evaluable patients, no responses were observed (95% confidence interval 0-10%). Best overall response was no change in 9 patients, for a median duration of 4 months. Linomide in this schedule was poorly tolerated, with 17% (6 patients) of patients being withdrawn and 23% (8 patients) having dose reductions due to adverse events, mostly influenza-like symptoms of myalgia, arthralgia and fatigue. Several cases of pericarditis and neuropathy were observed. In spite of selection of favourable prognosis patients and an optimal daily dosing schedule, linomide was not an effective treatment in renal cell carcinoma. In view of toxicity and lack of efficacy, there is no rationale in further testing the drug in this disease. (C) 1997 Published by Elsevier Science Ltd.

Interleukin 12 (IL-12) is a cytokine with important regulatory functions bridging innate and adaptive immunity. It has been proposed as an immune adjuvant for vaccination therapy of infectious diseases and malignancies. The inflammatory properties of IL-12 play an important role in the adjuvant effect. We studied the effect of s.c. injections of recombinant human IL-12 (rHuIL-12) in 26 patients with renal cell cancer and demonstrated dose-dependent systemic activation of multiple inflammatory mediator systems in humans. rHuIL-12 at a dose of 0.5 microg/kg induced degranulation of neutrophils with a significant increase in the plasma levels of elastase (p < 0.05) and lactoferrin (p = 0.01) at 24 h. Additionally, rHuIL-12 injection mediated the release of lipid mediators, as demonstrated by a sharp increase in the plasma secretory phospholipase A2 (sPLA2) level (p = 0.003). rHuIL-12, when administered at a dose of 0.1 microg/kg, showed minimal systemic effects. In conclusion, when IL-12 is used as an adjuvant, doses should not exceed 0.1 microg/kg, in order to avoid severe systemic inflammatory responses

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

PURPOSE: To evaluate relationships between various body-size measures and irinotecan (CPT-11) clearance and metabolism in cancer patients, and to provide future dosing recommendations for this agent. PATIENTS AND METHODS: Pharmacokinetic data were obtained from 82 adult patients (50 men, 32 women; median age, 54 years) receiving CPT-11 as a 90-minute intravenous infusion (dose range, 175 to 350 mg/m2). In each patient, plasma samples were collected at timed intervals in the first administration of a 3-week schedule, and CPT-11 and its metabolite, SN-38, were measured by a liquid chromatographic assay. RESULTS: The mean (± SD) CPT-11 clearance was 33.6 ± 10.8 L/h, with an interindividual variability (IIV) of 32.1%. When clearance was adjusted for body-surface area (BSA), the IIV was similar (34.0%). In addition, in a multiple linear regression analysis, none of the studied measures (BSA, lean body mass, [adjusted] ideal body weight, and body mass index) was a significant covariate (P > .13; r2 < .014) in our population. Similarly, BSA did not significantly contribute to variability in the relative extent of conversion to SN-38 (P = .26). CONCLUSION: BSA is not a predictor of CPT-11 clearance or SN-38 pharmacokinetics and does not contribute to reducing kinetic variability. These findings provide a rationale for the conduct of a comparative phase III study between BSA-based dosing and flat or fixed dosing of CPT-11.

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.

Nonlinear disposition of paclitaxel (Taxol) in cancer patients has been described in several studies, but the underlying mechanism is still a matter of speculation. Previously, we have shown in vitro that the paclitaxel formulation vehicle, Cremophor EL (CrEL), alters the blood distribution of paclitaxel as a result of entrapment of the compound in circulating CrEL micelles, thereby reducing the free drug fraction available for cellular partitioning. Based on these findings, we prospectively re-evaluated the linearity of paclitaxel disposition in patients using whole blood and plasma analysis, and sought to define a new pharmacokinetic model to describe the data. Seven patients with solid tumors were treated with paclitaxel infused over 3 h, each at consecutive 3-weekly dose levels of 225, 175 and 135 mg/m2 (CrEL dose level, 18.8, 14.6, and 11.3 ml/m2, respectively). Patient samples were collected up to 24 h after the start of infusion, and analyzed by high-performance liquid chromatography. Paclitaxel peak levels and areas under the curve in whole blood increased linearly with dose, whereas plasma levels showed substantial deviation from linearity. This was shown to be caused by a CrEL concentration-dependent decrease in paclitaxel uptake in blood cells, as reflected by the blood:plasma concentration ratios which altered significantly from 0.83 +/- 0.11 (at 135 mg/m2) to 0.68 +/- 0.07 (at 225 mg/m2). It is concluded that the nonlinear disposition of paclitaxel is related to paclitaxel dose-related levels of the formulation vehicle CrEL, leading to a disproportionate drug accumulation in the plasma fraction. The pharmacokinetic model developed accurately described the data, and will help guide future development and refinement of clinical protocols, especially in defining the exposure measure best linked to paclitaxel effects and toxicities.

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.

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.

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

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

Purpose: Testicular germ cell tumors (TGCTs) represent one of the few tumor types that are curable by antineoplastic therapy, probably due to the high sensitivity of this neoplasm to induction of apoptosis by chemotherapeutic agents and/or ionizing radiation. Here, we tested cell susceptibility to radiation-induced apoptosis in a panel of TGCT cell lines and attempted to correlate this with the known potentially relevant molecular determinants (p53 gene status and Bcl-2 family proteins) of apoptosis. Methods and Materials: Induction of apoptosis by γ-radiation was morphologically recognized in NT2, NCCIT, S2, and 2102 EP using Hoechst/PI staining and additionally confirmed by Western blot analysis of PARP cleavage. The p53 gene status was estimated by sequence analysis. Expression of p21/WAF/CIP was determined by Northern blot analysis and immunoblotting was used to monitor p53, Bax, Bcl-2, Bcl-x, and Bak protein levels. In vitro colony formation was studied to establish clonogenic survival curves. Results: NT2 and NCCIT appeared to be susceptible for radiation-induced apoptosis, contrasting 2102 EP and S2 which were highly resistant. Sequence analysis showed that NT2, S2, and 2102 EP are homozygous for wild-type p53 (wtp53), whereas NCCIT contains mutant p53 (mtp53). NT2 and 2102 EP cells showed radiation-induced p53 upregulation, while NCCIT (mtp53) and S2 (no p53 protein) cells did not. Consistently, γ-radiation-induced DNA damage resulted in a p53-dependent transactivation of the p21/WAF/CIP gene in NT2 and 2102 EP, but not in mtp53-containing NCCIT cells and p53 nonexpressing S2 cells. Constitutive expression of Bax, Bcl-2, Bcl-x, and Bak was not affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis. A discrepancy was found between apoptosis and reproductive death. Conclusions: The present study revealed that: i) the presence of wtp53 may not be absolutely required for the hypersensitivity for radiation-induced apoptosis in TGCT cell lines, ii) the molecular mechanism underlying the unique radiosensitivity was independent of the expression of Bcl-2 family proteins, and iii) cell susceptibility to apoptosis induction is not sufficiently informative to predict intrinsic radiosensitivity as determined by clonogenic survival.

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

The pharmacokinetics of oral dexniguldipine, a new multidrug-resistance-modifying agent under clinical evaluation, and its pyridine metabolite M-1 were determined in plasma, tumor, and renal tissue in Wag/Rij rats bearing a multidrug-resistant CC531 colon adenocarcinoma tumor under the renal capsule. The pharmacokinetics were studied in four experiments. After a single administration of dexniguldipine (30 mg/kg), tumors and kidneys were collected after 5 (experiment 1), 24 (experiment 2), and 48 h (experiment 3). In the fourth experiment, dexniguldipine was given once daily for 3 consecutive days at a dose of 30 mg/kg. In all experiments, plasma samples were collected at regular intervals. The concentrations of dexniguldipine and M-1 could be determined in plasma in most of the rats at up to 32 h after drug administration. The area under the curve (AUC) of dexniguldipine and M-1 varied by a factor of 2-6 in the four experiments. High tumor-tissue concentrations of dexniguldipine were observed. The concentrations were highest in the multiple-dose experiment (2014 +/- 1005 ng/g tissue). High degrees of correlation (> 0.8) were established between the concentrations of dexniguldipine measured in plasma and tumor as well as renal tissue. Overall, tumor-tissue concentrations of M-1 comprised one-third of the dexniguldipine concentrations measured.

In stage I non-seminomatous testicular cancer, the decision between surveillance and adjuvant chemotherapy rests heavily upon the valuation of quality of life. Decision analysis was used to assess at what relapse rate adjuvant chemotherapy is preferred when patients' and clinicians' evaluations are considered. Probabilities were obtained from the literature and from experts. Evaluations of the disease states were obtained from patients (n = 68) and clinicians (n = 50). Results from the model were compared with a treatment preference question, asking for the relapse rate directly. Adjuvant chemotherapy was preferred at relapse rates above 50% when patient evaluations were used. The evaluations of the disease states had a strong impact on the decision. Using clinician evaluations, adjuvant chemotherapy was preferred at relapse rates above 73%. The relapse rates from the treatment preference question were lower: 46% for patients and 35% for clinicians. The results indicate that when patient preferences are accounted for, adjuvant chemotherapy should be considered more often.

The presence of multidrug resistant cells, either acquired or de novo, severely limits treatment outcome in haematological malignancies. Although expression of the Mr 170,000 P-glycoprotein drug pump is likely to play a role in multidrug resistance (MDR) in haematological malignancies, it is now evident that other MDR mechanisms may be operational as well in leukaemias, lymphomas, and multiple myeloma. We determined the expression of a newly recognised drug resistance gene, the Multidrug Resistance-associated Protein (MRP) gene, in peripheral blood cells from healthy volunteers and from patients with haematological malignancies. Expression of MRP mRNA and its Mr 190,000 glycoprotein were estimated by RNase protection assay and immunocytochemistry, respectively. MRP appeared to be ubiquitously expressed at low levels in all nonmalignant haemopoietic cell types. However, some leukaemias showed elevated levels of MRP, probably due to transcriptional activation or increased mRNA stability. High to very high MRP expression levels were frequently found in chronic lymphocytic leukaemia and prolymphocytic leukaemia. Acute myelocytic leukemia often exhibited low but occasionally high MRP expression levels, while in the other acute and chronic leukaemias, lymphomas, and multiple myeloma, predominantly low, basal levels of MRP were found. We conclude that hyperexpression of MRP is observed in leukaemias, and that further studies are needed to assess the clinical relevance of MRP.

Sample preparation for the measurement of non-protein-bound platinum was evaluated by precipitation of plasma proteins with cold ethanol. The method was compared with the routinely used plasma ultrafiltration and with trichloroacetic acid (TCA) protein precipitation. After incubation of human plasma samples with cisplatin or carboplatin, unbound platinum concentrations were determined applying Amicon Diaflo ultrafiltration membranes and Millipore ultrafree-MC filters. For protein precipitation, 1 ml of cold (-20 degrees C) pure ethanol was added to 0.5 ml of human plasma and the supernatant was collected after 2 h, or 0.5 ml of cold 20% TCA was added to 0.5 ml of plasma. Platinum was analyzed by atomic absorption spectrophotometry (AAS). There was no significant difference between the ethanol and ultrafiltration methods in the unbound platinum concentration. The protein content in the supernatant (1.00 +/- 0.20%) was slightly higher than that in the Amicon (0.58 +/- 0.05%) and Millipore (0.55 +/- 0.04%) ultrafiltrates. On average, the TCA and ethanol method seemed to be equally appropriate. The ethanol precipitation method is concluded to be simple, convenient, and reproducible and has negligible costs.

PURPOSE To determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) used in addition to standard inpatient antibiotic therapy shortens the period of hospitalization due to chemotherapy-induced neutropenic fever. PATIENTS AND METHODS One hundred thirty-four patients with a hematologic (n = 47) or solid tumor (n = 87) who had severe neutropenia (< 0.5 x 10(9)/L) and fever (> 38.5 degrees C once or > 38 degrees C twice over a 12-hour observation period) were randomly assigned to receive GM-CSF 5 micrograms/kg/d (n = 65) or placebo (n = 69) in conjunction with broad-spectrum antibiotics for a minimum of 4 days and a maximum of 14 days. GM-CSF/placebo and antibiotics were stopped if the neutrophil count was greater than 1.0 x 10(9)/L and temperature less than 37.5 degrees C during 2 consecutive days, or for a leukocyte count > or = 10 x 10(9)/L, both followed by a 24-hour observation period (hospitalization period). RESULTS Compared with placebo, GM-CSF enhanced neutrophil recovery. Median neutrophil counts at day 4 were 2.5 x 10(9)/L (range, 0 to 25) in the GM-CSF arm and 1.3 x 10(9)/L (range, 0 to 9) in the placebo arm (P < .001). No significant difference was observed with regard to median number of days with less than 1.0 x 10(9)/L neutrophils (4 v 4) or days of fever (3 v 3). The median number of days patients were hospitalized while on study was comparable in the GM-CSF and placebo groups at 6 (range, 3 to 14) versus 7 (range, 4 to 14), respectively, according to an intention-to-treat analysis (P = .27). Quality-of-life scores in 90 patients demonstrated significant differences in favor of the placebo group. Hospital costs were significantly higher for GM-CSF-treated patients if GM-CSF was included in the price (median costs, $4,140 [US] for GM-CSF v $590 for placebo; P < .05). CONCLUSION These results indicate that GM-CSF does not affect the number of days for resolution of fever or the hospitalization period for this patient group, although a significant effect of GM-CSF was observed on neutrophil recovery.

The chemosensitizing potency of dexniguldipine hydrochloride (B8509-035) on epidoxorubicin was assessed in a multidrug-resistant (MDR) tumour model, the intrinsic MDR rat colon carcinoma CC531. In vitro in the sulphorhodamine B cell-viability assay the cytotoxicity of epidoxorubicin was increased approximately 15-fold by co-incubation with 50 ng/ml dexniguldipine. In vivo concentrations of dexniguldipine 5 h after a single oral dose of 30 mg/kg were 72 (+/- 19 SD) ng/ml in plasma and 925 (+/- 495 SD) ng/g in tumour tissue. Levels of the metabolite of dexniguldipine, M-1, which has the same chemosensitizing potential, were 26 (+/- 6 SD) ng/ml and 289 (+/- 127 SD) ng/g respectively. The efficacy of treatment with 6 mg/kg epidoxorubicin applied intravenously combined with 30 mg kg-1 day-1 dexniguldipine administered orally for 3 days prior to epidoxorubicin injection was evaluated on tumours grown under the renal capsule. Dexniguldipine alone did not show antitumour effects in vivo. Dexniguldipine modestly, but consistently, potentiated the tumour-growth-inhibiting effect of epidoxorubicin, reaching statistical significance in two out of four experiments. In conclusion, these experiments show that dexniguldipine has potency as an MDR reverter in vitro and in vivo in this solid MDR tumour model.