Effect of Vandetanib on Lung Tumorigenesis in Transgenic Mice Carrying an Activating Egfr Gene Mutation.

Vandetanib (ZactimaTM) is a novel, orally available inhibitor of both vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinase. In the present study, a line of transgenic mice with a mouse Egfr gene mutation (delE748-A752) corresponding to a human EGFR mutation (delE746-A750) was established. The transgenic mice developed atypical adenomatous hyperplasia to adenocarcinoma of the lung at around 5 weeks of age and died of lung tumors at approximately 17 weeks of age. In the mice treated with vandetanib (6mg/kg/day), these lung tumors disappeared and the phosphorylations of EGFR and VEGFR-2 were reduced in lung tissues to levels comparable to those of non-transgenic control mice. The median overall survival time of the transgenic mice was 28 weeks in the vandetanib-treated group and 17 weeks in the vehicle-treated group. Vandetanib significantly prolonged the survival of the transgenic mice (log-rank test, p＜ 0.01); resistance to vandetanib occurred at 20 weeks of age and the animals died from their lung tumors at about 28 weeks of age. These data suggest that vandetanib could suppress the progression of tumors harboring an activating EGFR mutation.

Clinical features and risk factors of panitumumab-induced interstitial lung disease: a postmarketing all-case surveillance study.

BACKGROUND: Drug-induced interstitial lung disease (ILD) is one of the most serious adverse reactions associated with the molecularly targeted drugs. Panitumumab has been approved for advanced or recurrent colorectal cancer. Although there were no adverse reaction reports of ILD in panitumumab monotherapy, 4 cases in combination chemotherapy were reported prior to its approval in Japan in 2010. Several studies also reported that the incidence of drug-induced ILD was higher in Japan than in other countries. The clinical features of ILD and the associated risk factors therefore need investigation. METHODS: We analyzed the data from 3085 unresectable, advanced or recurrent colorectal cancer patients enrolled in a postmarketing all-case surveillance study of panitumumab in Japan. ILD case reports were assessed based on the clinical and radiologic findings by a committee of external experts. Multivariate analysis using Cox's hazard model identified the risk factors. RESULTS: ILD incidence (1.3 %) and mortality rates (51.3 %) were similar to those of patients receiving another anti-epidermal growth factor receptor (EGFR) monoclonal antibody in Japan. No specific onset timing was determined. Although panitumumab-specific ILD findings were not observed in computed tomography images or clinical practice, panitumumab can induce ILD with diffuse alveolar damage, as do the other anti-EGFR targeting drugs. A history/complication of ILD, male sex, poor general condition, and 65 years or older were identified as ILD risk factors, and no history of previous drug treatment was an apparent risk factor. CONCLUSION: Panitumumab-induced ILD can occur at any time after initiation, and close and regular monitoring is needed.

Subpopulation of small-cell lung cancer cells expressing CD133 and CD87 show resistance to chemotherapy.

Tumors are presumed to contain a small population of cancer stem cells (CSCs) that initiate tumor growth and promote tumor spreading. Multidrug resistance in CSCs is thought to allow the tumor to evade conventional therapy. This study focused on expression of CD133 and CD87 because CD133 is a putative marker of CSCs in some cancers including lung, and CD87 is associated with a stem-cell-like property in small-cell lung cancer (SCLC). Six SCLC cell lines were used. The expression levels of CD133 and CD87 were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and flow cytometry. CD133+/- and CD87+/- cells were isolated by flow cytometry. The drug sensitivities were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Non-obese diabetic/severe combined immunodeficiency mice were used for the tumor formation assay. SBC-7 cells showed the highest expression levels of both CD133 and CD87 among the cell lines. CD133-/CD87-, CD133+/CD87-, and CD133-/CD87+ cells were isolated from SBC-7 cells; however, CD133+/CD87+ cells could not be obtained. Both CD133+/CD87- and CD133-/CD87+ subpopulations showed a higher resistance to etoposide and paclitaxel and greater re-populating ability than the CD133-/CD87- subpopulation. CD133+/CD87- cells contained more G0 quiescent cells than CD133-/CD87- cells. By contrast, CD133-/CD87- cells showed the highest tumorigenic potential. In conclusion, both CD133 and CD87 proved to be inadequate markers for CSCs; however, they might be beneficial for predicting resistance to chemotherapy.

Phase II study of irinotecan and amrubicin in patients with relapsed non-small cell lung cancer: Okayama Lung Cancer Study Group Trial 0402.

BACKGROUND: The survival advantage achieved by existing anti-cancer agents as second-line therapy for relapsed non-small cell lung cancer (NSCLC) is modest and further improvement of treatment outcome is desired. Combination chemotherapy with irinotecan and amrubicin for advanced NSCLC has not been fully evaluated. METHODS: The primary endpoint of this phase II clinical trial was objective response. Patients with NSCLC who had been treated previously with one or two chemotherapy agents were enrolled. Irinotecan and amrubicin were both administered on Days 1 and 8 of a 21-day cycle, at doses of 100 mg/m(2) and 40 mg/m(2), respectively. RESULTS: Between 2004 and 2006, 31 patients received a total of 101 courses; the median number of courses administered was three (range, one to six). Objective response was obtained in nine of the 31 patients (29.0% response rate; 95% confidence interval (CI), 12.1-46.0%). With a median follow-up time of 43.9 months, median survival time and the median progression-free survival time were 14.2 and 4.0 months, respectively. Myelosuppression was the most frequently observed adverse event, with grade 3/4 neutropenia in 51% of patients. Febrile neutropenia developed after nine courses (9%) and resulted in one treatment-related death. Cardiac toxicity and diarrhea, possibly specific for both agents, were infrequent and manageable. CONCLUSION: Combination chemotherapy with irinotecan and amrubicin is effective in patients with NSCLC but showed moderate toxicities in second- or third-line settings.

Induction of lung adenocarcinoma in transgenic mice expressing activated EGFR driven by the SP-C promoter.

To investigate the role of an activating epidermal growth factor receptor (EGFR) mutation in lung cancer, we generated transgenic mice expressing the delE748-A752 mutant version of mouse EGFR driven by the SP-C promoter, which is equivalent to the delE746-A750 mutation found in lung cancer patients. Strikingly, the mice invariably developed multifocal lung adenocarcinomas of varying sizes at between 5 and 6 weeks of age, and they died from tumor progression approximately 2 months later if left untreated. Daily oral administration of the EGFR tyrosine kinase inhibitor (TKI) gefitinib (5 mg/kg/day) reduced the total and phosphorylation levels of EGFR to those in wild-type mouse lung tissue; in addition, it abrogated tumor growth within 1 week and prolonged survival to >30 weeks. Interestingly, phosphorylated ErbB2, ErbB3, and thyroid transcriptional factor-1 increased in the transgenic mice compared with those in wild-type mice. They might play some roles in tumors progression in the transgenic mice. This model will be useful for studying the mechanisms of carcinogenesis, chemoprevention, and acquired resistance to EGFR TKIs in lung cancer patients carrying activating EGFR mutations.

Isolated metastasis of lung cancer to the thyroid gland.

A 67-year-old man with lung cancer developed an isolated metastasis to the thyroid gland. The patient had undergone a right upper lobectomy, followed by chemotherapy consisting of cisplatin and etoposide based on post-surgical diagnosis of small cell lung cancer. Four years later, he had an isolated metastasis to the thyroid gland. The patient underwent a metastasectomy and adjuvant chemotherapy including cisplatin and irinotecan. The cancer cells in resected thyroid tumor had large nuclei and cytoplasm, and expressed the neuroendocrine markers, CD56 and chromogranin A. Retrospectively, the primary lung cancer consisted of both small cell and large cell cancer, and the latter was consistent with the pathological finding of the thyroid tumor. This is the first report to document an isolated recurrence of the lung cancer to the thyroid.

Combination of SN-38 with gefitinib or imatinib overcomes SN-38-resistant small-cell lung cancer cells.

Irinotecan is one of the effective anticancer agents for small-cell lung cancer (SCLC) and 7-ethyl-10-hydroxy-campthothecin (SN-38) is an active metabolite of irinotecan. Gefitinib and imatinib are tyrosine kinase inhibitors which have clinical activities in several malignancies and they are also potent inhibitors of breast cancer resistance protein (BCRP) transporter, which confers the resistance of topoisomerase I inhibitors including SN-38 and topotecan. The cytotoxicity of SN-38, gefitinib and imatinib for the SN-38-resistant cells (SBC-3/SN-38) from human SCLC cells, SBC-3, was evaluated using AlamarBlue assay. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96-h exposure was used to evaluate the cytotoxicity. BCRP expression was determined by Western blotting and immunofluorescence staining. Intracellular topotecan accumulation was evaluated by flow cytometry. No differences were observed in the IC50 values (mean +/- SD) of the tyrosine kinase inhibitors between the SBC-3 cells and the SBC-3/SN-38 cells: 15+/-1.6 and 12+/-2.8 microM of gefitinib, respectively; 15+/-0.51 and 14+/-3.9 microM of imatinib, respectively. The SBC-3/SN-38 was 9.5-fold more resistant to SN-38 than the parental SBC-3. The SBC-3/SN-38 restored sensitivity to SN-38 when combined with 8 microM gefitinib or 8 microM imatinib, even though the IC50 values of SN-38 combined with gefitinib or imatinib in the SBC-3 cells did not change. BCRP was equally overexpressed in the SBC-3/SN-38 with and without gefitinib or imatinib. In addition, the BCRP expression on the SBC-3/SN-38 cell membrane with and without gefitinib seemed to be equal. Gefitinib increased intracellular accumulation of topotecan in the SBC-3/SN-38 cells. Gefitinib or imatinib reversed SN-38-resistance in these SCLC cells, possibly due to intracellular accumulation of SN-38 without any change in BCRP quantity. Irinotecan with gefitinib or imatinib might be effective for SCLC refractory to irinotecan.

Effects of vandetanib on lung adenocarcinoma cells harboring epidermal growth factor receptor T790M mutation in vivo.

Vandetanib is a novel multitarget tyrosine kinase inhibitor (TKI) that inhibits vascular endothelial growth factor receptor-2 (VEGFR-2), with additional inhibition of epidermal growth factor receptor (EGFR) and rearranged during transfection receptor signaling, which has shown promising results in clinical trials for advanced non-small cell lung cancer. However, the mechanisms of acquired resistance to vandetanib remain unclear. Therefore, we established in vitro vandetanib-resistant PC-9/VanR cells from PC-9, a vandetanib-sensitive lung adenocarcinoma cell line, by chronic exposure to this agent. PC-9/VanR cells were 50-fold more resistant to vandetanib than PC-9 cells in vitro. Compared with PC-9 cells, PC-9/VanR cells showed emergence of an EGFR T790M mutation, moderately elevated MET amplification, and similar VEGFR-2 inhibition by vandetanib. Note that phospho-MET in PC-9/VanR was suppressed following EGFR inhibition by an irreversible EGFR-TKI, indicating that MET signaling of PC-9/VanR was dependent on EGFR signaling and that MET amplification was not the primary mechanism of resistance to vandetanib. In contrast to the in vitro experiment, vandetanib effectively inhibited the growth of PC-9/VanR tumors in an in vivo xenograft model through the antiangiogenesis effects of VEGFR-2 inhibition. In conclusion, the multitarget TKI vandetanib induced or selected for the EGFR T790M mutation as observed previously with highly selective EGFR-TKIs. However, vandetanib retained significant efficacy in vivo against xenografts harboring the T790M mutation, providing a strong scientific rationale for investigating vandetanib in clinical settings where acquired resistance through emergence of EGFR T790M mutations limits the effectiveness of highly selective EGFR-TKIs.

Chemopreventive effects of gefitinib on nonsmoking-related lung tumorigenesis in activating epidermal growth factor receptor transgenic mice.

Twenty-five percent of all lung cancer cases are not attributable to smoking. Epidermal growth factor receptor (EGFR) mutations, which are involved in approximately 50% of nonsmoker lung cancer, are positively correlated with responsiveness to gefitinib, and inversely correlated with smoking history. Activating EGFR mutations play a critical role in the carcinogenesis of nonsmoking-related lung cancer. To investigate the chemopreventive effects of gefitinib on nonsmoking-related lung cancer, we generated transgenic mice expressing EGFR L858R in type II pneumocytes constitutively using the surfactant protein-C promoter. The transgenic mice invariably developed atypical adenomatous hyperplasia at age 4 weeks and multifocal adenocarcinoma of varying sizes at age 7 weeks. Notably, the expression levels of phosphorylated and total ErbB2, ErbB3, and thyroid transcription factor-1 were elevated in the transgenic mice compared with wild-type controls at age 3 weeks. Administration of gefitinib to 3-week-old transgenic mice for 1 week before carcinogenesis reduced the amount of phosphorylated EGFR in the lungs of the mice to the baseline level. Gefitinib (5 mg/kg/d; n = 5, 5, and 15) or vehicle (n = 5, 5, and 15) was administered to transgenic mice from age 3 to 8, 13, and 18 weeks, respectively. The numbers of lung tumors in the control and gefitinib-treated groups were 1.75, 5.8, 10.2, and 0 (P < 0.05), respectively. No fatal toxic events occurred in either group, and gefitinib inhibited tumorigenesis completely in this mouse model. These results suggest the utility of molecular targeted chemoprevention against nonsmoking-related lung cancer.