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The aim of this study was to examine the anti-tumour effects of dual vertical VEGF targeting consisting in the association between bevacizumab, a VEGF-depleting drug, and the VEGF receptor antityrosine kinase AZD2171. Mice bearing human head and neck CAL33 xenografted tumours were treated once daily for 11 d with either vehicle (controls), AZD2171 (2.5mg/kg/day, p.o.), bevacizumab (5mg/kg/day, i.p.) or the bevacizumab-AZD2171 combination. The AZD2171-bevacizumab combination produced additive effects on tumour growth and reduced the number of proliferating cells relative to control. Bevacizumab did not influence tumour vascular necrosis whilst AZD2171 (p=0.01) and the combination (p=0.01) increased it. The number of mature tumour cells decreased significantly with the combination treatment only (p=0.001), which induced the largest increase in the Bax/Bcl2 ratio (up to 25-fold) and a progressive 3-fold decrease in HIF1-alpha expression between 24h and 192h. The present data indicate that there is no redundancy in targeting the same angiogenic pathway with the presently tested clinically applicable drugs. The study provides a strong rationale for future clinical trials.

Epidermal growth factor receptor (EGFR) and HER-2 are associated with a poor prognosis in various cancers, including prostate cancer. Inhibition of these receptors may provide a treatment for hormone-refractory prostate cancer. The presence of HER-2 (Western blot) and EGFR (5830 fmol/mg protein, ligand-binding assay) was assessed in the hormone-refractory human prostate cancer cell line, DU-145. Cells were exposed to the selective EGFR-TKI (EGFR tyrosine kinase inhibitor) gefitinib ('Iressa; ZD1839) and/or the HER-2-targeted monoclonal antibody trastuzumab ('Herceptin'), for 96 h. Irradiation (RX) at 6 Gy the dose causing 50% growth inhibition, was applied 48 h after the start of drug treatment. There was a dose-related effect on cell survival for both ZD1839 and trastuzumab treatments. Combining ZD1839 and trastuzumab led to less than additive effects on cell survival. Chou and Talalay representations further characterised this less than additive effect on cell survival. The application of ZD1839 led to a marked elevation in the level of the negative regulator of cell division, p27. The ZD1839-trastuzumab combination had less of an impact on p27 expression compared with the effect of ZD1839 treatment alone. The lowest expression of the apoptotic-related protein, Bax, was observed in the presence of the drug combination. There was a significant interaction (synergism) between RX and either ZD1839 or trastuzumab treatments. In contrast, the drug combination with RX resulted in antagonistic cytotoxic effects. These results indicate an antagonistic interaction between EGFR and HER-2 targeting and provide molecular mechanisms supporting this observation. Data from DU-145 cells suggest that dual targeting of EGFR and HER-2 may be inappropriate for the treatment of hormone-refractory prostate cancer, especially in the context of their combination with RX.

The aim of this study was to analyse prognostic factors for time to treatment failure (TTF) and overall survival (OS) in patients with unresectable cancer of the pharynx. A twice daily (b.i.d.) radiotherapy with concomitant cisplatin-5-fluorouracil chemotherapy was administered to 77 consecutive patients (68 males, 9 females; median age: 56 years). The studied factors were: age, gender, tumour differentiation, tumour volume, initial hemoglobin level, karnofsky index (KI), primary tumour location, T, N, epidermal growth factor receptor (EGFR) level in the tumour (fmol/mg protein). KI and EGFR level were significant predictors in a multivariate analysis for TTF (P=0.004 and P=0.0001) and OS (P=0.004 and P=0.0001). In order to select subgroups with different outcomes, a stratification of patients was performed based on the EGFR value: patients with tumour EGFR levels35 fmol/mg protein, between 35 and 275 fmol/mg protein and275 fmol/mg protein had 95%, 51% and 16% 3 year OS rates, respectively (log rank test; P=0.0001). Interestingly, for patients exhibiting a complete response (CR) after concomitant b.i.d. chemo-radiotherapy, patients with EGFR levels35 fmol/mg protein were all alive at 3 years; in contrast, there was only 70 and 13% 3 year survival rates for patients with EGFR tumour levels between 35 and 275 fmol/mg protein and above 275 fmol/mg protein, respectively. EGFR determination appears to be a powerful prognostic parameter in unresectable pharyngeal cancer patients treated by concomitant chemo-radiotherapy.

The objective of this study was to attempt to identify patient co-variables which could influence interpatient variability in 5-fluorouracil (5-FU) clearance during a 5-day continuous venous infusion (CVI, cisplatin 100 mg/m2 day 1 then 5-FU 1 g/m2/day days 2-6). The analysis was performed using a NONMEM program according to a linear one-compartment model. A total of 186 cycles (2 samples per day, 8 a.m. and 5 p.m.) were analysed from 104 patients with various cancers (the majority of head and neck and oesophagus). The study co-variables were age, sex, body surface area, hepatic metastases, peripheral mononuclear cell dihydropyrimidine dehydrogenase activity (PMNC-DPD), liver enzymes, clock-time (8 a.m. versus 5 p.m.), elapsed time during CVI. The data showed that 5-FU clearance was significantly reduced by increased age, low PMNC-DPD, high serum alkaline phosphatase and elapsed time during infusion. These data may be useful for improving knowledge of predictive factors which can influence 5-FU exposure and thus predispose to toxic manifestations.

The purpose of this study was to investigate folate-related predictors of 5-fluorouracil (5-FU) cytotoxiticy in the presence or absence of l -folinic acid ( l -FA). Intracellular concentrations of the reduced folates (tetrahydrofolate + 5,10-methylenetetrahydrofolate) and folylpolyglutamate synthetase (FPGS) activity were determined in 14 human cancer cell lines expressing a spontaneous sensitivity to 5-FU. On these 14 cell lines grown without l - F A supplementation, a significant positive correlation was demonstrated between basal intracellular folate concentration and FPGS activity. 5-FU sensitivity (IC 50 range 0.6–25.4 μM) was not related to the basal intracellular folate concentration, whereas, significantly, it was linked to FPGS activity (range 2.5–11.1 pmol/min/mg protein): the higher the FPGS activity, the greater the 5-FU sensitivity. Under l -FA supplementation (0.01–300 μM), intracellular reduced folates increased continuously without evidence of saturation in all cell lines; the pattern of accumulation was independent of the FPGS activity. l -FA enhanced 5-FU cytoxicity by a factor of 1.9–6.4 in 12 of the 14 cell lines. In the 12 FA-sensitive cell lines, the l -FA concentrations allowing 90% of maximum 5-FU potentiation [ l -FA]90 ranged between 0.7 and 107.9 μM (median 1.9); in contrast, the intracellular concentrations of reduced folates allowing 90% of maximum 5-FU potentiation were much less variable (range 7.6–38.3, median 24.8 pmol/mg protein). In the presence of [ l -FA]90, 5-FU sensitivity remained significantly correlated to the basal FPGS activity. In addition, reduced folates were measured in 96 tumoral samples (50 head and neck, 16 colon, 30 liver metastases from colorectal cancer) taken before treatment. Almost all investigated tumours had folate concentrations below the median concentration required for optimal 5-FU potentiation in vitro : median levels (range, pmol/mg protein) were 3.8 (0–17.7) for head and neck, 5.8 (2.3–12.0) for colon and 12.1 (1.7–118.5) for liver metastases. Above all, these data establish the relevance of FPGS activity for predicting the efficacy of 5-FU modulated by FA or not and point to the potential clinical interest of FPGS determination in human tumours. © 1997 Elsevier Science Ltd.

Despite being one of the oldest anti-cancer drugs, fluorouracil (FU) is still being increasingly used in cancer chemotherapy. The source of variability for FU sensitivity in patients may be complex, although an overproduction of thymidylate synthase (TS) was the only mechanism of resistance identified in tumours from FU-resistant patients. Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme of FU catabolism. Thus, DPD activity may be a potential factor for controlling FU responsiveness. A panel of 19 human tumour cell lines, including digestive tract, breast and head and neck cancer cells, were investigated. Both TS and DPD activities were measured in parallel to FU responsiveness. None of the cell lines had been previously exposed to FU, and thus expressed a spontaneous sensitivity to FU. Sensitivity between cell lines showed marked differences, with IC50 values ranging from 45 ng/ml (colon cell line) to 5063 ng/ml (head and neck cell line). TS activity was measurable in all cell lines and varied within a 46-fold range. DPD activity was detected in all but four cell lines, showing a 100-fold range of variation. Cell lines most sensitive to FU exhibited the lowest DPD and TS activities and vice versa. Simple linear regression analysis showed that both TS (r2 = 0.22, P = 0.042) and DPD (r2 = 0.27, P = 0.022) activities were significantly correlated to FU effectiveness (log 10 IC50): the greater the enzyme activities, the higher the FU IC50. TS and DPD were demonstrated to be independent variables. A multiple regression analysis showed that the combination of TS and DPD activities explained 36% of the variability in FU IC50 (r2 = 0.36, P = 0.01). Two groups of cell lines could be identified, one group with both low TS and low DPD activities (G1), and the other with either high TS and/or high DPD activities (G2). Mean FU IC50 values were 193 and 930 ng/ml in G1 and G2, respectively, and this difference in FU sensitivity was highly significant (P = 0.009). The present study shows, for the first time, that DPD activity in tumour cells is an independent factor significantly related to FU sensitivity. These results should encourage DPD and TS coupled measurements in tumours of patients before FU treatment in order to establish their prognostic relevance. DPD and TS measurements could also be used during the treatment course to determine the implication of these enzymes in the development of tumour resistance to FU.

When multicentre breast cancer trials are performed, receptor analyses must be comparable both over time and in the participating laboratories. However, we show for the first time a high variability for the distribution of oestradiol receptor (ER) values measured by enzyme immunoassay (EIA) from 1987 to 1991. This variability could be explained by calibration changes in the immunoassay kits. We have also analysed the influence on ER-EIA levels of technical differences between laboratories apart from the assay itself. Many steps emerged as being critical, i.e. homogenisation buffer, homogenisation procedure and cytosol dilution. Finally, we show that addition of 4-monohydroxytamoxifen increases the apparent ER content measured by EIA in 92% of cytosols. Thus, many factors must be controlled to ensure high precision with ER-EIA assays. We have to be particularly cautious with the conformational changes that could occur during cytosol preparation and that could also pre-exist in the tumour samples. Quality controls of cytosol preparation are essential.

Fotemustine (Fote) is a new amino acid-linked chloroethyl nitrosourea which has been shown to be useful in disseminated malignant melanoma. The aim of the present study was to analyse the cytotoxic effects resulting from the combination of anti-oestrogens and Fote on human melanoma cell lines. The anti-oestrogens tested were tamoxifen (TMX, 5 × 10 −7 mol/l and 5 × 10 −6 mol/l) and 4OH TMX (5 × 10 −8 mol/l and 5 × 10 −7 mol/l). As a preliminary step, a series of nine human melanoma cell lines was screened in order to identify and quantify the presence of oestradiol receptors (ER) in these cell lines. This led to the selection of an ER-positive (+) cell line. The drugs alone or in combination were then tested against CAL 1 ER (+) and CAL 7 ER (−) melanoma cell lines. Different sequences of drug combinations were tested using clinically compatible drug concentrations. For CAL 1 cells, there was a growth inhibitory effect induced by the anti-oestrogens given alone. Overall, the presence of the anti-oestrogens resulted in higher cytotoxic effects than when cells were exposed to Fote alone. The lowest ic 50 Fote values as compared to Fote alone were generated by the sequences in which the anti-oestrogens were administered before Fote. Significantly, these associations with anti-oestrogens enabled the ic 50 values of Fote to be reduced by up to 80%. Globally, TMX and 4OH TMX had similar synergistic effects. TMX and 4OH TMX had a modest influence on Fote cytotoxic effects against CAL 7 ER-negative cells. These data may be useful for optimal planning of future clinical trials for malignant melanoma using anti-oestrogens and nitrosoureas.

The objective of the present in vitro study was to determine an optimal timing of the irradiation in the combination cisplatin (CDDP) and 5-fluorouracil-folinic-acid (5-FU-FA) allowing a maximal cytotoxic effect on a human cell line derived from a head and neck carcinoma (CAL 27 cells). The various tested chemoradiotherapy sequences were applied in parallel to human keratinocytes in culture (SVK 14 cells). This was done in order to define the best sequence allowing the achievement of an optimal selectivity of the cytotoxic effects. The drug sequence was: CDDP over 2 h then fresh medium was added including the tandem 5-FU-d,I FA applied 6 h after CDDP, for 5 days. Irradiation was applied only once and at various times within the drug sequence. The cytotoxicity effects of the different chemoradiotherapy combinations were assessed by the MTT semi-automated test. The part taken by the 5-FU-FA combinations in the overall cytotoxicity was examined; an effect was apparent on CAL 27 cells only. The evolution of the radiation effect (RE = cell survival after drugs/cell survival after drugs plus irradiation) was analysed as a function of the different times of irradiation within the given drug sequence. Clearly, the RE values were dependent upon time at which the radiation dose in the chemoradiotherapy regimen was administered. For CAL 27 cells, irradiation effects were maximal at the first irradiation time tested after the end of the CDDP exposure (i.e. t = 3.5 h). In contrast, this optimal chemoradiotherapy timing for better cytotoxicity on CAL 27 cells did not correspond to that of SVK 14 cells. Consequently, it was possible to establish that the best time for the selectivity index was located shortly after the CDDP exposure.

Dihydropyrimidine dehydrogenase (DPD) is the major catabolic enzyme of pyrimidines and fluoropyrimidines. The clinical course of 2 patients with suspected DPD deficiency is described. Both patients had significantly delayed clearance of fluorouracil (5-FU), elevated plasma uracil concentrations, and subsequent lethal toxicity. The prevalence of DPD deficiency in the general population is unknown, but given the large number of cancer patients treated with 5-FU, it may be of great clinical significance. Lymphocytes have been previously shown to be a useful marker of systemic DPD activity. Because DPD activity has not been previously reported in a large population of cancer patients using 5-FU as the substrate, we determined DPD activity in lymphocytes from 66 patients with cancer. DPD activity was determined by a sensitive high performance liquid chromatography method. The mean DPD activity (S.D.) in 66 patients with head and neck cancer was 0.189 (0.071) nmol/min/mg protein with wide interpatient variability (range 0.058–0.357). DPD activity was not correlated to age ( r = −0.164, P = 0.188). The mean DPD activity in men [0.192 (0.074)] was not significantly different from that in women [0.172 (0.057); t -test P = 0.418]. Likewise, there was no statistical difference in DPD activity in patients who had not received prior chemotherapy [0.195 (0.066)] to patients receiving one or more cycles of chemotherapy [0.186 (0.074); t -test P = 0.638].

The cytotoxic effects of prolonged exposure to low concentrations of doxorubicin or a doxorubicin bolus were examined in vitro on four human breast cancer cell lines to simulate the plasma concentration profile of weekly low-dose (WLD) doxorubicin in breast cancer patients. Cells were exposed to doxorubicin for various prolonged times (24, 72, 120 and 192 h) and with different drug concentrations (5, 10, 20, 50 and 80 nmol/l). In a series of parallel experiments, cell lines were placed in contact with the drug for short periods (1 h) before prolonged exposure to doxorubicin; the concentrations of these pulses were 150, 250 and 350 nmol/l. A constant decrease in tritiated thymidine incorporation was noted as a function of the drug concentration and the duration of the cell contact with the drug. Interestingly the lowest concentrations (5-10 nmol/l) produced marked cytotoxic effects. For equivalent concentration x time values, experiments including doxorubicin pulses resulted in greater cytotoxicity than continuous exposure alone, in a dose-related manner. This finding was related to differences in intracellular doxorubicin concentrations. Results suggest that the rather empirically designed WLD doxorubicin schedule can generate greater cytotoxic effects than continuous doxorubicin administration alone.

Twenty-five cell lines derived from nine different human cancers were tested for the cytotoxic activity of suramin. Two different initial cellular concentrations were used: C1 (800-2000 cells per well) and C2 (3000-7000 cells per well). Suramin concentrations ranged from 50 to 2500 micrograms/ml. Cytotoxicity was assessed by the MTT test. Epidermal growth factor receptors (EGFR) were assayed by competition analysis and Scatchard plots. In sixteen cell lines suramin had an unexpected growth stimulation effect at low concentration (50-125 micrograms/ml). IC50 varied from 21 micrograms/ml (osteosarcoma, OS2) to 1408 micrograms/ml (melanoma, CAL 24) and, within melanoma cell lines, it varied from 120 micrograms/ml (CAL 41) to 1408 micrograms/ml (CAL 24). The individual IC50 values were positively and significantly linked with the initial cellular density. Eighteen cell lines had measurable EGFR (six with two families of sites, twelve with one): Kd varied between 0.004 nmol/l for the highest affinity site (melanoma, CAL 7) to 1.852 nmol/l for the lowest affinity site (lung, CAL 12). There was no relation between presence or absence of EGF binding sites and distribution of IC50, but for cells with measurable EGFR there was a weak but significant correlation between the number of EGF binding sites per cell and the corresponding IC50 (r = -0.53, P = 0.021).