C.J. van Groeningen

Randomized Phase III Trial of High–Dose-Intensity Methotrexate, Vinblastine, Doxorubicin, and Cisplatin (MVAC) Chemotherapy and Recombinant Human Granulocyte Colony-Stimulating Factor Versus Classic MVAC in Advanced Urothelial Tract Tumors: European Organization for Research and Treatment of Cancer Protocol No. 30924

PURPOSE This randomized trial evaluated antitumor activity of and survival asociated with high-dose-intensity chemotherapy with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) plus granulocyte colony-stimulating factor (HD-MVAC) versus MVAC in patients with advanced transitional-cell carcinoma (TCC). PATIENTS AND METHODS A total of 263 patients with metastatic or advanced TCC who had no prior chemotherapy were randomized to HD-MVAC (2-week cycles) or MVAC (4-week cycles). RESULTS Using an intent-to-treat analysis, at a median follow-up of 38 months, on the HD-MVAC arm there were 28 complete responses (CRs) (21%) and 55 partial responses (PRs) (41%), for an overall response of 62% (95% confidence interval [CI], 54% to 70%). On the MVAC arm, there were 12 CRs (9%) and 53 PRs (41%), for an overall response of 50% (95% CI, 42% to 59%). The P value for the difference in CR rate was.009; and for the overall response, it was.06. There was no statistically significant difference in survival (P =.122) or time to progression (P =.114). Progression-free survival was significantly better with HD-MVAC (P=.037; hazard ratio.75; 95% CI.58 to.98). The median progression-free survival time was 9.1 months on the HD-MVAC arm versus 8.2 months on the MVAC arm. The 2-year progression-free survival rate was 24.7% for HD-MVAC (95% CI, 17.1% to 32.3%) versus 11.6% for MVAC (95% CI, 5.9% to 17.4%). CONCLUSION With HD-MVAC, it was possible to deliver twice the doses of cisplatin and doxorubicin in half the time, with fewer dose delays and less toxicity. Although a 50% difference in median overall survival was not detected, a benefit was observed in progression-free survival, CR rates, and overall response rates with HD-MVAC.

Intensive induction-sequential chemotherapy with BOP/VIP-B compared with treatment with BEP/EP for poor-prognosis metastatic nonseminomatous germ cell tumor: a Randomized Medical Research Council/European Organization for Research and Treatment of Cancer study

PURPOSE The aim of this randomized trial was to assess the potential therapeutic advantage of an intensive induction-sequential chemotherapy schedule (bleomycin, vincristine, cisplatin [BOP])/etoposide, ifosfamide, cisplatin, and bleomycin [VIP-B]), compared with a regimen based on bleomycin, etoposide, and cisplatin (BEP) (BEP/etoposide and cisplatin [EP]) for the treatment of patients with poor-prognosis metastatic nonseminomatous germ cell tumors (NSGCTs). PATIENTS AND METHODS Patients had one or more of the following: a retroperitoneal mass > or = 10 cm in diameter; mediastinal or supraclavicular mass > or = 5 cm in diameter; at least 20 lung metastases (any size); liver, bone, or brain metastases; and serum beta human chorionic gonadotropin (betaHCG) > or = 10,000 IU/L or alfa fetoprotein (AFP) > or = 1,000 IU/L. A total of 380 patients were accrued between May 1990 and June 1994 into this joint Medical Research Council (MRC)/European Organization for Research and Treatment of Cancer (EORTC) trial; of these, nine patients were deemed ineligible. RESULTS There was no significant difference between the two arms in the proportion of patients who achieved a complete response (CR) with chemotherapy alone, ie, 79 of 185 assessable patients (57%) with BEP/EP and 72 of 186 (54%) with BOP/VIP-B (P = 0.687). With a median follow-up of 3.1 years (maximum, 5.8), a total of 107 patients (28%) had progressive disease. There was no significant difference in time to first disease progression, or failure-free or overall survival between the two arms (P = 0.21, 0.101, and 0.190, respectively). The 1-year failure-free survival rates for BEP/EP and BOP/VIP-B were 60% (95% confidence interval [CI], 53% to 67%) and 53% (95% CI, 47% to 61%). Grade 3 or 4 myelosuppression, febrile neutropenia, and weight loss were more pronounced with BOP/VIP-B than with BEP/EP, and there were more toxic deaths with BOP/VIP-B than BEP/EP (18 [9%] v nine [5%]). CONCLUSION The intensive BOP/VIP-B therapy was associated with more toxicity, but there was no evidence of an improvement in response rate or survival compared with treatment with BEP/EP.

Thymidylate synthase and drug resistance

Thymidylate synthase is an important target for both fluorinated pyrimidines and for new folate analogues. Resistance to 5-fluorouracil (5FU) can be related to insufficient inhibition of thymidylate synthase. The 5FU-nucleotide FdUMP induces inhibition of thymidylate synthase which is enhanced and retained for longer in the presence of increased folate pools, for which leucovorin is a precursor. In a murine model system, 5FU treatment caused a 4-fold induction of thymidylate synthase levels which may have contributed to resistance. Addition of leucovorin to this treatment prevented this induction and increased the antitumour effect 2-3-fold. In the clinical setting, 5FU administration to patients resulted in approximately 50% inhibition of TS after 48 h. The combination with leucovorin resulted in a more pronounced inhibition after 48 h (approximately 70%). A significant relationship was observed with outcome of treatment; when thymidylate synthase levels were high and inhibition was low, no response was observed. A separate study showed that low thymidylate synthase levels appeared to be an independent prognostic factor for adjuvant therapy.

Clinical activity and benefit of irinotecan (CPT-11) in patients with colorectal cancer truly resistant to 5-fluorouracil (5-FU).

The aim of this prospective study was to assess the efficacy, clinical benefit and safety of CPT-11 (irinotecan) in patients with stringently-defined 5-fluorouracil-resistant metastatic colorectal cancer (CRC). 107 patients with documented progression of metastatic CRC during 5-FU were treated with CPT-11 350 mg/m2 once every 3 weeks in a multicentre phase II study. Tumour response and toxicity were assessed using WHO criteria. Changes in performance status (PS), weight and pain were also measured. The WHO response rate was 13/95 (13.7%, 95% CI 7.5% to 22.3%) eligible patients with a median duration of response of 8.5 months (37 weeks, range: 18-53+). There was also a high rate of disease stabilisation (44.2%) with a median duration of 4.8 months. The probability of being free of progression at 4 months was 50%. Median survival from first administration of CPT-11 was 10.4 months or 45 weeks (range: 3-66+ weeks). There was weight stabilisation or gain in 81% (73/90) of patients, a favourable outcome in PS in 91% (82/90) (improvement of WHO PS 2 or stabilisation of PS 0-1), and pain relief in 54% (26/48). There were no toxic deaths. Neutropenia was short-lasting and non-cumulative. Diarrhoea grade > or = 3 occurred in 7% of cycles and 28/107 (26%) of patients. CPT-11 350 mg/m2 once every 3 weeks has an encouraging degree of activity in progressive metastatic CRC truly resistant to 5-FU with a relatively high rate of tumour growth control translated into clinical benefit. The toxicity profile of CPT-11 is becoming better understood and has been considerably improved.

Prolonged retention of high concentrations of 5-fluorouracil in human and murine tumors as compared with plasma

SummaryConcentrations of 5-fluorouracil (5-FU) and its active metabolite 5-fluoro-2′-deoxy-5′-monophosphate (FdUMP) were measured in biopsy specimens of tumor tissue, normal mucosa, metastatic liver nodules, and normal liver tissue obtained from 39 patients and in two murine colon tumors (colon 26 and colon 38) after a single injection of 5FU at a therapeutic dose (500 mg/m2 and 100 mg/kg, respectively). These data were compared with plasma concentrations. Peak plasma concentrations (300–500 μm) of 5FU were comparable in human and murine plasma. The half-life of plasma elimination (during the period from 15 to 120 min) in both mouse and man ranged from 10 to 20 min, whereas at between 2 and 8 h, plasma concentrations varied from 0.1 to 1 μm, the half-life being about 100 min. In both species, 5FU could be measured in plasma at concentrations ranging from 0.01 to 1 μm for several days after 5FU treatment. 5FU concentrations in tissue samples obtained from 14 patients were measured during the time range of 1–6 h, those in samples taken from 7 patients, during the interval of 19–27 h; and those in samples obtained from 18 patients, within the interval of 40–48 h after injection. 5FU tumor concentrations varied between 0.78–21.6, 0.44–6.1, and 0.17–10.8 μmol/kg wet wt., respectively. Some of the 48-h samples were obtained from patients who had received leucovorin plus 5FU; coadministration of leucovorin did not alter 5FU tissue concentrations. At between 4 and 48 h, the tissue concentration/plasma concentration ratio was at least 10. 5FU concentrations in murine tumors were measured for up to 10 days after 5FU administration, with plateau 5FU tumor concentrations being about 50 μmol/kg wet wt. in colon 38 and about 200 μmol/kg wet wt. in colon 26 at 2 h after treatment; after 4 days, values of 0.5 and 4.8 μmol/kg, respectively, were obtained and after 10 days, respective concentrations of 0.1 and 0.07 μmol/kg were detected. The FdUMP concentrations measured in colon 26 and colon 38 tumors were 214 and 46 pmol/g, respectively, at 2 h after 5FU administration, and these values subsequently decreased to about 15 pmol/g in both tumors. In human tumors the initial FdUMP concentration ranged from 10 to 1000 pmol/g; at later time points the level of FdUMP was just above the detection limit of the assay. In liver metastases, high 5FU concentrations seemed to be related to high levels of FdUMP, which was likely of importance for the antitumor effect. The prolonged retention of 5FU should be taken into consideration in the design of biochemical modulation studies.

Gemcitabine-radiotherapy in patients with locally advanced pancreatic cancer

A feasibility study was performed to assess the toxicity and efficacy of a combination of gemcitabine-radiotherapy in patients with locally advanced pancreatic cancer (LAPC). 24 patients (15 females and 9 males) with measurable LAPC were included; the median age of the patients was 63 years (range 39-74 years). The performance status ranged from 0 to 2. Gemcitabine was administered at a dose of 300 mg/m(2), concurrent with radiotherapy, three fractions of 8 Gy, on days 1, 8 and 15. When compliance allowed, gemcitabine alone was continued thereafter, at 1000 mg/m(2), weekly times 3, every 4 weeks, depending on the response and toxicity. All patients were evaluable for toxicity and response. The objective response rate was 29.2% (1 complete remission+6 partial remissions); 12 patients had stable disease. However, 2 of the radiological partial remissions were shown to be complete remissions by pathology assessment. Median duration of response was 3 months (range 1-35+months). Median time to progression was 7 months (range 2-37+months). Median survival was 10 months (range 3-37+months). Dose reduction or omission of gemcitabine was necessary in 10 patients. Non-haematological toxicity consisted of 87.5% nausea and vomiting grade I-II, diarrhoea 54%, ulceration in stomach and duodenum 37.5% (20.8% ulceration with bleeding); 1 patient developed a fistula between the duodenum and aorta, 5 months after treatment. Anaemia grade III-IV was observed in 8.3% of the patients. Neutropenia grade III-IV was observed in 8.3%, thrombocytopenia grades III-IV in 16.7%. In 1 patient who underwent resection postchemoradiation, no viable tumour cells were found. In addition, in the patient who suddenly died of a fistula between the duodenum and aorta, no viable tumour cells were detectable at autopsy. Although the toxicity of this treatment was occasionally severe, the response and survival are encouraging and warrant further studies of this combination.

Sequence dependent effect of paclitaxel on gemcitabine metabolism in relation to cell cycle and cytotoxicity in non-small-cell lung cancer cell lines

Gemcitabine and paclitaxel are active agents in the treatment of non-small-cell lung cancer (NSCLC). To optimize treatment drug combinations, simultaneously and 4 and 24 h intervals, were studied using DNA flow cytometry and multiple drug effect analysis in the NSCLC cell lines H460, H322 and Lewis Lung. All combinations resulted in comparable cytotoxicity, varying from additivity to antagonism (combination index: 1.0–2.6). Gemcitabine caused a S (48%) and G1 (64%) arrest at IC-50 and 10 × IC-50 concentrations, respectively. Paclitaxel induced G2/M arrest (70%) which was maximal within 24 h at 10 × IC-50. Simultaneous treatment increased S-phase arrest, while at the 24 h interval after 72 h the first drug seemed to dominate the effect. Apoptosis was more pronounced when paclitaxel preceded gemcitabine (20% for both intervals) as compared to the reverse sequence (8%, P = 0.173 for the 4 h and 12%, P = 0.051 for the 24 h time interval). In H460 cells, paclitaxel increased 2-fold the accumulation of dFdCTP, the active metabolite of gemcitabine, in contrast to H322 cells. Paclitaxel did not affect deoxycytidine kinase levels, but ribonucleotide levels increased possibly explaining the increase in dFdCTP. Paclitaxel did not affect gemcitabine incorporation into DNA, but seemed to increase incorporation into RNA. Gemcitabine almost completely inhibited DNA synthesis in both cell lines (70–89%), while paclitaxel had a minor effect and did not increase that of gemcitabine. In conclusion, various gemcitabine–paclitaxel combinations did not show sequence dependent cytotoxic effects; all combinations were not more than additive. However, since paclitaxel increased dFdCTP accumulation, gemcitabine incorporation into RNA and the apoptotic index, the administration of paclitaxel prior to gemcitabine might be favourable as compared to reversed sequences.

Gemcitabine–cisplatin: A schedule finding study

PURPOSE To evaluate the tolerability of four alternating cisplatin-gemcitabine schedules. A secondary aim was to evaluate the clinical efficacy of this combination. PATIENTS AND METHODS Forty-one patients with advanced solid tumors received alternating sequences with a 4- and 24-hour interval of cisplatin and gemcitabine. Gemcitabine 800 mg/m2 was administered as a 30-min infusion on day 1, 8 and 15, and cisplatin 50 mg/m2 over 1 hour on day 1 and 8; in case of the 24-hour time interval the second drug was administered one day later. Four cisplatin-gemcitabine schedules were studied: gemcitabine four hour before cisplatin (10 patients), or vice versa (14 patients) and gemcitabine twenty-four hours before cisplatin (9 patients) or vice versa (8 patients). The sequence of drug administration was reversed in the second cycle of therapy in each individual patient, enabling the evaluation of sequence-dependent side effects. Twenty-six patients had received prior chemotherapy, of which twenty-one platinum-based. RESULTS The main toxicity was myelosuppression. Overall, grade 3 and 4 thrombocytopenia was observed in 27 out of 41 patients (66%) and was not schedule dependent. No serious bleeding occurred. Leukopenia was significantly different between the 4 alternating schedules (P = 0.01); gemcitabine 24 hours before cisplatin was significantly less toxic compared to both cisplatin 4 hours and 24 hours before gemcitabine (P = 0.01 and P = 0.003, respectively). Furthermore, paired analysis of the 4-hour and 24-hour data sets showed that leukopenia was significantly more serious when cisplatin preceded gemcitabine (P = 0.005). Although most patients received prior treatment, both prior chemotherapy and radiotherapy were not related to toxicity. Overall, grade 3 and 4 leukopenia occurred in 19 out of 41 patients (46%). Anemia (Hb < or = 6.0 mmol/l) was not sequence dependent and was observed in 63% of patients. Myelotoxicity was cumulative between cycles and caused frequent omission of gemcitabine on day 15. Overall, in 51% of administered cycles there was no omission of gemcitabine. A mean of 3.5 therapy cycles was administered. Non-hematological toxicity was moderate, consisting mainly of grade 1 and 2 nausea/vomiting and fatigue, and was not schedule dependent. Recently, we described that the schedule in which cisplatin was administered 24 hours before gemcitabine produced the best pharmacological profile. Based on this and because toxicity was manageable, the schedule cisplatin 24 hours prior to gemcitabine was chosen for phase II evaluation. Nine out of thirty-six evaluable patients had an objective response. These responses were observed in head and neck squamous-cell carcinoma (HNSCC), non-small-cell lung cancer (NSCLC), melanoma, adenocarcinoma of unknown origin, ovarian and esophageal carcinoma. CONCLUSIONS Myelosuppression was the most important toxicity. Leukopenia was schedule dependent: gemcitabine before cisplatin was less toxic than the reversed sequence, in this respect. Some encouraging responses were seen in patients with esophageal cancer. Currently, a phase II study with cisplatin 24 hours before gemcitabine is ongoing in patients with advanced upper gastro-intestinal tumors.

Gemcitabine uptake in glioblastoma multiforme: potential as a radiosensitizer

Glioblastoma multiforme (GBM), the most frequent malignant brain tumor, has a poor prognosis, but is relatively sensitive to radiation. Both gemcitabine and its metabolite difluorodeoxyuridine (dFdU) are potent radiosensitizers. The aim of this phase 0 study was to investigate whether gemcitabine passes the blood-tumor barrier, and is phosphorylated in the tumor by deoxycytidine kinase (dCK) to gemcitabine nucleotides in order to enable radiosensitization, and whether it is deaminated by deoxycytidine deaminase (dCDA) to dFdU. Gemcitabine was administered at 500 or 1000 mg/m(2) just before surgery to 10 GBM patients, who were biopsied after 1-4 h. Plasma gemcitabine and dFdU levels varied between 0.9 and 9.2 microM and 24.9 and 72.6 microM, respectively. Tumor gemcitabine and dFdU levels varied from 60 to 3580 pmol/g tissue and from 29 to 72 nmol/g tissue, respectively. The gene expression of dCK (beta-actin ratio) varied between 0.44 and 2.56. The dCK and dCDA activities varied from 1.06 to 2.32 nmol/h/mg protein and from 1.51 to 5.50 nmol/h/mg protein, respectively. These enzyme levels were sufficient to enable gemcitabine phosphorylation, leading to 130-3083 pmol gemcitabine nucleotides/g tissue. These data demonstrate for the first time that gemcitabine passes the blood-tumor barrier in GBM patients. In tumor samples, both gemcitabine and dFdU concentrations are high enough to enable radiosensitization, which warrants clinical studies using gemcitabine in combination with radiation.

5-Fluorouracil induced Fas upregulation associated with apoptosis in liver metastases of colorectal cancer patients

BACKGROUND In vitro, thymidylate synthase (TS) inhibition by 5-fluorouracil (5-FU) induces thymineless apoptosis possibly via Fas receptor Fas ligand interactions and cell-cycle arrest. In colorectal cancer patients we evaluated whether 5-FU administration also resulted in apoptosis and cell-cycle arrest and which proteins might be involved. PATIENTS AND METHODS Biopsy specimens were taken from 36 patients 2, 22 or 46 hours after administration of 500 mg/m2 5-FU, and from 12 control patients who did not receive 5-FU. In frozen tissue-sections from liver metastases immunohistochemistry was performed with antibodies directed against p53, p21, E2F2, Rb, Ki67 and TS (cell-cycle related) and bax, BCL-2, BCL-x, mcl-1, PARP, caspase-3, Fas receptor and Fas ligand (apoptosis related). Apoptosis was determined by M30 immunostaining, which recognises a cleavage product of cytokeratin 18. RESULTS Fas receptor expression was 50% higher (P = 0.036) 46 hours after 5-FU administration compared to the control group. This was associated with a 12% increase (P < 0.02) in M30 positive tumour cells and with elevation of caspase-3 and PARP expression. The expression of Ki67 and E2F2 was 30% lower after 46 hours compared to the control group, whereas TS was 56% lower after 2 hours and 32% higher again after 46 hours. No differences in the expression of the other proteins were found. CONCLUSIONS These results suggest that 5-FU decreases proliferation status and induces apoptosis possibly via the Fas pathway. Since Fas mediated cell killing is important for cytotoxic T cells this indicates that clinical studies combining immunotherapy for activation of T cells and chemotherapy using 5-FU might be very effective.