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Purpose: Epidermal growth factor receptor (EGFR) mutations, especially deletional mutations in exon 19 (DEL) and L858R, predict gefitinib sensitivity in patients with non–small cell lung cancer (NSCLC). In this study, we validated EGFR mutation detection using high-resolution melting analysis (HRMA) and evaluated the associations between EGFR mutations and clinical outcomes in advanced NSCLC patients treated with gefitinib on a larger scale. Experimental Design: The presence of DEL or L858R was evaluated using HRMA and paraffin-embedded tissues and/or cytologic slides from 212 patients. In 66 patients, the results were compared with direct sequencing data. Results: HRMA using formalin-fixed tissues had a 92% sensitivity and a 100% specificity. The analysis was successfully completed in 207 patients, and DEL or L858R mutations were detected in 85 (41%) patients. The response rate (78% versus 8%), time-to-progression (median, 9.2 versus 1.6 months), and overall survival (median, 21.7 versus 8.7 months) were significantly better in patients with EGFR mutations (P < 0.001). Even among the 34 patients with stable diseases, the time-to-progression was significantly longer in patients with EGFR mutations. Patients with DEL (n = 49) tended to have better outcomes than those with L858R (n = 36); the response rates were 86% and 67%, respectively (P = 0.037), and the median time-to-progression was 10.5 and 7.4 months, respectively (P = 0.11). Conclusions: HRMA is a precise method for detecting DEL and L858R mutations and is useful for predicting clinical outcomes in patients with advanced NSCLC treated with gefitinib.

Introduction: EMT phenotype is known to relate to drug resistance and immune tolerance of cancer. However, mechanism underling these phenomena, including the mechanism of the induction and reversion of EMT, are not fully understood. Methods: EGFR mutated human lung adenocarcinoma cell lines, HCC-827 and PC-9, were treated with TGF-ß and/or FGF-2 to induce EMT. EMT was reversed by treating the induced EMT cells with either of PP242 (mTOR inhibitor), metformin and DMSO. The phenotypic alterations according to the induction and reversion of EMT were accessed by a wound healing assay for mobility, flow cytometry for cell cycle, MTT and apoptosis assays for drug sensitivity to cisplatin and gefitinib, and RT-PCR and fluorescence IHC for PD-L1 expression. Results: The treatment with combination of TGF-ß/FGF-2 showed an efficient increase in expressions of mesenchymal markers and slug in both cell lines, and a decrease in E-cadherin expression in HCC827. In immunoblotting analyses, the Smad3 pathway in PC-9, and the Smad3, MEK/Erk and mTOR pathways in HCC-827 were involved in the induction of the EMT. The induced EMT was accompanied by enhanced cell motility and accumulation in the G0/G1 phases in both cell lines. The induced EMT also made the cells less sensitive to gefitinib in both cell lines, and to cisplatin in HCC-827. Increased PD-L1 expression was observed in both cell lines. Treatment of the induced EMT cells with PP242, metformin and DMSO reverted the altered expressions of epithelial and mesenchymal markers, cell motility and cell cycle. These agents reverted the EMT to different extents and through different pathways, depending on the cell lines. Reversion of the EMT using each of the 3 agents partly restored drug sensitivity, and suppressed PD-L1 expression. Conclusion: Inducing EMT by treatment with TGF-ß/FGF-2 is an effective way to acquire drug resistance and up-regulation of PD-L1. Reversion of the EMT, therefore, has a potential to overcome drug resistance and immune tolerance of cancer. Citation Format: Ryota Kurimoto, Shunichiro Iwasawa, Takahiro Ebata, Tsukasa Ishiwata, Ikuo Sekine, Yuji Tada, Koichiro Tatsumi, Shuhei Koide, Atsushi Iwama, Yuichi Takiguchi. Alteration of drug sensitivity according to the induction and reversion of epithelial-to-mesenchymal transition (EMT) in human lung adenocarcinoma cell lines harboring an EGFR mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 239.

Background:Metformin, one of the most widely prescribed oral agents for treating diabetes mellitus, has been speculated to have antineoplastic effects. The effects, however, have not been fully understood. Experimental Design:A human non-small-cell lung cancer cell line, PC9 (with an EGFR mutation of the exon 19 E746-A750 deletion), was used to evaluate antineoplastic effects of metformin in vitro and in vivo. For in vitro experiments, the cells were exposed to metformin, gefitinib or cisplatin to observe inhibitory effects on cell proliferation. At 24, 48 and 72 hours after the start of the culture with the agents, cell cycle analysis, measurements of caspase 3, 8 and 9 activities, and evaluation with Hoechst staining and APOPercentage TM assay, respectively, were performed. For in vivo experiments in SCID mouse with a xenograft subcutaneous tumor, gefitinib (150 mg/kg, oral gavage) or metformin (250 mg/kg, i.p.) was daily administered to the mice. Results:Gefitinib at a concentration of 0.03 μM, cisplatin of 1.5 μg/ml and metformin of 10 mM exerted equivalent inhibitory effects on cell proliferation in vitro. At these particular concentrations, metformin did not induce significant apoptosis over control as assessed by Hoechst staining, APOPercent assay and caspase 3 activity, whereas gefitinib and cisplatin did induce apoptosis dramatically. Metformin failed to alter cell cycle distribution, whereas gefitinib and cisplatin caused G1/G0 an S phase accumulation, respectively. Metformin did not inhibit growth of already grown subcutaneous tumors, whereas the tumors shrank dramatically with gefitinib treatment. After almost complete disappearance of the tumors by gefitinib, however, they regrew by gefitinib withdrawal. Metformin did significantly suppress the regrowth of the tumors after gefitinib withdrawal. Conclusions: Metformin has inhibitory effects on growth of a human non-small cell lung cancer cell line with a sensitive EGFR mutation in vitro and in vivo, and its mechanism may not depend on apoptosis or cell cycle alteration. Although the agent does not suppress growth of already grown tumors, it significantly suppresses tumor regrowth after gefitinib withdrawal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2735. doi:1538-7445.AM2012-2735

[Background] Metformin has a potential to inhibit tumor growth in various cancer cell lines in vitro and in vivo. [Purpose] To elucidate anti-proliferative actions of the agent on various types of human lung cancer cell lines, and to evaluate combined effects with cisplatin. [Methods] Cell lines used consisted of four human lung cancer cell lines, RERF-LC-AI (squamous cell carcinoma), A549 (adenocarcinoma), IA-5 (large cell carcinoma) and WA-hT (small cell carcinoma). Colonogenic assay was employed to evaluate colonogenicity with exposure to metformin for 1, 24 hours and 10 days. In cell surviving assay, 100,000 cells per plate were cultured for 4 days with metformin and/or cisplatin, and survived cells were counted. [Results] Metformin inhibited colonogenecity in every cell line in a dose-dependent manner when cells were exposed to the agent for 10 days, whereas 1- and 24-hour exposure yielded not significant or only mild effects, with the degree varied depending on cell lines. With cell surviving assay at the metformin concentrations where 70% (IC70) and 30% (IC30) of the cells survived for each cell line, apoptosis was not increased over control groups in every cell line except for WA-hT that showed significant increase in apoptosis with metformin. Significant alteration in cell cycle distribution with the agent was not observed in all of the 4 cell lines. For evaluating combination treatment consisting of metformin and cisplatin, cisplatin at doses of IC50 and IC10 was combined with metformin. Inhibitory effects of metformin on cell proliferation were slightly suppressed with cisplatin at its dose of IC50 in all except for in A549. High dose (IC10) of cisplatin almost completely countervailed or even reversed the metformin effects in all cell lines, again except for A549 where a modest but significant sub-additive effect was observed. [Conclusions] Metformin has anti-proliferative effects on various types of human lung cancer cell lines. Although apoptosis was observed only in a small-cell lung cancer cell line, its effects may not depend on apoptosis in other 3 cell lines. Alteration of cell cycle distribution was not observed in all cell lines. Metformin and cisplatin were antagonistic especially with higher dose cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2737. doi:1538-7445.AM2012-2737

Background: Malignant pleural mesothelioma (MPM) is one of the most lethal cancers with a rapidly increasing incidence worldwide. Although cisplatin and pemetrexed are recognized as key drugs, and their combination chemotherapy is a standard therapy for MPM, their drug-to-drug interactions, cross-resistance, and resistance mechanisms are not well understood. Methods: The interaction between cisplatin and pemetrexed was evaluated by colonogenic assays followed by isobologram analysis in 4 human MPM cell lines. The cell lines were exposed to the agents using a stepwise dose-escalation method to establish drug-resistant sublines. Thymidylate synthase (TS) mRNA expression was evaluated by RT-PCR, and gene expression profiles were assessed by cDNA microarray analysis and gene ontology analysis using Ingenuity Pathway Analisis (IPA) in the drug-resistant sublines. Results: Cisplatin and pemetrexed had synergistic effects in 3 cell lines and an additive effect in the fourth cell line. The former 3 cell lines showed similar sensitivity to pemetrexed in the parental cells and their cisplatin-resistant sublines, whereas the fourth cell line exhibited cross-resistance. In contrast, cisplatin had diverse effects on pemetrexed-resistant sublines. High TS expression did not correlate with natural pemetrexed resistance and its elevatedexpression correlated with acquired pemetrexed resistance in 2 sublines. Microarray analysis disclosed that the genes which related to “cellular development”,” small molecule biochemistry,” “cellular movement,” “cell death, and “cellular growth and proliferation” affected drug-resistant mechanisms deeply, and that this tendency was common to cisplatin-resistant sublines and pemetrexed-resistant sublines. Conclusion: Cisplatin and pemetrexed showed synergistic activity and no cross-resistance in 3 of the 4 MPM cell lines, suggesting the clinical relevance of their combination chemotherapy. TS expression did not necessarily correlate with pemetrexed resistance. Multiple mechanisms of molecular and cellular functions may underlie acquired resistance to cisplatin and pemetrexed in MPM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3821. doi:1538-7445.AM2012-3821

A considerable proportion of lung adenocarcinoma (LADC) cases develop through activation of oncogenes, i.e., somatic mutations in either the EGFR (10–40%) or KRAS (10–20%) genes, or fusion of the ALK gene (5%) with the EML4 or KIF5B genes, in a mutually exclusive manner. Tyrosine kinase inhibitors that target EGFR and ALK proteins are particularly effective for treating LADCs carrying EGFR mutations and ALK fusions, respectively. In this study, whole transcriptome sequencing (RNA sequencing) of 30 Japanese LADCs and three adjacent non-cancerous lung tissues was performed to identify novel chimeric fusion transcripts that could be potential targets for therapy. Analysis of more than 2 × 107 paired-end reads obtained by RNA sequencing and subsequent validation by Sanger sequencing of the reverse transcription (RT)-PCR products identified EML4-ALK fusions in two cases and a few other gene fusions in each single case. The two EML4-ALK positive LADC cases were negative for EGFR and KRAS mutations and also negative for other gene fusions. Therefore, the authenticity of ALK fusions as a driver mutation was validated. The prevalence and oncogenic property of novel gene fusions identified are being studied. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B93.

Here we show that TP53 SNP is involved in chemotherapeutic responses in non-small cell lung cancer (NSCLC) patients. To identify polymorphisms in DNA repair genes that affect responses to platinum-based doublet chemotherapy, 640 NSCLC patients, who received platinum-based doublet chemotherapy and whose responses were evaluated by the Response Evaluation Criteria in Solid Tumors, were subjected to an association study between the response and genotypes for 30 SNPs in 27 DNA repair genes. Candidate SNPs were selected by screening of a discovery set of 201 cases and their associations were validated in an independent set of 439. Homozygotes for the TP53–72Pro allele of the TP53-Arg72Pro SNPs showed a better response rate (54.3%) than those for the TP53–72Arg allele (29.1%), and TP53–72Pro allele homozygotes had significantly longer progression-free and overall survivals than TP53–72Arg allele homozygotes. Minor allele carriers for a SNP, Lys940Arg, in the PARP1 (poly ADP-ribose polymerase 1) gene showed a better response rate to the regimen using paclitaxel (45.8%) than to the regimen using gemcitabine (10.5%). These results indicate that polymorphisms in the TP53 and PARP1 genes are involved in inter-individual differences in the response to platinum-based doublet chemotherapy in NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C109.