SOMATIC MUTATIONS IN PANCREATIC DUCTAL ADENOCARCINOMA USING AMPLICON SEQUENCING FROM FORMALIN FIXED PARAFFIN EMBEDDED TISSUE (#416)
INTRODUCTION: Cancer is believed to be a disease of the genome that develops due to underlying genetic and epigenetic changes. Understanding these changes can allow identification of actionable (druggable) mutations enabling tailoring of therapy for an individual patient, a central concept in personalised medicine. Formalin-fixed paraffin embedded tissues offer a rich resource for retrospective analysis but the processing of the tissue often results in fragmented and cross-linked DNA. Recent developments in Next Generation Sequencing technologies now allows for the rapid high-throughput sequencing of short amplicons (amplicon sequencing) overcoming many of the problems with FFPE DNA. Here we report on our initial experience of targeted amplicon sequencing in PC for actionable mutations.
METHODS: DNA was extracted from tumour and normal FFPE blocks using Qiagen Allprep DNA/RNA kit. The extracted DNA was quantified using the Qubit fluorometer and dsDNA HS assay kit. Using 50ng of DNA a library was constructed of 190 amplicons which were then sequenced using the Ion Ampliseq Cancer Panel v1 and the Ion Torrent Personal Genome Machine (PGM) sequencer. The target genes include whole-exon coverage of KRAS, BRAF and EGFR and “hot-spots” in 43 other oncogenes and tumour suppressor genes.
RESULTS: We compared the results of amplicon sequencing with whole-exome sequencing (SOLID 4 system, Applied Biosystems) from matched FFPE and snap frozen tumour tissue respectively. Amplicon sequencing was able to detect all single-nucleotide variants in the target regions seen in the fresh tissue. DNA from a further >40 tumours has been isolated and entered the sequencing pipeline.
CONCLUSIONS: Amplicon sequencing of DNA from FFPE tissue is feasible and shows good correlation with findings in snap-frozen tissue. Amplicon sequencing has potential clinical utility in identifying actionable tumour genomic alterations. We will present our final analysis of targeted sequencing from over 40 pancreatic adenocarcinomas.