Abstract 2279: Bringing next generation sequencing (NGS) to the clinic: Analytical validation of a comprehensive NGS-based cancer gene test.
Authors: Yelensky Roman , Frampton Garrett M. , Fichtenholtz Alex , Downing Sean , He Jie , Juhn Frank , Brennan Kristina , Wang Kai , Otto Geoff , Jarosz Mirna , Parker Alex , Ross Jeffrey S. , Curran John , Cronin Maureen T. , Stephens Philip J. , Lipson Doron 
Published: August 2014

Abstract
Background: As the number of clinically relevant cancer genes grows and the amount of tissue available for analysis decreases, next-generation sequencing (NGS) becomes increasingly attractive as a diagnostic tool, as it can detect all classes of genomic alterations in all cancer genes in a single test. However, for NGS to demonstrate its full utility in the clinic, robust analytical validation approaches and performance benchmarks against gold standard methodologies are required.
Methods: We have developed a comprehensive NGS-based diagnostic test to enable accurate detection of clinically-relevant genomic alterations across 4604 exons of 289 cancer genes (∼1.5 Mb) in routine FFPE specimens, including needle biopsies. To validate the test, we created synthetic reference standards spanning key determinants of detection accuracy for somatic alterations, such as a range of stromal admixture, indel length, and amplitude of copy change: For base substitutions, we mixed 2 pools of 10 normal cell-lines, thereby testing 1035 different variants in a broad 10%-100% allele frequency range. For indels, 28 tumor cell lines with 44 alterations (1-40bp) were mixed into 41 variably sized pools, creating a validation set of 163 events. For copy alterations, 7 matched tumor/normal cell-lines bearing a total of 12 amplifications (copy number 8-15) and 7 homozygous deletions were mixed in 5 tumor content ratios 20%-75%. The test was applied blinded to all pools and alteration calls compared to expectation based on constituents in each mix. We then verified that observed performance translated to FFPE by examining test concordance on 185 tumors characterized clinically for mutations in 8 genes including KRAS, BRAF, and EGFR (Sanger, Sequenom, and PCR) and 131 specimens characterized for CNAs in ERBB2, PTEN, AR and CCND1 (IHC and FISH).
Results: On synthetic standards, performance reached &gt;99% sensitivity for substitutions (1,035/1,035) and &gt;98% for indels (161/163) at ≥10% allele-frequency with PPV&gt;99%. Sensitivity for CNAs was &gt;95% for focal amplifications (46/48) and &gt;99% for homozygous deletions (28/28) at ≥30% tumor fraction, with PPV&gt;99%. Robust performance translated to FFPE: concordance was 98% for both substitutions and indels (164/168) and CNAs (41/42) relative to prior calls.
Conclusions: We present a rigorous validation approach and performance benchmarks for a comprehensive NGS-based test for use in clinical oncology. Given the ability of NGS to detect a much broader range of genomic alterations than currently available technologies, particularly on limited tissue, we suggest that this type of testing can be a direct component of patient care and potentially expand targeted treatment options.
Citation Format: Roman Yelensky, Garrett M. Frampton, Alex Fichtenholtz, Sean Downing, Jie He, Frank Juhn, Kristina Brennan, Kai Wang, Geoff Otto, Mirna Jarosz, Alex Parker, Jeffrey S. Ross, John Curran, Maureen T. Cronin, Philip J. Stephens, Doron Lipson. Bringing next generation sequencing (NGS) to the clinic: Analytical validation of a comprehensive NGS-based cancer gene test. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2279. doi:10.1158/1538-7445.AM2013-2279