Abstract 813: Diagnostic yield of targeted next-generation sequencing for personalized cancer therapeutics.
Authors: Hagemann Ian S. , Al-Kateb Hussam , Cottrell Catherine E. , Lockwood Christina , Nguyen TuDung , Spencer David , Bredemeyer Andrew , Head Richard , Varghese Nobish , Nagarajan Rakesh , Seibert Karen , Duncavage Eric J. , Kulkarni Shashikant , Pfeifer John D. 
Published: August 2014

Abstract
We have implemented a clinical assay to perform next-generation sequencing (NGS) of twenty-five genes relevant to multiple cancer types. We report findings from 210 consecutive malignancies submitted to Genomics and Pathology Services at Washington University in St. Louis (GPS@WUSTL). DNA was extracted from formalin-fixed, paraffin-embedded tumor selected by histopathologic review. Up to 1 μg of DNA (minimum, 0.2 μg) was used for library preparation using liquid-phase cRNA capture probes targeting all exons of BRAF, CTNNB1, CHIC2, CSF1R, DNMT3A, EGFR, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MAPK1, MAP2K2, MET, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RET, RUNX1, TP53, and WT1 (302 kb). Libraries were sequenced by Illumina technology to obtain 101bp or 150bp paired-end reads. Mean coverage was 1596; 50x depth was achieved at &gt;95% of positions. The analysis pipeline was validated for variant allele frequency &gt;10%. Variants were called and interpreted using the Clinical Genomicist Workstation. A list of clinically actionable variants (“hot spots”) was curated by literature review. Of 210 cases, 94 (45%) were non-small cell lung cancer (NSCLC), 20 (9.5%) were pancreatic cancer (PCA), and 17 (8.1%) were colorectal cancer (CRC), the balance representing 38 diverse other malignancies. FFPE tissue cores yielded a median of 2.4 μg DNA (interquartile range 1.0-4.9 μg). Sequencing identified a mean of 5.4 non-synonymous coding and/or splice site variants per case (single-nucleotide variants and indels). Of these, a mean of 2.8/case were not known polymorphisms and likely represented somatic mutations. Somatic variants most commonly occurred in TP53 (233 variants in 107 cases), KRAS (52 variants in 51 cases), and EGFR (40 variants in 33 cases). Sequencing identified 74 clinically actionable (i.e., predictive or prognostic) mutations in 61/210 = 29% of cases. In NSCLC, actionable mutations were present in KRAS codon 12 (29/94 cases), EGFR exons 19 (5 cases), 20 (1 case) and 21 (5 cases), and the PIK3CA helical domain (4 cases). In PCA, there were no variants currently recognized as actionable, but one potentially targetable MET T1010I variant was found. In CRC, actionable mutations were present in KRAS codon 12 or 13 (6/17 cases), PIK3CA (3 cases) and KIT (1 case). Average turnaround time was 29 days. In conclusion, NGS-based diagnostics can provide clinically relevant information using readily available FFPE tissue. Actionable variants were identified in 29% of cases. The existence of targeted therapies suggests that such diagnostics have the potential to improve patient outcomes.
Citation Format: Ian S. Hagemann, Hussam Al-Kateb, Catherine E. Cottrell, Christina Lockwood, TuDung Nguyen, David Spencer, Andrew Bredemeyer, Richard Head, Nobish Varghese, Rakesh Nagarajan, Karen Seibert, Eric J. Duncavage, Shashikant Kulkarni, John D. Pfeifer. Diagnostic yield of targeted next-generation sequencing for personalized cancer therapeutics. [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 813. doi:10.1158/1538-7445.AM2013-813