Abstract 3645: Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples

Abstract Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants by the gold standard Sanger sequencing has been well established, however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging. To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes implicated in tumorigenesis (e.g. TP53, KRAS and NRAS). We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations in Sanger sequencing studies. To demonstrate the workflow of these panels with MVF, we analyzed DNA from lung cancer FFPE samples. We initially determined variants of TP53 and KRAS in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing (NGS). We confirmed the identity and variant allele frequency of these variants by Sanger sequencing coupled with MVF. Furthermore, we were able to confirm these results in 1 ng, 0.5 ng or 0.1 ng of DNA from these samples. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD). We show that this workflow can detect as little as 3% of a minor variant in an FFPE sample. Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available. For Research Use only - Not for use in diagnostic procedures. Citation Format: Arpad Gerstner, Edgar Schreiber, Stephen Jackson, Kamini Varma. Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3645.