Detection Of Somatic Mutations And Gene Amplifications Using Amplicon Sequencing With Biopsy Samples From Patients With Advanced Gastric Cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Next-generation sequencing (NGS) is a powerful tool for individualized cancer treatment by detecting alterations of target genes. We are now conducting a prospective study to investigate the profile of targeted somatic mutations using Ion AmpliseqTM Cancer Panel in advanced solid tumors (ABC-T study). This panel allows characterization of 739 (version 1) or 2855 (version 2) mutations in a single day with high sensitivity and high accuracy using 10 ng of DNA; however, neither gene copy number alteration nor gene rearrangements can be reliably detected. Methods: Genomic DNA was extracted from FFPE biopsy samples, and 10 ng of double-strand DNAs were applied to amplicon sequence using Ion AmpliseqTM Cancer Panel (version 1 or 2). A multi-disciplinary institutional cancer board (“Expert panel”) was organized to review safety of the biopsy and the quality of the sequencing. Furthermore, we established a diagnostic system for detecting gene amplification. The preliminary results focusing on gastric cancer (GC) are presented. Results: Among the 141 patients enrolled from July 2012 to July 2013, 42 GC patients were analyzed. No serious adverse events were observed with biopsy procedures. Both DNA extraction and targeting sequence were successfully performed in 40 patients (95%) including one patient underwent re-biopsy. The most frequently detected mutations were TP53 (38%), STK11 (28%), PIK3CA (13%), and KRAS (10%). We indicated that the coverage depth of the HER2 exon was correlated with the copy number of HER2 analyzed by quantitative Real Time PCR in cancer cells with HER2 amplification. DNA samples from the biopsy specimens contain the genomic DNA of both tumor and stroma cells. Therefore, we adjsted the coverage depth of the HER2 using a semi-quantitative tumor-stroma ratio determined by histopathological examination since lower tumor contents obscures the copy number gain occurred in the tumor cells. Finally, HER2 amplification status recognized by high coverage depth of the HER2 exon was fully concordant with those of 4 FISH-positive and 9 FISH-negative cases. The adjusted coverage depth of the HER2 exon exhibited correlation with the estimated copy number gain determined by conventional HER2 FISH. Conclusions: Our preliminary results suggested that the NGS-based multiplex analysis is safe and plausible for the screening of gene amplification as well as single nucleotide variants and indels of genomic DNA extracted from FFPE biopsy samples of GC. Citation Format: Wataru Okamoto, Takeshi Kuwata, Shingo Matsumoto, Yoichi Naito, Hideaki Takahashi, Kohei Shitara, Yasutoshi Kuboki, Hideaki Bando, Yoko Yamada, Izumi Miki, Takeharu Yamanaka, Atsushi Ohtsu, Atsushi Ochiai, Hiroyasu Esumi, Takayuki Yoshino, Katsuya Tsuchihara. Detection of somatic mutations and gene amplifications using amplicon sequencing with biopsy samples from patients with advanced gastric cancer. [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 2208. doi:10.1158/1538-7445.AM2014-2208