Abstract LB-284: Toward clinical sequencing: analysis of FFPE samples of 121 gastric cancer by a benchtop sequencer
Authors: Yamashita Satoshi , Kuboki Yasutoshi , Niwa Tohru , Nagatsuma Akiko , Shitara Kohei , Fuse Nozomu , Doi Toshihiko , Ochiai Atsushi , Ohtsu Atsushi , Ushijima Toshikazu 
Published: May 2015

Abstract
Background: Clinical sequencing is becoming more important in cancer therapy, and analysis of formalin-fixed, paraffin-embedded (FFPE) samples is often required. In this study, we optimized pipeline in sequencing analysis of FFPE samples using a benchtop sequencer and profiled mutations and amplifications of cancer-related genes in advanced gastric cancer.
Methods: FFPE samples of cancer and corresponding non-cancer were collected from patients with stage III/IV gastric cancer who underwent gastrectomy. Sequence libraries for almost all the exons of 409 cancer-related genes were prepared using the Ion AmpliSeq Comprehensive Cancer Panel. Sequence variations were analyzed using the Ion Proton Sequencer and the CLC Genomic Workbench software. Gene amplification in cancer was evaluated by ratio between reading depth of an individual gene divided by total reading depth in a cancer sample and that in a corresponding non-cancer sample.
Results: We selected 129 pairs of samples whose template DNA had &gt;32 copies in cancer samples and &gt;5 copies in non-cancer samples, and sequence data were successfully obtained for 121 of the 129 pairs. If sequence variations with &gt;10% frequency of reads were counted, a large number of variations was observed even in non-cancer samples (mean = 6.5). Therefore, the unfiltered results using FFPE samples were likely to contain false positive variants. Selection of variation (i) observed on both forward and reverse read, count &gt;5; ii) total variant count &gt;40) markedly reduced the variation in non-cancer samples (mean = 0.8). After the filtering, the mean number of variation in cancer samples was 4.9 and it was similar to the result of frozen samples (mean = 4.8).
Next, profiling of mutation and amplification in the 121 gastric cancer was performed. The most frequently mutated genes were TP53 (36.4%). Mutations in oncogenes such as PIK3CA (7.4%), ROS1 (5.0%), ERBB2 (4.1%), MET (1.7%) and ALK (1.7%) were also detected. In addition, frequent mutations in genes not reported as gastric cancer-related genes such as SYNE1 (10.7%), CSMD3 (9.1%) and LRP1B (9.1%) were found. Amplification was detected for oncogenes such as SRC (20.7%, max 3.3-fold), ERBB2 (14.9%, 54-fold), CCNE1 (13.2%, 10-fold), CCND1 (9.1%, 8.6-fold), EGFR (7.4%, 22-fold), KRAS (7.4%, 22-fold) and MET (6.6%, 7.7-fold).
Conclusion: We successfully optimized the pipeline for sequencing analysis of FFPE samples, and profiled gene mutations and amplifications in advanced gastric cancer. The pipeline will help us to drive clinical sequencing using FFPE samples.
Citation Format: Satoshi Yamashita, Yasutoshi Kuboki, Tohru Niwa, Akiko Nagatsuma, Kohei Shitara, Nozomu Fuse, Toshihiko Doi, Atsushi Ochiai, Atsushi Ohtsu, Toshikazu Ushijima. Toward clinical sequencing: analysis of FFPE samples of 121 gastric cancer by a benchtop sequencer. [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 LB-284. doi:10.1158/1538-7445.AM2014-LB-284