Yasunori Murata

Relationship of circulating tumor cells to the effectiveness of cytotoxic chemotherapy in patients with metastatic non-small-cell lung cancer.

The aim of this study was to investigate the relationship of the number of circulating tumor cells (CTCs) with the effectiveness of cytotoxic chemotherapy in patients with metastatic non-small-cell lung cancer (NSCLC). We prospectively evaluated CTCs in the peripheral blood of patients with previously untreated metastatic NSCLC. From May 2008 through August 2010, 33 patients (23 men and 10 women; median age, 64 years; range, 46-74 years) were enrolled. All patients received combination chemotherapy with gemcitabine and carboplatin. The CTCs were captured from samples of peripheral blood with a semiautomated system using an antibody against epithelial cell adhesion molecule. Blood samples with one or more CTC per 7.5 ml were defined as positive. Of total 33 patients, 12 (36.4%) had positive CTCs and 5 (15.2%) had five or more CTCs before chemotherapy. There were no differences in response rates to cytotoxic chemotherapy between CTC-positive patients and CTC-negative patients. On the other hand, the rate of progressive disease in cytotoxic chemotherapy was significantly higher in CTC-positive patients (66.7%) than in CTC-negative patients (23.8%, p = 0.02). In conclusion, the number of CTCs could be a useful predictive factor for the effectiveness of cytotoxic chemotherapy in patients with metastatic NSCLC.

Phase II trial of the combination of carboplatin and irinotecan in elderly patients with small-cell lung cancer

AIM The aim of the present phase II study was to assess the antitumour activity and safety of the combination of irinotecan and carboplatin in elderly patients with small-cell lung cancer (SCLC). MATERIAL AND METHODS Patients with previously untreated SCLC were eligible if they had a performance status of 0-2, were 70 years or older, and had adequate organ function. Patients were treated with carboplatin at an area under the plasma concentration versus time curve of 5 min/ml on day 1 and with irinotecan at 50mg/m(2) on days 1 and 8 every 3 weeks. RESULTS Thirty patients (26 men and 4 women; median age, 76 years; age range, 70-86 years) were enrolled. Eight patients had limited disease (LD) and 22 patients had extensive disease (ED). The overall response rate was 83.3% (95% confidence interval: 65.3-94.4%). Response rates did not differ significantly between patients with LD (87.5%) and those with ED (81.8%; p=0.71). The median survival time was 14 months overall and was significantly longer in patients with LD (26 months) than in patients with ED (11 months; p=0.025). The median progression free survival time was 6 months overall and was significantly longer in patients with LD (12 months) than in patients with ED (6 months; p=0.016). Grade 3-4 toxicities included neutropenia in 83% of patients, thrombocytopenia in 47%, anaemia in 60%, infection in 23%, and diarrhoea in 20%. There were no treatment-related deaths. CONCLUSIONS This chemotherapy is safe and effective for elderly patients with SCLC.

Acquired Resistance Mechanisms to Combination Met-TKI/EGFR-TKI Exposure in Met-Amplified EGFR-TKI Resistant Lung Adenocarcinoma Harboring an Activating EGFR Mutation

Met-amplified EGFR-tyrosine kinase inhibitor (TKI)-resistant non–small cell lung cancer (NSCLC) harboring an activating EGFR mutation is responsive to concurrent EGFR-TKI and Met-TKI treatment in a preclinical model. Here, we determined that Met-amplified gefitinib-resistant cells acquire dual resistance to inhibition of EGFR and Met tyrosine kinase activities. PC-9 lung adenocarcinoma cells harboring 15-bp deletions (Del E746_A750) in EGFR exon 19 were treated with increasing concentrations of the Met-TKI PHA665752 and 1 μmol/L gefitinib for 1 year; three resistant clones were established via Met amplification. The three dual-resistance cell lines (PC-9DR2, PC-9DR4, and PC-9DR6, designated as DR2, DR4, and DR6, respectively) exhibited different mechanisms for evading both EGFR and Met inhibition. None of the clones harbored a secondary mutation of EGFR T790M or a Met mutation. Insulin-like growth factor (IGF)/IGF1 receptor activation in DR2 and DR4 cells acted as a bypass signaling pathway. Met expression was attenuated to a greater extent in DR2 than in PC-9 cells, but was maintained in DR4 cells by overexpression of IGF-binding protein 3. In DR6 cells, Met was further amplified by association with HSP90, which protected Met from degradation and induced SET and MYND domain-containing 3 (SMYD3)-mediated Met transcription. This is the first report describing the acquisition of dual resistance mechanisms in NSCLC harboring an activating EGFR mutation to Met-TKI and EGFR-TKI following previous EGFR-TKI treatment. These results might inform the development of more effective therapeutic strategies for NSCLC treatment. Mol Cancer Ther; 15(12); 3040–54. ©2016 AACR.

Association of pharmacokinetics and pharmacogenomics with safety and efficacy of gefitinib in patients with EGFR mutation positive advanced non-small cell lung cancer.

OBJECTIVES Gefitinib is a potent epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and is a key drug for patients with EGFR mutation-positive advanced non-small cell lung cancer (NSCLC). The pharmacokinetics of orally administered gefitinib varies greatly among patients. We prospectively evaluated the association of pharmacokinetics and pharmacogenomics with the safety and efficacy of gefitinib in patients with EGFR mutation-positive advanced NSCLC. PATIENTS AND METHODS Pharmacokinetics was evaluated with samples of peripheral blood obtained on day 1 before treatment and 1, 3, 5, 8, and 24h after gefitinib (250 mg per day) was administered and on days 8 and 15 as the trough values. The plasma concentration of gefitinib was analyzed with high-performance liquid chromatography. The genotypes of ABCG2, ABCB1, CYP3A4, CYP3A5, and CYP2D6 genes were analyzed with direct sequencing. RESULTS The subjects were 35 patients (21 women; median age, 72 years; range, 53 to 90 years) with stage IV adenocarcinoma harboring EGFR mutations. The median peak plasma concentration (Cmax) was 377 (range, 168-781)ng/mL. The median area under the curve (AUC) of the plasma concentration of gefitinib from 0 to 24h was 4893 (range, 698-13991) ng/mL h. The common adverse events were skin toxicity (68% of patients), diarrhea (46%), and liver injury (63%). One patient died of drug-induced interstitial lung disease (ILD). The overall response rate was 82.9% (95% confidence interval, 66.4%-93.4%). The median progression-free survival time was 10 months, and the median survival time was 25 months. The pharmacokinetics and pharmacogenomics were not associated with significantly different toxicities, response rates, or survival times with gefitinib. However, the AUC and Cmax were highest and the trough value on day 8 was the second highest in one patient who died of drug-induced ILD. CONCLUSION Elevated gefitinib exposure might be associated with drug-induced ILD.

Evaluation of the Efficacy and Safety of the Combination of Gemcitabine and Nedaplatin for Elderly Patients with Advanced Non-Small-Cell Lung Cancer

Objective: The aim of the present study was to retrospectively assess the safety and efficacy of the combination of gemcitabine and nedaplatin in elderly patients with advanced non-small-cell lung cancer (NSCLC). Methods: Patients ≧75 years with previously untreated NSCLC who underwent chemotherapy consisting of gemcitabine (800 mg/m2 on days 1 and 8) and nedaplatin (80 mg/m2 on day 1) every 3 weeks were retrospectively analyzed. Results: Of the 35 patients, 28 were men and 7 were women, with a mean age of 78 years (range 75–87); 10 patients had stage IIIB disease and 25 patients had stage IV disease. The overall response rate was 45.7% (95% confidence interval 28.8–63.4). The median survival time was 14 months (range 3–44). Grade 3–4 toxicities included neutropenia in 74.3%, thrombocytopenia in 48.6%, anemia in 34.3%, hepatic dysfunction in 11.4%, and infection in 2.9%. There were no treatment-related deaths. There were no differences in response rate and survival between patients aged 75–79 years and patients ≧80 years, although grade 3–4 thrombocytopenia and anemia were significantly more frequent in patients ≧80 years. Conclusion: Our results suggest that the combination of gemcitabine and nedaplatin is effective and well tolerated for selected elderly patients with advanced NSCLC.

Distinct Afatinib Resistance Mechanisms Identified in Lung Adenocarcinoma Harboring an EGFR Mutation

EGFR tyrosine kinase inhibitors (TKI) are associated with significant responses in non–small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations. However, acquired resistance to reversible EGFR-TKIs remains a major obstacle. In particular, although the second-generation irreversible EGFR-TKI afatinib is currently used for treating NSCLC patients, the mechanisms underlying acquired afatinib resistance remain poorly understood. Here, heterogeneous mechanisms of acquired resistance were identified following long-term exposure to increasing doses of afatinib in EGFR-mutant lung adenocarcinoma PC-9 cells. Notably, three resistant cell lines, PC-9AFR1, PC-9AFR2, and PC-9AFR3 (AFR1, AFR2, and AFR3, respectively) employed distinct mechanisms for avoiding EGFR inhibition, with increased EGFR expression being detected in all resistant cell lines. Moreover, an activating EGFR mutation was partially lost in AFR1 and AFR2 cells. AFR1 cells exhibited afatinib resistance as a result of wild-type KRAS amplification and overexpression; however, these cells showed a progressive decrease and eventual loss of the acquired KRAS dependence, as well as resensitization to afatinib, following a drug holiday. Meanwhile, AFR2 cells exhibited increased expression of insulin-like growth factor-binding protein 3 (IGFBP3), which promoted insulin-like growth factor 1 receptor (IGF1R) activity and subsequent AKT phosphorylation, thereby indicating a potential bypass signaling pathway associated with IGFR1. Finally, AFR3 cells harbored the secondary EGFR mutation T790M. Our findings constitute the first report showing acquired wild-type KRAS overexpression and attenuation of afatinib resistance following a drug holiday. Implications: The heterogeneous mechanisms of afatinib resistance should facilitate the development of more effective therapeutic strategies for NSCLC patients. Mol Cancer Res; 15(7); 915–28. ©2017 AACR.

Abstract 1208: Combination effect of afatinib and BI836845, a humanized IGF ligand-neutralizing antibody, on EGFR-TKI-resistant NSCLC cells

Insulin-like growth factor (IGF) signaling is thought to have a role in the cancer progression and survival, and many carcinomas are known to overexpress IGF-1 receptor (IGF1R). Furthermore, it was clarified that IGF1R signaling contributes to EGFR-TKI resistance in NSCLC. IGF1R is thought to be a therapeutic target for cancer chemotherapy. Previously, we screened the combination effect of afatinib and various anticancer drugs on a panel of NSCLC cell lines including gefitinib-resistant and afatinib-resistant PC-9 cells which we established. As the result, the combination of afatinib and small molecule IGF1R-TKIs (BMS754807, NVP-ADW742) has shown the most cytotoxicity on wide variety cancer cells. However, these IGF1R-TKIs have cross-reactivity to insulin receptor and, as a result, are known to cause hyperglycemia. Moreover, a phase III trial of the combination chemotherapy between erlotinib and IGF1R specific antibody, figitumumab, in advanced nonadenocarcinoma NSCLC patients (ADVIGO 1018)was closed early because of futility and an increased incidence of serious adverse events. BI836845 is a humanized IGF ligand-neutralizing antibody which is neutralizing both IGF-1 and IGF-2. The proliferation of several cell lines was reported to be potently inhibited by BI 836845 with lower than 100 μg/ml EC50 values. This antibody is thought to be a substitute approach to inhibit IGF1R signaling pathway other than previous IGF1R-TKIs. We evaluated the combination effect of afatinib and BI836845 on the same NSCLC panel by MTS assay. BI836845 had only a limited cytotoxicity by itself. The combination of afatinib and BI836845 had additive or synergistic cytotoxicity on most of the NSCLC cell lines including EGFR-TKIs-resistant cells except the cells overexpressing c-Met or expressing K-ras mutation. This combination significantly inhibited both EGFR and IGF1R autophosphorylation and their downstream signaling, especially Akt/mTOR pathway, as compared with each singular usage. These combination effects were attenuated when BI836845 combined with erlotinib. Since BI836845 cross-react to rodent IGFs, we examined pharmacological activity of this combination using SCID mice xenograft mode. The mice bearing tumor were given 6 mg/kg afatinib (5 times/week, p.o.) and 200 mg/kg BI836845 (1 time/week, i.p.) for 8 weeks. This combination synergistically inhibited afatinib-resistant tumor (PC-9Afa1, PC-9Afa2) and gefitinib-resistant tumor (PC-9ZD, expressed T790M mutant EGFR). This BI836845 administration completely inhibited IGFs activity in the mice serum without alteration of blood sugar level and insulin concentration. Though growth rate of the treated mice was slightly reduced, body weight loss and other serious adverse events were not observed. From these observations, this combination chemotherapy is thought to be a potential therapeutic strategy for NSCLC. Citation Format: Tohru Ohmori, Toshimitsu Yamaoka, Satoru Arata, Motoi Ohba, Yasunori Murata, Yasunari Kishida, Sojiro Kusumoto, Masanao Nakashima, Takashi Hirose, Tsukasa Ohnishi, Kazuto Nishio. Combination effect of afatinib and BI836845, a humanized IGF ligand-neutralizing antibody, on EGFR-TKI-resistant NSCLC cells. [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 1208.

Abstract 2277: TNF transactivation of EGFR protects EGFR-TKI, gefitinib induced pulmonary epithelial cell apoptosis and injury in TNF transgenic mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: TNF is a cytokine with many biological properties including both anti- and pro-apoptotic signaling pathways. However, the molecular switch, which determines TNF regulation of these two different functions, is not well characterized. EGFR and other ErbB family members could be activated by direct interaction with EGF-like ligands initiating formation of homo- and/or hetero-dimers and increased kinase activity. In addition to direct EGFR ligands, EGFR can be transactivated by various extracellular stimuli, such as agonists for GPCR and cytokine receptors. Previously, we have reported that EGFR and ErbB2 were transactivated via Src kinase family activation with TNF exposure and blockade of EGFR enhanced intestinal epithelial cells apoptosis. Furthermore, TNF administration enhanced apoptotic cells in colonic grand of EGFRwa2 mice. Hypothesis: The induction of pulmonary epithelial cell apoptosis is a persistent finding in lung tissue from patients with emphysema and pulmonary fibrosis. We hypothesized that EGFR tyrosine kinase activity is required for protection from pulmonary cell apoptosis in TNF-rich condition. Methods: We employed C57BL/6 mice as a control and surfactant protein-C (SP-C)/TNF transgenic (TG) mice (6-8 weeks old), which showed over-expression of TNF in lung tissue by induction of SP-C production. The lung tissue sections were stained by H&E and trichrome stain. Apoptosis and signal transduction was determined by TUNEL assay and Western-blot analysis, respectively. The accumulation of collagen was measured by Sircol assay. Results: Control mice and TG mice was administrated gefitinib 100mg/kg for 14days. Gefitinib increased lymphocyte infiltration into interstitial spaces of the TG mice lung, significantly. Although TG mice lung was severely injured by gefitinib, fibroblast proliferation was not observed and the accumulation of collagen was not detected. In TG mice, gefitinib increased apoptotic response 10-fold compared to 1% tween80 treated TG mice in interstitial spaces. To test the role of EGFR in apoptosis induction in TG mice, the protein was isolated from whole lung tissue. The tyrosine kinase activity of EGFR was increased and the downstream of AKT and ERK were activated in TG mice. Conversely, treating with gefitinib on TG mice, EGFR phosphorylation was inhibited and, AKT and ERK were inactivated. One of the pro-apoptotic signals, p38 MAPK was activated via MKK3/6 by gefitinib treatment in TG mice, suggesting activation of ASK1/p38 MAPK signals leading to pulmonary epithelial cell apoptosis. Conclusions: The dysregulated production of TNF is a mediator of inflammation and tumor growth. Therefore, the mechanisms of TNF-regulated anti-/pro- apoptotic responses are important in understanding the role of TNF in the pathogenesis of pulmonary disorders, such as inflammation and cancer. Citation Format: Toshimitsu Yamaoka, Yasunari Oki, Yasunori Murata, Sojiro Kusumoto, Hiroo Ishida, Takao Shirai, Etsuko Toya, Motoi Ohba, Ken-ichi Fujita, Satoru Arata, Tohru Ohmori, Tsukasa Ohnishi, Hironori Sagara, Yasutsuna Sasaki. TNF transactivation of EGFR protects EGFR-TKI, gefitinib induced pulmonary epithelial cell apoptosis and injury in TNF transgenic mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2277. doi:10.1158/1538-7445.AM2014-2277

1237PASSOCIATION OF PHARMACOKINETICS OR PHARMACOGENOMICS WITH TOXICITY OF ERLOTINIB IN PATIENTS WITH RECURRENT ADVANCED NON-SMALL CELL LUNG CANCER

ABSTRACT Aim: Erlotinib is a potent epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and is a key drug for patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR mutation. The orally administered erlotinib showed large interindividual variability in its pharmacokinetics. The aim of this study was to evaluate the association of pharmacokinetics or pharmacogenomics with toxicity of erlotinib in patients with advanced NSCLC. Methods: The evaluation of pharmacokinetics was performed using samples obtained on day 1 at 0, 1, 2, 4, 6, 8, 24 hour and day 8 and day 15 after starting of erlotinib 150 mg administration. Plasma concentrations of erlotinib were analyzed by high-performance liquid chromatography. The genotypes of ABCG2, ABCB1, ABCC2, CYP3A4, CYP3A5, CYP1A1, and CYP1A2 were analyzed by direct sequencing. Results: From January 2010 through June 2012, 25 patients with advanced NSCLC (18 men and 7 women; median age, 68 years; range, 31 to 80 years) were enrolled. 17 patients had adenocarcinoma, 8 had squamous cell carcinoma, and 1 was other. Four patients were EGFR mutation positive, 14 were negative, and 8 were unknown. There were no statistically significant associations of pharmacokinetics or pharmacogenomics with toxicity, such as skin toxicity, diarrehea, stomatitis, liver injury, and interstitial lung disease (ILD) of erlotinib. However, one patient who died of drug induced ILD was homozygous for ABCG2 C > A and showed the highest AUC. Additionally, Cmax was trending towards a higher value in 4 patients with liver injury than in those without (p = 0.054). Conclusions: The homozygote of ABCG2 C > A could be associated with elevated erlotinib exposure and drug induced ILD. Further studies of the association of pharmacokinetics or pharmacogenomics with toxicity of EGFR tyrosine kinase inhibitor are needed. Disclosure: All authors have declared no conflicts of interest.

Abstract 5648: The characteristics of an acquired dual resistance to EGFR & MET inhibitors in non-small cell lung cancer, harboring active mutation of EGFR.

BACKGROUND. The selective inhibitors of EGFR tyrosine kinase, gefitinib and erlotinib, prevent binding of ATP to the ATP-binding pocket in a competitive manner and then leading to the loss of catalytic activity. They provided dramatic clinical responses and survival benefits for patients with pulmonary adenocarcinoma, harboring somatic mutations in the EGFR kinase domain. Despite initial responses, acquired resistance develops inevitably after a median of ∼10 months. The secondary EGFR mutation, T790M and the amplification of the MET gene are the two main molecular mechanisms responsible for acquired resistance to EGFR-TKI. AIMS. We established the acquired resistant cells to gefitinib from pulmonary adenocarcinoma, PC-9 (15 bp deletion in exon 19) cells with continuous exposure of gefitinib. The MET amplification was determined in this acquired resistant cell line, so named as PC-9MET. In PC-9MET cells, the secondary mutation of T790M was not detected. These cells are sensitive to the combination of EGFR and MET inhibitors. In this study, for understanding the extreme bypass signaling pathways, we challenged to establish the resistant PC-9 cells to both EGFR and MET inhibitors, which named PC-9DR (Dual Resistant). METHODS. For screening new bypass signals, we employed the phospho-receptor tyrosine kinase array. Cell proliferation and signal transduction was determined by MTT assay and Western-blot analysis, respectively. RESULTS. Like as the establishment of PC-9MET by step-wised dose escalation of gefitinib, PC-9MET cells were also treated with the increasing concentration of PHA665752, which is a MET-TKI, under gefitinib 1μM exposure. After about 12 month, PHA665752 was reached to 1μM and finally PC-9DR cells were survived in the condition of gefitinib 1μM and PHA665752 1μM and showed cross-resistance to the combination of erlotinib/crizotinib and also afatinib/crizotinib. Using both direct-sequence and cycleave PCR technology, PC-9DR cells were not detected the T790M in EGFR. The expression levels of EGFR, ErbB3 and MET were not different between PC-9MET and PC-9DR. However, PC-9DR cells were still activated in EGFR, ErbB3 and MET, even exposed with both gefitinib and PHA665752 and the downstream of AKT and ERK were also activated. For identifying the novel bypass survival signals, the phospho-receptor tyrosine kinase array was performed. In PC-9MET and PC-9DR cells, IGF-IRβ, RET and FGFR3 were slightly activated than PC-9, however there were no changed between PC-9MET and PC-9DR. It might be suggested these activated multiple signal pathways were up-regulated with MET amplification and did not contribute for the acquired resistance to EGFR/MET-TKI. CONCLUSIONS. Further studies might imply to discover attractive targets for a significant portion of patients with EGFR-TKI resistance. Citation Format: Toshimitsu Yamaoka, Yoko Ichihashi, Yasunori Murata, Yasunari Oki, Tomohide Sugiyama, Hiroo Ishida, Takao Shirai, Masanao Nakashima, Kentaro Okuda, Takashi Hirose, Tsukasa Ohnishi, Tohru Ohmori. The characteristics of an acquired dual resistance to EGFR & MET inhibitors in non-small cell lung cancer, harboring active mutation of EGFR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5648. doi:10.1158/1538-7445.AM2013-5648