Systematic review and meta-analysis of randomised, phase II/III trials adding bevacizumab to platinum-based chemotherapy as first-line treatment in patients with advanced non-small-cell lung cancer.

BACKGROUND: Previous studies have demonstrated the efficacy and safety of bevacizumab in the treatment of non-small-cell lung cancer (NSCLC). METHODS: Summary data from randomised trials comparing first-line bevacizumab plus platinum-based chemotherapy with chemotherapy alone for inoperable locally advanced, recurrent or metastatic NSCLC were meta-analysed. Pooled hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS), and pooled odds ratio (OR) for adverse events were calculated. The chi-squared tests evaluated interactions between treatment effects, and prognostic factors and patient characteristics. RESULTS: Data of 2194 patients (1313 bevacizumab; 881 controls) from four phase II and III trials: AVF-0757g, JO19907,ECOG 4599 and AVAiL, were analysed. Compared with chemotherapy alone, bevacizumab significantly prolonged OS(HR 0.90; 95% confidence interval [CI] 0.81, 0.99; P=0.03), and PFS (0.72; 95% CI 0.66, 0.79; P<0.001). Bevacizumab showed a significantly greater effect on OS in patients with adenocarcinoma versus other histologies (P=0.03), and patients with body weight loss ≤5% versus >5% (P=0.04). Bevacizumab significantly increased the risk of grade ≥3 proteinuria, hypertension,haemorrhagic events, neutropenia, and febrile neutropenia [corrected]. CONCLUSIONS: Bevacizumab significantly prolonged OS and PFS when added to first-line platinum-based chemotherapy in patients with advanced NSCLC; no unexpected toxicity was evident.

Induction chemotherapy followed by gefitinib and concurrent thoracic radiotherapy for unresectable locally advanced adenocarcinoma of the lung: a multicenter feasibility study (JCOG 0402).

BACKGROUND: We conducted a feasibility study of induction chemotherapy followed by gefitinib and thoracic radiotherapy (TRT) for unresectable locally advanced adenocarcinoma of the lung. PATIENTS AND METHODS: Patients received induction chemotherapy with cisplatin (80 mg/m(2), days 1 and 22) and vinorelbine (25 mg/m(2), days 1, 8, 22, and 29) followed by gefitinib (250 mg daily, beginning on day 43, for 1 year) and TRT (60 Gy/30 fractions, days 57-98). The primary end point was feasibility, which was defined as the proportion of patients who completed 60 Gy of TRT and received >75% of the planned dose of gefitinib without developing grade 2 or worse pneumonitis. RESULTS: Of the 38 enrolled patients, 23 patients [60.5% ; 80% confidence interval (CI) 48.8-71.3] completed treatment without experiencing grade 2 or worse pneumonitis. During the chemoradiation phase, grade 3-4 alanine aminotransferase elevations were observed in 37.1% of the patients. The overall response rate was 73.0% . The median survival time was 28.5 months (95% CI 22.5-38.2), and the 2-year survival rate was 65.4% . CONCLUSIONS: Although the results did not meet our criterion for feasibility, the toxicity was acceptable. This treatment warrants further evaluation among patients with locally advanced non-small-cell lung cancer harboring epidermal growth factor receptor mutations.

Randomised, phase III trial of epoetin-ß to treat chemotherapy-induced anaemia according to the EU regulation.

BACKGROUND: Erythropoietin-stimulating agents (ESAs) effectively decrease the transfusion requirements of patients with chemotherapy-induced anaemia (CIA). Recent studies indicate that ESAs increase mortality and accelerate tumour progression. The studies also identify a 1.6-fold increased risk of venous thromboembolism. The ESA labelling was thus revised in Europe and the United States in 2008. This is the first randomised, phase III trial evaluating the efficacy and safety of epoetin-ß (EPO), an ESA, dosed in accordance with the revised labelling, which specifies that ESAs should be administered to CIA patients with a haemoglobin level of ≤ 10 g dl⁻¹ and that a sustained haemoglobin level of > 12 g dl⁻¹ should be avoided. METHODS: A total of 186 CIA patients (8.0 g dl⁻¹ ≤ haemoglobin ≤ 10.0 g dl⁻¹) with lung or gynaecological cancer were randomised to receive EPO 36,000 IU or placebo weekly for 12 weeks. RESULTS: The proportion of patients receiving transfusions or with haemoglobin < 8.0 g dl⁻¹ between week 5 and the end of the treatment period as the primary end point was significantly lower in the EPO group (n=89) than in the placebo group (n=92; 10.0% vs 56.4%, P < 0.001). The proportion receiving transfusions was significantly lower in the EPO group (4.5% vs 19.6%, P=0.002). Changes in quality of life were not different. No significant differences in adverse events - for example, the incidence of thromboembolic events was 1.1% for each group - or the 1-year overall survival were observed between groups. CONCLUSION: Weekly EPO administered according to the revised labelling approved by the European Medicines Agency is effective and well tolerated for CIA treatment. Further investigations are needed on the effect of ESAs on mortality.

A phase II trial of dose-dense chemotherapy, followed by surgical resection and/or thoracic radiotherapy, in locally advanced thymoma: report of a Japan Clinical Oncology Group trial (JCOG 9606).

BACKGROUND: This study aimed to evaluate the safety and efficacy of dose-dense weekly chemotherapy, followed by resection and/or thoracic radiotherapy. METHODS: Patients with histologically documented thymoma with unresectable stage III disease received 9 weeks of chemotherapy: cisplatin 25 mg m(-2) on weeks 1-9; vincristine 1 mg m(-2) on weeks 1, 2, 4, 6 and 8; and doxorubicin 40 mg m(-2) and etoposide 80 mg m(-2) on days 1-3 of weeks 1, 3, 5, 7 and 9. Patients went on to surgery and post-operative radiotherapy of 48 Gy; those with unresectable disease received 60 Gy radiotherapy. RESULTS: total of 23 patients were entered. The main toxicities of the chemotherapy regimen were neutropenia and anaemia, and 57% of patients completed the planned 9 weeks of therapy. There were no toxic deaths. Of the 21 eligible patients, 13 (62%) achieved a partial response (95% confidence interval: 38-82%). Thirteen patients underwent a thoracotomy and nine (39%) underwent complete resection. Progression-free survival at 2 and 5 years was 80 and 43%, respectively. Overall survival at 5 and 8 years was 85 and 69%, respectively. Survival did not seem to be affected by resection. CONCLUSION: In thymoma patients, weekly dose-dense chemotherapy has activity similar to that of conventional regimens. Although some patients could achieve complete resection, the role of surgery remains unclear.

Association between gain-of-function mutations in PIK3CA and resistance to HER2-targeted agents in HER2-amplified breast cancer cell lines.

BACKGROUND: The mechanism of resistance to human epidermal growth factor receptor 2 (HER2)-targeted agents has not been fully understood. We investigated the influence of PIK3CA mutations on sensitivity to HER2-targeted agents in naturally derived breast cancer cells. MATERIALS AND METHODS: We examined the effects of Calbiochem (CL)-387,785, HER2 tyrosine kinase inhibitor, and trastuzumab on cell growth and HER2 signaling in eight breast cancer cell lines showing HER2 amplification and trastuzumab-conditioned BT474 (BT474-TR). RESULTS: Four cell lines with PIK3CA mutations (E545K and H1047R) were more resistant to trastuzumab than the remaining four without mutations (mean percentage of control with 10 microg/ml trastuzumab: 58% versus 92%; P = 0.010). While PIK3CA-mutant cells were more resistant to CL-387,785 than PIK3CA-wild-type cells (mean percentage of control with 1 microM CL-387,785: 21% versus 77%; P = 0.001), CL-387,785 retained activity against BT474-TR. Growth inhibition by trastuzumab and CL-387,785 was more closely correlated with changes in phosphorylation of S6K (correlation coefficient, 0.811) than those of HER2, Akt, or ERK1/2. Growth of most HER2-amplified cells was inhibited by LY294002, regardless of PIK3CA genotype. CONCLUSIONS: PIK3CA mutations are associated with resistance to HER2-targeted agents. PI3K inhibitors are potentially effective in overcoming trastuzumab resistance caused by PIK3CA mutations. S6K phosphorylation is a possibly useful pharmacodynamic marker in HER2-targeted therapy.

A randomised trial of intrapericardial bleomycin for malignant pericardial effusion with lung cancer (JCOG9811).

Safety and efficacy of intrapericardial (i.p.c.) instillation of bleomycin (BLM) following pericardial drainage in patients with malignant pericardial effusion (MPE) remain unclear. Patients with pathologically documented lung cancer, who had undergone pericardial drainage for MPE within 72 h of enrolment, were randomised to either arm A (observation alone after drainage) or arm B (i.p.c. BLM at 15 mg, followed by additional i.p.c. BLM 10 mg every 48 h). The drainage tube was removed when daily drainage was 20 ml or less. The primary end point was survival with MPE control (effusion failure-free survival, EFFS) at 2 months. Eighty patients were enrolled, and 79 were eligible. Effusion failure-free survival at 2 months was 29% in arm A and 46% in arm B (one-sided P=0.086 by Fisher's exact test). Arm B tended to favour EFFS, with a hazard ratio of 0.64 (95% confidence interval: 0.40-1.03, one-sided P=0.030 by log-rank test). No significant differences in the acute toxicities or complications were observed. The median survival was 79 days and 119 days in arm A and arm B, respectively. This medium-sized trial failed to show statistical significance in the primary end point. Although ipc BLM appeared safe and effective in the management of MPE, the therapeutic advantage seems modest.

A phase-II trial of dose-dense chemotherapy in patients with disseminated thymoma: report of a Japan Clinical Oncology Group trial (JCOG 9605).

BACKGROUND: To evaluate the safety and efficacy of dose-dense weekly chemotherapy in the treatment of advanced thymoma. METHODS: Subjects comprised patients with histologically documented chemotherapy-naïve thymoma with stage-IVa or IVb disease. Thymic carcinoma, carcinoid or lymphoma cases were excluded. Patients received 9 weeks of chemotherapy: cisplatin (25 mg m(-2)) on weeks 1-9; vincristine (1 mg m(-2)) on weeks 1, 2, 4, 6 and 8; and doxorubicin (40 mg m(-2)) and etoposide (80 mg m(-2)) on days 1-3 of weeks 1, 3, 5, 7 and 9. Chemotherapy courses were supported by granulocyte colony-stimulating factor. Post-protocol local therapy was allowed. RESULTS: From July 1997 to March 2004, 30 patients were entered. Three were ineligible due to different histology. Chemotherapy-associated toxicity was mainly haematological and was well tolerated, with no deaths due to toxicity, and 87% of patients completed the planned 9-week regimen. Overall response rate was 59%, with 16 of the 27 eligible patients achieving partial response. Median progression-fee survival (PFS) was 0.79 years (95% confidence interval: 0.52-1.40 years), and PFS at 1 and 2 years was 37 and 15%, respectively. Overall survival rates at 2 and 5 years were 89 and 65%, respectively. CONCLUSION: In stage-IV thymoma patients, weekly dose-dense chemotherapy offers similar activity to conventional regimens.

Homozygous CDA*3 is a major cause of life-threatening toxicities in gemcitabine-treated Japanese cancer patients.

Among 242 Japanese pancreatic cancer patients, three patients (1.2%) encountered life-threatening toxicities, including myelosuppression, after gemcitabine-based chemotherapies. Two of them carried homozygous CDA*3 (CDA208G>A [Ala70Thr]), and showed extremely low plasma cytidine deaminase activity and gemcitabine clearance. Our results suggest that homozygous *3 is a major factor causing gemcitabine-mediated severe adverse reactions among the Japanese population.

Phase III trial of docetaxel plus gemcitabine versus docetaxel in second-line treatment for non-small-cell lung cancer: results of a Japan Clinical Oncology Group trial (JCOG0104).

BACKGROUND: This trial evaluated whether a combination of docetaxel and gemcitabine provides better survival than docetaxel alone in patients with previously treated non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Eligibility included pathologically or cytologically proven NSCLC, failure of one platinum-based regimen, performance status of zero or one, 20-75 years old, and adequate organ function. Patients received docetaxel 60 mg/m(2) (day 1) or docetaxel 60 mg/m(2) (day 8) and gemcitabine 800 mg/m(2) (days 1 and 8), both administered every 21 days until disease progression. RESULTS: Sixty-five patients participated in each arm. This trial was terminated early due to an unexpected high incidence of interstitial lung disease (ILD) and three treatment-related deaths due to ILD in the combination arm. Docetaxel plus gemcitabine compared with docetaxel-alone patients experienced similar grade and incidence of toxicity, except for ILD. No baseline factor was identified for predicting ILD. Median survival times were 10.3 and 10.1 months (one-sided P = 0.36) for docetaxel plus gemcitabine and docetaxel arms, respectively. CONCLUSION: Docetaxel alone is still the standard second-line treatment for NSCLC. The incidence of ILD is higher for docetaxel combined with gemcitabine than for docetaxel alone in patients with previously treated NSCLC.

Quality of life and disease-related symptoms in previously treated Japanese patients with non-small-cell lung cancer: results of a randomized phase III study (V-15-32) of gefitinib versus docetaxel.

BACKGROUND: This report describes quality of life (QoL) findings of a randomized study comparing gefitinib with docetaxel in patients with advanced/metastatic pretreated non-small-cell lung cancer. PATIENTS AND METHODS: This open-label, phase III study randomized 490 Japanese patients to gefitinib (250 mg/day) or docetaxel (60 mg/m(2)/3 weeks), with survival as the primary outcome. Preplanned QoL analyses included Functional Assessment of Cancer Therapy-Lung (FACT-L), Trial Outcome Index (TOI) and Lung Cancer Subscale (LCS) improvement rates, and mean change from baseline. RESULTS: Gefitinib showed statistically significant benefits over docetaxel in QoL improvement rates (FACT-L 23% versus 14%, P = 0.023; TOI 21% versus 9%, P = 0.002) and mean change from baseline score [mean treatment difference: FACT-L 3.72 points, 95% confidence interval (CI) 0.55-6.89, P = 0.022; TOI 4.31 points, 95% CI 2.13-6.49, P < 0.001], although differences did not meet the clinically relevant six-point change. There were no significant differences between treatments in LCS improvement rates (23% versus 20%, P = 0.562) or mean change from baseline score (0.63 points, 95% CI -0.07 to 1.34, P = 0.077). CONCLUSIONS: Gefitinib improved aspects of QoL over docetaxel, with superior objective response rate and a more favorable tolerability profile and no statistically significant difference in overall survival (although noninferiority was not statistically proven).

A new statistical screening approach for finding pharmacokinetics-related genes in genome-wide studies.

Biomedical researchers usually test the null hypothesis that there is no difference of the population mean of pharmacokinetics (PK) parameters between genotypes by the Kruskal-Wallis test. Although a monotone increasing pattern with a number of alleles is expected for PK-related genes, the Kruskal-Wallis test does not consider a monotonic response pattern. For detecting such patterns in clinical and toxicological trials, a maximum contrast method has been proposed. We show how that method can be used with pharmacogenomics data to a develop test of association. Further, using simulation studies, we compare the power of the modified maximum contrast method to those of the maximum contrast method and the Kruskal-Wallis test. On the basis of the results of those studies, we suggest rules of thumb for which statistics to use in a given situation. An application of all three methods to an actual genome-wide pharmacogenomics study illustrates the practical relevance of our discussion.

Emerging ethnic differences in lung cancer therapy.

Although global clinical trials for lung cancer can enable the development of new agents efficiently, whether the results of clinical trials performed in one population can be fully extrapolated to another population remains questionable. A comparison of phase III trials for the same drug combinations against lung cancer in different countries shows a great diversity in haematological toxicity. One possible reason for this diversity may be that different ethnic populations may have different physiological capacities for white blood cell production and maturation. In addition, polymorphisms in the promoter and coding regions of drug-metabolising enzymes (e.g., CYP3A4 and UGT1A1) or in transporters (e.g., ABCB1) may vary among different ethnic populations. For example, epidermal growth factor receptor (EGFR) inhibitors are more effective in Asian patients than in patients of other ethnicities, a characteristic that parallels the incidence of EGFR-activating mutations. Interstitial lung disease associated with the administration of gefitinib is also more common among Japanese patients than among patients of other ethnicities. Although research into these differences has just begun, these studies suggest that possible pharmacogenomic and tumour genetic differences associated with individual responses to anticancer agents should be carefully considered when conducting global clinical trials.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is activated by cis-diamminedichloroplatinum(II).

We have investigated the effect of cis-diamminedichloroplatinum(II) (CDDP) on signal transduction pathways. CDDP treatment did not cause any change in the binding of [3H]-phorbol dibutyrate to PC-9 (human lung adenocarcinoma cell line) cells, a measure of protein kinase C activation. However, 2-h CDDP treatment (20 micrograms/ml) caused approximately 200% increase in 1,2-sn-diacylglycerol (DAG) production and approximately 50% decrease in inositol 1,4,5-triphosphate production. To explore the different source of DAG, we analyzed phospholipids labeled with [14C]choline by TLC and revealed that [14C]choline-labeled phosphatidylcholine (PC) was decreased to 50% by CDDP treatment. This suggested that PC turnover was increased by CDDP-treatment. PC-specific phospholipase C (PC-PLC) activity was increased to 2.5-fold (2.58 +/- 0.28 nmol/mg protein per min) by 2 h CDDP (20 micrograms/ml) treatment compared with control (1.05 +/- 0.24 nmol/mg protein per min). Treatment of CDDP also stimulated PC-PLC in the crude membrane extract from PC-9 cells. CDDP had no effect on the activities of phospholipase A2 and D. Trans-DDP, which has far less cytotoxicity than its stereoisomer, CDDP, did not cause any change in PC-PLC activity. A significant inhibition of DNA synthesis (less than 80%) occurred 4 h after 2 h CDDP (20 micrograms/ml) treatment. These results demonstrated that CDDP-induced PC-PLC activation was an early event in CDDP-induced cytotoxicity and suggested that the effects of CDDP on signal transduction pathways had an important role in CDDP-induced cytotoxicity.

Randomized trial of cyclophosphamide, doxorubicin, and vincristine versus cisplatin and etoposide versus alternation of these regimens in small-cell lung cancer.

Between April 1985 and May 1988, we conducted a randomized study comparing two standard chemotherapy regimens with the same regimens given on an alternating basis in patients with small-cell lung cancer. The patients were randomly assigned to receive cyclophosphamide at a dose of 800 mg/m2 intravenously (IV) on day 1, doxorubicin at 50 mg/m2 IV on day 1, and vincristine at 1.4 mg/m2 IV on day 1 (CAV); cisplatin at 80 mg/m2 IV on day 1 and etoposide at 100 mg/m2 IV on days 1, 3, and 5 (PE); or CAV alternating with PE (CAV/PE). Each regimen was repeated every 3-4 weeks. Three hundred patients were entered in the study, and 288 of them were eligible for analysis (97 for CAV, 97 for PE, and 94 for CAV/PE). The response rates for PE (78%) and CAV/PE (76%) were significantly higher than the rate for CAV (55%), while the complete response rates were similar (14%, 16%, and 15%, respectively). Nine (23%) of 39 patients who failed to respond to the initial CAV regimen responded to PE when they were crossed over. In contrast, only one (8%) of 13 patients responded to CAV after failing to respond to the PE regimen, suggesting that these two regimens were partially non-cross-resistant. The response duration on CAV/PE was significantly longer than that with CAV (P = .004). The survival time with CAV/PE (11.8 months) was superior to that with CAV (9.9 months) (P = .027) or that with PE (9.9 months) (P = .056). In patients with limited disease, the survival in the alternating arm was significantly superior to the survival in the CAV arm (P = .014) or the survival in the PE arm (P = .023). The toxic effects were acceptable in all three chemotherapy regimens. These results favor the alternating chemotherapy over either standard chemotherapy, such as CAV and PE, although the differences are not dramatic.

Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines.

BACKGROUND: Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. PURPOSE: To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. METHODS: Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. RESULTS: Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. CONCLUSIONS: These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors.

Antitumor activities of a new indolocarbazole substance, NB-506, and establishment of NB-506-resistant cell lines, SBC-3/NB.

The novel anticancer glucosyl derivative of indolo-carbazole (NB-506), an inhibitor of DNA topoisomerase I, exhibited strong in vitro cytotoxicity against various human cancer cell lines. In order to elucidate its cytotoxic mechanisms, we established nine NB-506-resistant sublines with different resistance ratios from human small cell lung cancer cells (SBC-3/P) by stepwise and brief exposure (24 h) to NB-506. Among them, SBC-3/NB#9 was 454 times more resistant to NB-506 than the parent cell line. The SBC-3/NB#9 cells showed cross-resistance only to topoisomerase I inhibitors, such as 11,7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecia and 7-ethyl-10-hydroxy-camptothecin, and not to other anticancer drugs, such as vincristine, vinblastine, Adriamycin, etoposide, and teniposide. These results indicate that the difference on the effect of topoisomerase I was considered to be related to a resistance mechanism. The topoisomerase I activities of nuclear extracts eluted from SBC-3/NB#9 cells was only one-tenth of the parent cell activity. A Western blotting study indicated that this lower activity was due to a lower amount of DNA topoisomerase I. Furthermore, we found correlations between topoisomerase I activity and sensitivity to NB-506 in sublines with different degrees of resistance. Accumulation of 3H-labeled NB-506 by SBC-3/NB#9 cells was only one-fifth of that by the parent cells, whereas intracellular accumulation of 3H-labeled camptothecin by both cell lines did not differ. The reduction of accumulation was specific to NB-506, and this result may explain why the resistance ratio for NB-506 was higher than those for 11,7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-hydroxy-camptothecin.

Modulation of cisplatin sensitivity and accumulation by amphotericin B in cisplatin-resistant human lung cancer cell lines.

To ascertain whether resistance to cis-diamminedichloroplatinum(II) (cisplatin) could be overcome, we determined the effects of amphotericin B (AmB), an antifungal agent, on cisplatin cytotoxicity, cisplatin-induced DNA interstrand cross-links formation, and cellular accumulation of cisplatin in human lung cancer cell lines, PC-9, PC-14, PC-7, and H69 and their corresponding respective cisplatin-resistant sublines PC-9/CDDP, PC-14/CDDP, PC-7/CDDP, and H69/CDDP in vitro. In PC-9/CDDP but not PC-9 cells, augmentation of cytotoxicity was observed when a nontoxic concentration (10 micrograms/ml) of AmB was combined with cisplatin, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II), and cis-diammine(glycolato)platinum(II). Sensitizing effects of AmB of varying magnitudes on cisplatin cytotoxicity also were observed in all the other cell lines except PC-14. AmB-induced increases in cisplatin-induced interstrand cross-links formation were observed, the magnitudes of which corresponded to the magnitudes of AmB-augmented cisplatin cytotoxicity. Increased intracellular cisplatin accumulation was observed in the presence of AmB in all the cells that were sensitized to cisplatin by AmB. Therefore, the increases in cisplatin accumulation were considered to be responsible, at least in part, for the mechanism of the sensitizing effect. Further experiments using other human lung cancer cell lines showed that cells that were more resistant to cisplatin were more sensitized to cisplatin by AmB than cells that were cisplatin-sensitive.

In vivo antitumor activity of multiple injections of recombinant interleukin 2, alone and in combination with three different types of recombinant interferon, on various syngeneic murine tumors.

We have used several transplantable experimental murine tumors to evaluate the potentiation of antitumor activity by a combination of human recombinant interleukin 2 (rHIL2) and recombinant interferons (rIFNs). The combination of rHIL2 and either human hybrid recombinant alpha-interferon A/D (rIFN-alpha A/D) or mouse recombinant beta-interferon (rIFN-beta) induced the s.c. adenocarcinoma 755, which had been established for 8 days, to regress, although rHIL2 or the rIFNs alone hardly inhibited the tumor's growth. Eight injections of the rHIL2-rIFN-alpha A/D combination cured 38% of the tumor-bearing mice. The rHIL2-rIFN-beta combination achieved a complete cure only when given in more than 13 injections. The administration of rHIL2 and mouse recombinant gamma-interferon (rIFN-gamma) markedly inhibited tumor growth of the s.c. established adenocarcinoma 755, but did not cure any of the mice. Other tumors, B16-F10 melanoma, and colon tumors 38 and 26 responded almost as well to a rHIL2-rIFN-alpha A/D or -beta combination, but not to a rHIL2-rIFN-gamma combination. The growth of Lewis lung carcinoma was inhibited to a lesser extent by all combinations, for which there were no long-term survivors. The combination therapy of rHIL2 and rIFN-beta produced a marked regression of the tumor in beige mice which have low natural killer activity, suggesting the activated natural killer cells not to be responsible for the therapeutic effect. And T-cell immunity may be important in the regression of s.c. established tumors, because of the lesser potentiation of antitumor activity in athymic mice. These results demonstrate that combination therapies of rHIL2 and rIFN-alpha A/D or -beta can function synergistically in the various s.c. established murine tumor systems and give further evidence in support of their clinical potential.

Decreased accumulation as a mechanism of resistance to cis-diamminedichloroplatinum(II) in human non-small cell lung cancer cell lines: relation to DNA damage and repair.

The mechanism of resistance to cis-diamminedichloroplatinum(II) (CDDP) is still controversial, although several kinds of processes have been proposed. For elucidation of the mechanism of CDDP resistance in CDDP-resistant human non-small cell lung cancer cells (PC-9/0.5, 7.1-fold resistant), we examined the formation of DNA interstrand cross-links (ICL), one kind of DNA damage induced by CDDP, its repair, and intracellular accumulation of CDDP. We measured the frequency of CDDP-induced ICL by means of the alkaline elution technique and the amount of intracellular platinum for intracellular accumulation of CDDP by means of atomic absorption spectrophotometry in PC-9 (parental cell) and PC-9/0.5 cells. Formation of ICL in PC-9 cells exposed to 5 micrograms of CDDP per ml for 6 h was 5.85 times that in PC-9/0.5 cells. On the other hand, the ability to repair CDDP-induced ICL was identical in both cell lines. Intracellular accumulation studies revealed that PC-9 retained 5.07 times as much platinum as that in PC-9/0.5 after 3-h exposure to CDDP. It was conjectured that the decrease in the intracellular accumulation of CDDP might be the main cause of CDDP resistance in PC-9 cells, since the decreased accumulation was paralleled by the decreased level of ICL.

Mechanism of cross-resistance to a camptothecin analogue (CPT-11) in a human ovarian cancer cell line selected by cisplatin.

We established a cisplatin-resistant human ovarian cancer cell line (HAC2/0.1) from the parent cell line (HAC2/P) by continuous exposure of HAC2/P to 0.1 microgram of cisplatin/ml. Drug sensitivity determined by colony assay revealed that HAC2/0.1 was 2.4 times as resistant to cisplatin as the parental cell line. HAC2/0.1 was 12.1 and 2.0 times as resistant to (4s)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)-carbony loxy]dione hydrochloride trithydrate (CPT-11) and 7-ethyl-10-hydroxy-CPT (SN-38; an active metabolite of CPT-11), respectively, than HAC2/P. We studied the mechanism of cross-resistance to CPT-11 in HAC2/0.1. The glutathione (GSH) content was higher in HAC2/0.1 than in HAC2/P. The activity of DNA topoisomerase I and the accumulation of CPT-11 and SN-38 were also the same. On the other hand, the conversion of CPT-11 to SN-38 in HAC2/0.1 was about 3-fold less than in HAC2/P. Treatment of the parent and resistant cell lines with buthionine sulfoxamine (BSO) decreased the GSH content of both cell lines and decreased the 50% inhibitory concentrations of all the tested drugs for HAC2/0.1. The accumulation of CPT-11 in HAC2/0.1 but not in HAC2/P was increased by BSO treatment. On the other hand, in HAC2/P the 50% inhibitory concentrations of SN-38 and CPT-11 were not influenced by BSO treatment. The 50% inhibitory concentration of CPT-11 for HAC2/0.1 was not reduced by BSO treatment to the level for HAC2/P, even though the GSH content had been reduced more than in HAC2/P. These results show that there is no clear relationship between GSH and resistance to CPT-11. The decreased conversion of CPT-11 to SN-38 is considered to be the main cause of resistance to CPT-11 in this cell line.