Abstract 965: Massively parallel sequencing of cancer FFPE specimens matches diagnostic accuracy of methods in current clinical use and reveals additional actionable mutations
Authors: Yelensky Roman , Dogan Snjezana , Borsu Laetitia , Frampton Garrett , Lipson Doron , Bastian Boris C. , Klimstra David S. , Ladanyi Marc , Cronin Maureen , Hedvat Cyrus V. , Berger Michael F. 
Published: August 2012

Abstract
As genomic data accumulate at an ever-increasing rate, it is becoming evident that a comprehensive description of molecular aberrations that define individual patients’ tumors will prove useful to determine optimal therapeutic strategies. Several platforms are currently in use for clinical molecular diagnostics testing, including PCR, Sanger sequencing, restriction fragment length polymorphism analysis, and mass spectrometric genotyping (Sequenom). Massively parallel sequencing (MPS) technology has the potential to expand upon Sequenom genotyping because it allows for the identification of mutations across entire exons (rather than single base pairs) as well as copy gains, losses and gene fusions. However, for MPS to be considered a viable clinical strategy, it must first be shown to be compatible with formalin-fixed, paraffin embedded (FFPE) tissue across a range of tumor types, sizes, and cellularities. Also, it must exhibit high concordance with mutation profiles derived using the current best diagnostic methods available. To explore the clinical utility of MPS, we selected 71 surgically resected FFPE tumors that had previously been tested for approximately 100 oncogenic mutations in 8 oncogenes by Sequenom genotyping and subjected them to targeted DNA sequencing of 189 cancer-related genes. DNA was extracted from four 10-micron unstained sections from the diagnostic FFPE block (yielding a minimum of 250 nanograms per case), followed by sequencing library construction and hybridization-based capture of 3230 exons and 37 intronic intervals. Deep sequencing was performed, yielding an average coverage of &gt;750X for uniquely-mapping reads. Sequence data were analyzed for single nucleotide variants and small insertions and deletions. High concordance was noted between Sequenom and MPS: 62 and 65 mutations were called by the two technologies, respectively, at mutually tested sites, with 60 mutation calls in common. Notably, mutant allele frequencies in these concordant calls ranged as low as 4% by MPS, highlighting the sensitivity of detection enabled by both approaches. The few discordant mutation calls exhibited no or weak evidence in the other dataset, possibly due to local tumor heterogeneity. Further, MPS revealed 73 sequence variants at additional sites of known recurrent somatic mutations and 30 loss-of-function variants in key tumor suppressor genes not tested by Sequenom. Many of these variants represent plausibly actionable mutations that could influence treatment decisions. Thus, we conclude that: (1) MPS exhibits high concordance with current best methods and is a viable strategy for clinical diagnostics, and (2) MPS captures additional variants not typically interrogated in the current clinical setting but with potential implications for the selection of approved and/or experimental targeted therapies.
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 965. doi:1538-7445.AM2012-965