Abstract B29: Feasibility of amplicon sequencing using a pan-cancer gene panel with pre-treatment biopsy samples of (Japanese) patients with advanced solid tumors: Analyses of Biopsy Samples for Cancer Genomics (ABC) study.
Authors: Takahashi Hideaki , Shitara Kohei , Kuwata Takeshi , Naito Yoichi , Matsumoto Shingo , Okamoto Wataru , Niho Seiji , Bando Hideaki , Kuboki Yasutoshi , Yamada Yoko , Miki Izumi , Yamanaka Takeharu , Watanabe Atsushi , Kojima Motohiro , Ishii Genichiro , Fujii Satoshi , Umemura Shigeki , Ikeda Masafumi , Kohno Takashi , Sato Akihiro , Ohtsu Atsushi , Esumi Hiroyasu , Ochiai Atsushi , Yoshino Takayuki , Tsuchihara Katsuya 
Published: August 2014

Abstract
Background: Next-generation sequencing (NGS) is a powerful tool to individualize cancer treatment by detecting alterations in target genes. Safe, robust, and feasible diagnostic systems are urgently required.
Methods: Recruited patients had advanced solid tumors and were planning to receive anticancer drugs at National Cancer Center Hospital East. Genomic DNA was extracted from pretreatment FFPE biopsy samples, and 10 ng of double strand (ds) DNA was applied to the amplicon sequence using Ion AmpliSeq™ Cancer Panel ver1.0, targeting 739 COSMIC-registered mutations of 46 oncogenes and tumor suppressor genes. A multidisciplinary institutional cancer board (“Expert panel”) was organized to review the safety of the biopsy and quality of the sequencing. The panel also gave qualified reports to clinicians. Results are presented for the prespecified feasibility evaluation with over 100 full analysis sets.
Results: From July 2012 to February 2013, 105 patients were enrolled. Primary tumor sites were stomach (n=28), colorectal (n=20), lung (n=12), breast (n=11), liver (n=9), and others (n=25). Most patients were enrolled before receiving first-line chemotherapy (74.3%). No serious adverse events were observed with biopsy procedures. Adequate biopsy samples for DNA extraction were obtained from 92 patients, and successful sequencing was performed in 90 patients using only 10 ng dsDNA, for a sequence success rate for all enrolled patients and for DNA extracted samples of 85.7% (90/105), and 97.8% (90/92), respectively. Mutation analyses were performed in 93 patients, which included archival tissue or re-biopsied samples from three patients. The median amount of extracted dsDNA was 3334 ng; more than 100 ng dsDNA was retrieved from 92 of 93 samples (99%), which provided enough material for the Cancer Panel and for other additional analyses. The mean number of mutations detected was 1.6 per patient. Potentially targetable mutations were detected in 44% of the patients; these included PIK3CA (15%), KRAS (15%), CTNNB1 (7.5%), EGFR (3.2%), GNAS (3.2%), BRAF (2.1%), ERBB2 (2.1%), KIT (1.1%), and NRAS (1.1%). Proportions of patient with these mutations in major types of malignancies were as follows: breast (77%), colorectal (66%), liver (63%), lung (45%), and stomach (29%). These mutation profiles resulted in treatment of one colon cancer patient with an ERBB2 mutation and amplification with trastuzumab, while other colon cancer patients with KRAS mutations (p.Q61P or p.A146T) received alternative treatments prior to anti-EGFR therapy. Detailed analyses of the sequence data suggested that apparent amplifications of the target genes correlated with the sequence coverage.
Conclusions: An NGS-based multiplex mutation analysis was safely and effectively performed with FFPE biopsy samples. Potentially targetable mutations were detected in about half of the patients with major types of malignancies except for gastric cancer. Application of gene amplification detection using sequence data may expand the utility of the multiplex sequencing for stratifying cancer patients; additional gene amplification analysis should be conducted. Evaluation of the clinical utility of this method is warranted.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B29.
Citation Format: Hideaki Takahashi, Kohei Shitara, Takeshi Kuwata, Yoichi Naito, Shingo Matsumoto, Wataru Okamoto, Seiji Niho, Hideaki Bando, Yasutoshi Kuboki, Yoko Yamada, Izumi Miki, Takeharu Yamanaka, Atsushi Watanabe, Motohiro Kojima, Genichiro Ishii, Satoshi Fujii, Shigeki Umemura, Masafumi Ikeda, Takashi Kohno, Akihiro Sato, Atsushi Ohtsu, Hiroyasu Esumi, Atsushi Ochiai, Takayuki Yoshino, Katsuya Tsuchihara. Feasibility of amplicon sequencing using a pan-cancer gene panel with pre-treatment biopsy samples of (Japanese) patients with advanced solid tumors: Analyses of Biopsy Samples for Cancer Genomics (ABC) study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B29.