Hiroshi Nokihara

Epidermal Growth Factor Receptor Gene Mutations and Increased Copy Numbers Predict Gefitinib Sensitivity in Patients With Recurrent Non–Small-Cell Lung Cancer

PURPOSE To evaluate epidermal growth factor receptor (EGFR) mutations and copy number as predictors of clinical outcome in patients with non-small-cell lung cancer (NSCLC) receiving gefitinib. PATIENTS AND METHODS Sixty-six patients with NSCLC who experienced relapse after surgery and received gefitinib were included. Direct sequencing of exons 18 to 24 of EGFR and exons 18 to 24 of ERBB2 was performed using DNA extracted from surgical specimens. Pyrosequencing and quantitative real-time polymerase chain reaction were performed to analyze the allelic pattern and copy number of EGFR. RESULTS Thirty-nine patients (59%) had EGFR mutations; 20 patients had deletional mutations in exon 19, 17 patients had missense mutations (L858R) in exon 21, and two patients had missense mutations (G719S or G719C) in exon 18. No mutations were identified in ERBB2. Response rate (82% [32 of 39 patients] v 11% [three of 27 patients]; P < .0001), time to progression (TTP; median, 12.6 v 1.7 months; P < .0001), and overall survival (median, 20.4 v 6.9 months; P = .0001) were significantly better in patients with EGFR mutations than in patients with wild-type EGFR. Increased EGFR copy numbers (> or = 3/cell) were observed in 29 patients (44%) and were significantly associated with a higher response rate (72% [21 of 29 patients] v 38% [14 of 37 patients]; P = .005) and a longer TTP (median, 9.4 v 2.6 months; P = .038). High EGFR copy numbers (> or = 6/cell) were caused by selective amplification of mutant alleles. CONCLUSION EGFR mutations and increased copy numbers were significantly associated with better clinical outcome in gefitinib-treated NSCLC patients.

EGFR Mutations Predict Survival Benefit From Gefitinib in Patients With Advanced Lung Adenocarcinoma: A Historical Comparison of Patients Treated Before and After Gefitinib Approval in Japan

PURPOSE This study evaluated whether the presence of epidermal growth factor receptor (EGFR) mutations is a predictive marker for survival benefit from gefitinib and/or a prognostic marker in patients with advanced lung adenocarcinoma. PATIENTS AND METHODS Overall survival (OS) was compared between patients with advanced lung adenocarcinoma who began first-line systemic therapy before and after gefitinib approval in Japan (January 1999 to July 2001 and July 2002 to December 2004, respectively). Deletional mutations in exon 19 or the L858R mutation in exon 21 of EGFR were evaluated using high-resolution melting analysis. RESULTS EGFR mutations were detected in 136 (41%) of the 330 patients included in this study. OS was significantly longer among the EGFR-mutant patients treated after gefitinib approval compared with the OS of patients treated before gefitinib approval (median survival time [MST], 27.2 v 13.6 months, respectively; P < .001), whereas no significant survival improvement was observed in patients without EGFR mutations (MST, 13.2 v 10.4 months, respectively; P = .13). A significant interaction between the presence of EGFR mutations and a survival improvement was seen (P = .045). Among patients treated before gefitinib approval, those with EGFR mutations lived longer than those without EGFR mutations (MST, 13.6 v 10.4 months, respectively; P = .034). The response rates to first-line cytotoxic chemotherapy were not significantly different between patients with and without EGFR mutations (31% v 28%, respectively; P = .50). CONCLUSION EGFR mutations significantly predict both a survival benefit from gefitinib and a favorable prognosis in patients with advanced lung adenocarcinoma.

Hepatocyte Growth Factor Expression in EGFR Mutant Lung Cancer with Intrinsic and Acquired Resistance to Tyrosine Kinase Inhibitors in a Japanese Cohort

Introduction: This study was performed to determine the incidence rates of resistance factors, i.e., high-level hepatocyte growth factor (HGF) expression, epidermal growth factor receptor (EGFR) T790M secondary mutation, and MET amplification, in tumors with intrinsic and acquired EGFR tyrosine kinase inhibitor (TKI) resistance in EGFR mutant lung cancer. Methods: Ninety-seven specimens from 93 EGFR mutant lung cancer patients (23 tumors with acquired resistance from 20 patients, 45 tumors with intrinsic resistance from 44 patients [nonresponders], 29 sensitive tumors from 29 patients) from 11 institutes in Japan were analyzed. HGF expression, EGFR T790M secondary mutation, and MET amplification were determined by immunohistochemistry, cycleave real-time polymerase chain reaction, and fluorescence in situ hybridization, respectively. Results: High-level HGF expression, EGFR T790M secondary mutation, and MET amplification were detected in 61, 52, and 9% of tumors with acquired resistance, respectively. High-level HGF expression was detected in 29% of tumors with intrinsic resistance (nonresponders), whereas EGFR T790M secondary mutation and MET amplification were detected in 0 and 4%, respectively. HGF expression was significantly higher in tumors with acquired resistance than in sensitive tumors (p < 0.001, Students t test). Fifty percent of tumors with acquired resistance showed simultaneous HGF expression with EGFR T790M secondary mutation and MET amplification. Conclusions: High-level HGF expression was detected more frequently than EGFR T790M secondary mutation or MET amplification in tumors with intrinsic and acquired EGFR-TKI resistance in EGFR mutant lung cancer in Japanese patients. These observations provide a rationale for targeting HGF in EGFR-TKI resistance in EGFR mutant lung cancer.

Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial

BACKGROUND Alectinib, a potent, highly selective, CNS-active inhibitor of anaplastic lymphoma kinase (ALK), showed promising efficacy and tolerability in the single-arm phase 1/2 AF-001JP trial in Japanese patients with ALK-positive non-small-cell lung cancer. Given those promising results, we did a phase 3 trial to directly compare the efficacy and safety of alectinib and crizotinib. METHODS J-ALEX was a randomised, open-label, phase 3 trial that recruited ALK inhibitor-naive Japanese patients with ALK-positive non-small-cell lung cancer, who were chemotherapy-naive or had received one previous chemotherapy regimen, from 41 study sites in Japan. Patients were randomly assigned (1:1) via an interactive web response system using a permuted-block method stratified by Eastern Cooperative Oncology Group performance status, treatment line, and disease stage to receive oral alectinib 300 mg twice daily or crizotinib 250 mg twice daily until progressive disease, unacceptable toxicity, death, or withdrawal. The primary endpoint was progression-free survival assessed by an independent review facility. The efficacy analysis was done in the intention-to-treat population, and safety analyses were done in all patients who received at least one dose of the study drug. The study is ongoing and patient recruitment is closed. This study is registered with the Japan Pharmaceutical Information Center (number JapicCTI-132316). FINDINGS Between Nov 18, 2013, and Aug 4, 2015, 207 patients were recruited and assigned to the alectinib (n=103) or crizotinib (n=104) groups. At data cutoff for the second interim analysis, 24 patients in the alectinib group had discontinued treatment compared with 61 in the crizotinib group, mostly due to lack of efficacy or adverse events. At the second interim analysis (data cutoff date Dec 3, 2015), an independent data monitoring committee determined that the primary endpoint of the study had been met (hazard ratio 0·34 [99·7% CI 0·17-0·71], stratified log-rank p<0·0001) and recommended an immediate release of the data. Median progression-free survival had not yet been reached with alectinib (95% CI 20·3-not estimated) and was 10·2 months (8·2-12·0) with crizotinib. Grade 3 or 4 adverse events occurred at a greater frequency with crizotinib (54 [52%] of 104) than alectinib (27 [26%] of 103). Dose interruptions due to adverse events were also more prevalent with crizotinib (77 [74%] of 104) than with alectinib (30 [29%] of 103), and more patients receiving crizotinib (21 [20%]) than alectinib (nine [9%]) discontinued the study drug because of an adverse event. No adverse events with a fatal outcome occurred in either treatment group. INTERPRETATION These results provide the first head-to-head comparison of alectinib and crizotinib and have the potential to change the standard of care for the first-line treatment of ALK-positive non-small-cell lung cancer. The dose of alectinib (300 mg twice daily) used in this study is lower than the approved dose in countries other than Japan; however, this limitation is being addressed in the ongoing ALEX study. FUNDING Chugai Pharmaceutical Co, Ltd.

Randomized phase II study of first-line carboplatin-paclitaxel with or without bevacizumab in Japanese patients with advanced non-squamous non-small-cell lung cancer.

PURPOSE This multicenter, randomized, open-label, phase II study (JO19907) compared the efficacy and safety of first-line carboplatin-paclitaxel (CP) alone with bevacizumab-CP in Japanese patients with advanced non-squamous non-small-cell lung cancer (NSCLC). METHODS Chemonaïve patients with stage IIIB, IV or recurrent non-squamous NSCLC were eligible for participation. Patients were randomly assigned in a 2:1 ratio to receive bevacizumab-CP or CP alone. Chemotherapy was repeated for up to 6 cycles or until disease progression or unacceptable toxicity. Bevacizumab recipients who completed ≥3 cycles of chemotherapy could continue bevacizumab as monotherapy until disease progression or unacceptable toxicity. The primary endpoint was progression-free survival (PFS). RESULTS After confirming the tolerability of bevacizumab-CP in a small number of patients, 180 patients were recruited, of whom 121 were assigned to bevacizumab-CP and 59 to CP alone. Hazard ratio (HR) for PFS was 0.61 with bevacizumab-CP versus CP alone (p=0.0090; median 6.9 versus 5.9 months). Objective response rate was significantly higher with bevacizumab-CP than with CP alone (60.7% versus 31.0%; p=0.0013). Median overall survival was >22 months in both treatment groups (HR 0.99; p=0.9526). No new safety signals were detected. CONCLUSION Study JO19907 met its primary endpoint, demonstrating that the addition of bevacizumab to first-line CP significantly improves PFS in Japanese patients with advanced non-squamous NSCLC. This prolonged PFS by bevacizumab did not translate into OS benefit with the extremely longer underlying survival compared to historical data. No new safety signals were identified in this population. (Japan Pharmaceutical Information Center [JAPIC] registration number: CTI-060338).

Epidermal Growth Factor Receptor Mutation Detection Using High-Resolution Melting Analysis Predicts Outcomes in Patients with Advanced Non–Small Cell Lung Cancer Treated with Gefitinib

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.

Randomized Pharmacokinetic and Pharmacodynamic Study of Docetaxel: Dosing Based on Body-Surface Area Compared With Individualized Dosing Based on Cytochrome P450 Activity Estimated Using a Urinary Metabolite of Exogenous Cortisol

PURPOSE Docetaxel is metabolized by cytochrome P450 (CYP3A4) enzyme, and the area under the concentration-time curve (AUC) is correlated with neutropenia. We developed a novel method for estimating the interpatient variability of CYP3A4 activity by the urinary metabolite of exogenous cortisol (6-beta-hydroxycortisol [6-beta-OHF]). This study was designed to assess whether the application of our method to individualized dosing could decrease pharmacokinetic (PK) and pharmacodynamic (PD) variability compared with body-surface area (BSA) -based dosing. PATIENTS AND METHODS Fifty-nine patients with advanced non-small-cell lung cancer were randomly assigned to either the BSA-based arm or individualized arm. In the BSA-based arm, 60 mg/m(2) of docetaxel was administered. In the individualized arm, individualized doses of docetaxel were calculated from the estimated clearance (estimated clearance = 31.177 + [7.655 x 10(-4) x total 6-beta-OHF] - [4.02 x alpha-1 acid glycoprotein] - [0.172 x AST] - [0.125 x age]) and the target AUC of 2.66 mg/L . h. RESULTS In the individualized arm, individualized doses of docetaxel ranged from 37.4 to 76.4 mg/m(2) (mean, 58.1 mg/m(2)). The mean AUC and standard deviation (SD) were 2.71 (range, 2.02 to 3.40 mg/L . h) and 0.40 mg/L . h in the BSA-based arm, and 2.64 (range, 2.15 to 3.07 mg/L . h) and 0.22 mg/L . h in the individualized arm, respectively. The SD of the AUC was significantly smaller in the individualized arm than in the BSA-based arm (P < .01). The percentage decrease in absolute neutrophil count (ANC) averaged 87.1% (range, 59.0 to 97.7%; SD, 8.7) in the BSA-based arm, and 87.4% (range, 78.0 to 97.2%; SD, 6.1) in the individualized arm, suggesting that the interpatient variability in percent decrease in ANC was slightly smaller in the individualized arm. CONCLUSION The individualized dosing method based on the total amount of urinary 6-beta-OHF after cortisol administration can decrease PK variability of docetaxel.

Prospective Study of the Accuracy of EGFR Mutational Analysis by High-Resolution Melting Analysis in Small Samples Obtained from Patients with Non–Small Cell Lung Cancer

Purpose: Epidermal growth factor receptor (EGFR) mutations, especially in-frame deletions in exon 19 (DEL) and a point mutation in exon 21 (L858R), predict gefitinib sensitivity in patients with non–small cell lung cancer (NSCLC). In this study, we verified the accuracy of EGFR mutation analysis in small samples by high-resolution melting analysis (HRMA), which is a rapid method using PCR amplification with a dye to analyze the melting curves in NSCLC. Experimental Design: We designed a prospective study to compare the sensitivity and specificity of HRMA and DNA sequencing with laser capture microdissection. Eligible patients with lung lesions were screened by bronchoscopy or percutaneous needle biopsy to histologically confirm the diagnosis, followed by surgical resection of the NSCLC. Small diagnostic specimens were analyzed for EGFR mutations by HRMA, and the surgically resected specimens were examined for mutations by HRMA and DNA sequencing. Results: The analyses for EGFR mutations were conducted in 52 eligible cases of the 92 enrolled patients. EGFR mutations were detected in 18 (34.6%) patients. The results of HRMA from surgically resected specimens as well as DNA sequencing revealed 100% sensitivity and specificity. On the other hand, the sensitivity and specificity of HRMA from the small diagnostic specimens were 83.3% and 100%, respectively. Conclusions: In this study, we showed that HRMA is a highly accurate method for detecting DEL and L858R mutations in patients with NSCLC, although it is necessary to consider the identification of patients with a false-negative result when the analysis is conducted using small samples.

The Clinical Relevance of the miR-197/CKS1B/STAT3-mediated PD-L1 Network in Chemoresistant Non-small-cell Lung Cancer

Programmed cell death ligand-1 (PD-L1) has recently gained considerable attention for its role in tumor immune escape. Here, we identify a miR-197/CKS1B/STAT3-mediated PD-L1 network in chemoresistant non-small-cell lung cancer (NSCLC), independent of immunoinhibitory signals. miR-197 is downregulated in platinum-resistant NSCLC specimens, resulting in the promotion of chemoresistance, tumorigenicity, and pulmonary metastasis in vitro and in vivo. Mechanistic investigations reveal that a miR-197-mediated CKS1B/STAT3 axis exerts tumor progression regulated by various oncogenic genes (Bcl-2, c-Myc, and cyclin D1), and PD-L1 is a putative biomarker of this axis. Furthermore, we demonstrate that a miR-197 mimic sensitizes PD-L1high drug-resistant cells to chemotherapy. These results indicate that the biological interaction between PD-L1 and chemoresistance occurs through the microRNA regulatory cascade. More importantly, expression levels of miR-197 are inversely correlated with PD-L1 expression (n = 177; P = 0.026) and are associated with worse overall survival (P = 0.015). Our discoveries suggest that the miR-197/CKS1B/STAT3-mediated network can drive tumor PD-L1 expression as a biomarker of this cascade, and miR-197 replacement therapy may be a potential treatment strategy for chemoresistant NSCLC.

Druggable Oncogene Fusions in Invasive Mucinous Lung Adenocarcinoma

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur. Experimental Design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products. Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use. Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs. Clin Cancer Res; 20(12); 3087–93. ©2014 AACR.