Kazuhiko Kataoka

Ultra-Sensitive Detection of the Pretreatment EGFR T790M Mutation in Non–Small Cell Lung Cancer Patients with an EGFR-Activating Mutation Using Droplet Digital PCR

Purpose: The resistance to the EGFR tyrosine kinase inhibitors (TKI) is a major concern in non–small cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort. Experimental Design: ddPCR was established as follows: wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild type. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR. Results: Our data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The T790M mutation was detected more frequently in patients with a larger tumor size (P = 0.019) and those with common EGFR-activating mutations (P = 0.022), as compared with the others. Conclusions: The ultra-sensitive ddPCR assay revealed that pretreatment T790M was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment T790M mutation on treatment with EGFR-TKIs. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.

Rapidly enlarging esophageal duplication cyst

Abstract: We report a rare esophageal duplication cyst, in a 12-year-old girl. The cyst had enlarged rapidly within 2 years. In December 1997, on admission, computed tomography and magnetic resonance imaging demonstrated a cystic mass in the pleural cavity. We resected the cyst and the adjacent lung. Histopathological examination revealed an esophageal duplication cyst. Her presenting symptoms of fever and cough may have been related to infection of the cyst.

Prospective Analysis of Oncogenic Driver Mutations and Environmental Factors: Japan Molecular Epidemiology for Lung Cancer Study

PURPOSE Oncogenic driver mutations are critical for lung cancer development and serve as therapeutic targets. However, their associations with environmental factors are not fully understood. We aimed to elucidate the relationship between tumor developmental biology and exposure to environmental factors. PATIENTS AND METHODS This was a prospective, multicenter, molecular epidemiology study. Eligible patients were those with newly diagnosed stages I to IIIB non-small-cell lung cancer (NSCLC) who underwent surgery. The tumors were examined for somatic mutations in 72 cancer-associated genes by targeted deep sequencing, estrogen receptor β (ERβ) expression using immunohistochemical staining, and infection with any of 37 types of human papillomavirus (HPV) using a polymerase chain reaction-based microarray system. Detailed information on patient demographics and environmental factors was obtained from a comprehensive questionnaire. RESULTS From July 2012 to December 2013, 957 patients were enrolled, and molecular analyses were performed on 876 samples (from 441 ever- and 435 never-smokers). Oncogenic driver mutations in P53 and KRAS increased proportionally with smoking status, whereas mutations in EGFR and SMAD4 decreased. KRAS mutations in smokers and SMAD4 mutations were observed more frequently in proportion to body mass index. TP53 and NFE2L2 mutations were observed more frequently in advanced NSCLC stages. As for never-smokers, no environmental factors were significantly associated with mutational changes. EGFR mutations and TP53 mutations were observed more frequently in women and in men, respectively. Mutations in these two genes were also potentially associated with ERβ expression. Only three patients (0.3%) were HPV positive. CONCLUSION The mutational spectrum is associated with smoking, body mass index, and other environmental factors, as well as with ERβ expression. Little association was observed between HPV and NSCLC.

Flow cytometric analysis on perforin induction in peripheral blood mononuclear cells with interleukin-2 or OK-432

Perforin is a protein present in the cytoplasmic granules of killer cells and is considered to be an important effector molecule. We assessed the perforin appearance via flow cytometry in human peripheral blood mononuclear cells stimulated in vitro for 3 days by recombinant interleukin-2 (rIL-2) or OK-432, a biological response modifier. The relationship between the lymphocyte subsets and perforin was investigated via two-color assay. CD4-positive cells had almost no perforin, and most of the CD16-positive cells did. Regarding the relationship with CD8, some of the bright positive cells (which were likely T cells) and most of the dull positive cells (likely NK cells) had perforin. Mean fluorescence was greatest in perforin-positive cells incubated with rIL-2, less in cells incubated with OK-432, and minimal in cells incubated in a medium without additives. Immunohistochemical staining with antiperforin antibody revealed that blast-transformed and enlarge cells were stained positively and that the intensity of staining of each cell alone was enhanced in cells incubated with OK-432 or rIL-2. If the fluorescence intensity of perforin-positive cells correlates with the amount of perforin in those cells, then the appearance of perforin was enhanced with OK-432, more enhanced with rIL-2, and consistent for cytotoxicity against K562 and Daudi cells. IL-2 was induced by OK-432, suggesting that the indirect effect of this IL-2 may play a role in OK-432-perforin induction. The results suggest that perforin may be an effector molecule in killer cells induced by rIL-2 or OK-432.

A Feasibility Study of Postoperative Adjuvant Therapy of Carboplatin and Weekly Paclitaxel for Completely Resected Non-small Cell Lung Cancer

Introduction: Recent clinical trials have shown significant survival benefits from postoperative adjuvant therapy for respectable nonsmall cell lung cancer (NSCLC). However, evaluation of adjuvant chemotherapy with carboplatin combination is still uncertain. The purpose of the study was to test the feasibility of adjuvant chemotherapy with carboplatin and separate weekly paclitaxel after complete resection of pStage IB, II, IIIA NSCLC in a multicenter study. Methods: The study was conducted from 2001 to 2006 in the outpatient setting. A total of 61 patients were enrolled. Patients received adjuvant chemotherapy with 4 cycles of carboplatin (AUC 5) on day 1 and paclitaxel (70 mg/m2) on day 1, 8, and 15 every 4 weeks. Primary endpoints were toxicity and chemotherapy compliance. Secondary endpoints were disease-free survival and overall survival. Results: More than 65% of eligible patients had pStage IIIA. The median number of chemotherapy cycles was 4 (range 1–4). Grade 3 or 4 toxicities of neutropenia were 34% (grade 4: 2%). Other hematologic adverse effects were extremely less frequent. Regarding the nonhematologic adverse effect, hair loss was frequent; however, peripheral neuralgia was less frequent. Treatment-related death was not registered. During median follow-up of 21 months, 24 patients developed recurrent disease. Estimated disease-free survival and overall survival at 2 years was 51.2% and 84.6%, respectively. Conclusions: Postoperative carboplatin and weekly paclitaxel showed favorable feasibility and acceptable toxicity in comparison with the cisplatin-containing regimen. Consequently, it is desirable that this regimen would be validated in a phase III clinical trial for NSCLC after curative resection.

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~ 0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, < 0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M).

Randomized feasibility study of S-1 for adjuvant chemotherapy in completely resected Stage IA non–small-cell lung cancer: results of the Setouchi Lung Cancer Group Study 0701

The feasibility of the S-1 administration schedules (the 4-week versus the 2-week) showed no significant difference for adjuvant chemotherapy among pathological-Stage IA non–small-cell lung cancer patients.

A proposal for a comprehensive risk scoring system for predicting postoperative complications in octogenarian patients with medically operable lung cancer: JACS1303

OBJECTIVES Although some retrospective studies have reported clinicopathological scoring systems for predicting postoperative complications and survival outcomes for elderly lung cancer patients, optimized scoring systems remain controversial. METHODS The Japanese Association for Chest Surgery (JACS) conducted a nationwide multicentre prospective cohort and enrolled a total of 1019 octogenarians with medically operable lung cancer. Details of the clinical factors, comorbidities and comprehensive geriatric assessment were recorded for 895 patients to develop a comprehensive risk scoring (RS) system capable of predicting severe complications. RESULTS Operative (30 days) and hospital mortality rates were 1.0% and 1.6%, respectively. Complications were observed in 308 (34%) patients, of whom 81 (8.4%) had Grade 3-4 severe complications. Pneumonia was the most common severe complication, observed in 27 (3.0%) patients. Five predictive factors, gender, comprehensive geriatric assessment75: memory and Simplified Comorbidity Score (SCS): diabetes mellitus, albumin and percentage vital capacity, were identified as independent predictive factors for severe postoperative complications (odds ratio = 2.73, 1.86, 1.54, 1.66 and 1.61, respectively) through univariate and multivariate analyses. A 5-fold cross-validation was performed as an internal validation to reconfirm these 5 predictive factors (average area under the curve 0.70). We developed a simplified RS system as follows: RS = 3 (gender: male) + 2 (comprehensive geriatric assessment 75: memory: yes) + 2 (albumin: <3.8 ng/ml) + 1 (percentage vital capacity: ≤90) + 1 (SCS: diabetes mellitus: yes). CONCLUSIONS The current series shows that octogenarians can be successfully treated for lung cancer with surgical resection with an acceptable rate of severe complications and mortality. We propose a simplified RS system to predict severe complications in octogenarian patients with medically operative lung cancer. Trial Registration Number JACS1303 (UMIN000016756).

Restrictive ventilatory impairment is associated with poor outcome in patients with cT1aN0M0 peripheral squamous cell carcinoma of the lung

Background Patients with squamous cell carcinoma (SqCC) of the lung sometimes have a comorbid pulmonary disease such as pulmonary emphysema or an interstitial lung disease (ILD), both of which negatively affect patient outcome. The aim of this study was to determine the outcome of patients in a multicenter database who underwent surgery for cT1aN0M0 peripheral SqCC lung cancer. Methods The medical records of a total of 228 eligible patients from seven institutions were reviewed to evaluate the impact of concomitant impaired pulmonary function and other clinicopathological factors on overall survival (OS) and relapse-free survival (RFS). Results Six patients with positive or unclear tumor margins were excluded. Of the 222 remaining study patients, 42 (18.9%) and 97 (43.7%) patients were found to have coexisting restrictive or obstructive ventilatory impairment, respectively. Over a median follow-up period of 30.6 months, the 5-year OS and RFS were 69.0% and 62.6%, respectively. By multivariate analysis, ILDs identified on high-resolution computed tomography (HRCT), pulmonary function test results indicating a restrictive ventilatory impairment, and wedge resection were found to be independent risk factors for poor OS. An increased level of serum squamous cell carcinoma antigen (SCC-Ag) (>1.5 ng/mL) and the same risk factors for poor OS were independent risk factors for recurrence. Among patients who underwent anatomical lung resection (lobectomy and segmentectomy, n=173), a restrictive ventilatory impairment was an independent risk factor for poor OS, and increased serum SCC-Ag level, ILDs on HRCT, and restrictive ventilatory impairment were independent risk factors for poor RFS by multivariate analysis. Factors such as visceral pleural invasion, and lymphatic or vascular invasion were not significantly associated with outcome. Conclusions A restrictive ventilatory impairment negatively affects the outcome of patients with cT1aN0M0 peripheral SqCC lung cancer.

Abstract 1376: Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients

BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency ( CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [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 1376.