Targeted Resequencing Of Genes Frequently Mutated In Esophageal Adenocarcinoma Using Highly Multiplexed Ion Torrent Ampliseq (Tm) Technology And Semiconductor Sequencing

Abstract Introduction: Whole genome and whole exome sequencing by large collaborative research groups is providing us with an unprecedented view of the mutations that occur in cancer. Identifying clinically-relevant and actionable mutations however remains a challenge that will most likely be met by individual groups focused on specific clinical questions and scenarios. Such studies will rely on targeted resequencing of selected genes in large numbers of well-annotated clinical specimens. These specimens are most often formalin-fixed and paraffin-embedded (FFPE) and some of the most valuable samples are small biopsies or fine needle aspirates (FNA's). Translational and clinical researchers require rapid and flexible resequencing approaches that are compatible with the small amounts of highly degraded nucleic acids derived from these challenging samples. Materials & Methods: Using Illumina whole exome sequence data from >60 esophageal adenocarcinoma (EAC) specimens, we identified a set of 20 genes that are frequently mutated in this tumor. PCR amplicons no larger than 140bp were designed to cover all exons from these genes, using the Ion Torrent AmpliSeqTM Designer pipeline. Primers were pooled for a 1285-plex multiplex PCR preamplification step starting with 1-20ng of input DNA from both FFPE and frozen tissue and the resulting amplicon products were used to generate sequencing libraries. Semiconductor sequencing was performed on the Ion Personal Genome MachineTM (PGMTM) sequencer. Metrics evaluated included reads on target, base accuracy, uniform coverage of bases and sequencing base read depth. Mutations detected in EAC samples were compared between the two sequencing platforms. Data from dysplastic and metaplastic esophageal biopsies are now being generated to identify mutations that occur in the development of EAC. Results: 1285 PCR primer pairs were designed that covered 66103bp of the coding region of the 20 target genes. Sequencing data covered >99% with at least 100 reads indicating minimal drop-out during preamplification. Average base read depth was >1900X and no significant differences were observed between FFPE and frozen tissue derived DNA. Initial studies demonstrate 100% concordance of mutations (n=42) detected in the 20 target genes and mutant allele frequencies are highly correlated between the two sequencing approaches (slope =0.96; R2 =0.85; range 4-78%). Conclusion: We have used the Ion Torrent AmpliSeqTM technology to detect mutations in EAC specimens and precursor lesions with high sensitivity and accuracy. The flexible primer design pipeline combined with AmpliSeqTM technology provides a fast and easy approach to targeted resequencing in particularly challenging clinical specimens including FFPE biopsies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-410. doi:1538-7445.AM2012-LB-410