Abstract 967: Massively parallel sequencing of clinical FFPE cancer specimens enables comprehensive genomic assessment of patient eligibility for targeted therapy
Authors: Yelensky Roman , Jarosz Mirna , Downing Sean , Wang Kai , Lipson Doron , Juhn Frank , Brennan Kristina , Frampton Garrett , Parker Alex , Curran John , Stephens Philip , Ross Jeffrey , Cronin Maureen 
Published: August 2012

Abstract
Molecular diagnostics are growing in importance to clinical oncology as an increasing number of therapies targeting specific molecular alterations become available. This trend has led to a proliferation of focused biomarker tests delivered using a variety of technologies, which are generally restricted in their breadth of mutational status assessment and constrained by scarce tissue material. We deployed massively parallel sequencing (or “NGS”) technology to develop a pan-cancer, comprehensive genomic profiling assay interrogating 189 relevant cancer genes, using minimal DNA from fixed surgical or biopsy specimens. With this assay we identify all classes of DNA alterations (base substitutions, insertions and deletions, copy number alterations and rearrangements at specific loci) in a single test with high sensitivity and specificity. To assess the potential for comprehensive genomic profiling to inform clinical decisions, we sequenced 58 archival FFPE tumor specimens, including 16 breast, 16 colon, 14 lung, and 12 renal cell cancers to an average of depth of 850x and considered the resultant mutation profiles in the context of current treatment guidelines and clinical trial information. In total, 121 mutations and 30 copy number alterations were identified, of which less than one third could be identified by traditional “hotspot” analysis. The observed mutation frequencies were consistent with published studies for these tissue types: 56% of breast cancers carried TP53 mutations, 38% had PIK3CA mutations, and 13% harbored HER2 amplifications. In colon cancers, 75% and 63% carried APC and TP53 mutations, while 38%, 31%, and 25% harbored KRAS, BRAF, and PIK3CA mutations respectively. Finally, 57% and 42% of NSCLCs carried mutations in TP53 and KRAS, while 17% of RCCs had mutations in VHL. Nearly 70% of the 58 cases carried one or more mutations that could plausibly confer sensitivity or resistance to approved or experimental targeted therapies. Of these potentially clinically actionable mutations, 57% occurred at known sites of oncogene activation, 27% involved gene amplification or deletion, 14% resulted in tumor suppressor loss, and one resulted in the EML4-ALK fusion gene. Our results demonstrate technical feasibility of comprehensive cancer gene characterization through massively parallel sequencing in clinical FFPE specimens. The prevalence and diversity of potentially actionable mutations we observed highlights the benefits of adopting this approach. As massively-parallel sequencing is the only practical means to detect all classes of somatic mutation in a small, clinically relevant sample, we suggest that this type of testing will be an essential ingredient in bringing targeted therapies to cancer patient care.
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 967. doi:1538-7445.AM2012-967