[1810] Spectrum of Non-Synonymous Mutations in Cancer Identified by Clinical Targeted Next-Generation Sequencing

Ian S Hagemann, Hussam Al-Kateb, Catherine E Cottrell, Christina Lockwood, TuDung T Nguyen, David Spencer, Andrew Bredemeyer, Richard Head, Savita Shrivastava, Rakesh Nagarajan, Karen Seibert, Eric J Duncavage, Shashikant Kulkarni, John D Pfeifer. Washington University, St. Louis, MO

Background: We have implemented a clinical assay using next-generation sequencing (NGS) to analyze twenty-five genes relevant to multiple cancer types. We report findings from 155 consecutive malignancies submitted to Genomics and Pathology Services at Washington University in St. Louis (GPS@WUSTL).
Design: DNA was extracted from up to six 1 mm cores of tumor formalin-fixed, paraffin-embedded (FFPE) tissue after histopathologic review. Up to 1 µg of DNA (minimum, 0.2 µg) was subjected to library preparation using liquid-phase cRNA capture probes targeting a 300 kb region consisting of 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. Libraries were sequenced using Illumina technology to obtain 101 bp or 150 bp paired-end reads. Mean depth was >5660x, and 50x coverage was achieved at >95% of positions. The analysis pipeline was validated for variant allele frequencies >10%. Variants were called, prioritized, and interpreted using a custom software package. A list of actionable variants was prepared by literature review.
Results: Of 155 cases, 79 (50%) were non-small cell lung cancer (NSCLC), 19 (12%) were pancreatic malignancies, and 15 (9.4%) were colorectal cancer, with the balance representing 22 diverse other malignancies. FFPE tissue cores yielded a median of 2.7 μg DNA (interquartile range, 1.19-5.55 μg). Sequencing identified a mean of 4.6 non-synonymous variants per case (single-nucleotide variants and indels). Of these, a mean of 2.5/case were not known polymorphisms and most likely represented somatic mutations. Somatic variants most commonly occurred in TP53 (127/155 cases), KRAS (45 cases), and EGFR (20 cases). Sequencing identified 57 clinically actionable (i.e., predictive or prognostic) mutations in 46/155 = 30% of cases. In NSCLC, actionable mutations were present in KRAS codon 12 (22/78 cases), EGFR exon 21 (5 cases), and the PIK3CA helical domain (4 cases). In pancreatic cancer, there were no variants currently recognized as actionable. In colorectal cancer, actionable mutations were present in KRAS codon 12 or 13 (6/15 cases) or PIK3CA (3 cases). Average turnaround time was 29 days.
Conclusions: NGS-based diagnostics can provide clinically relevant information using readily available FFPE tissue. Actionable variants were identified in 30% of cases. Targeted therapies exist for many of these mutations, suggesting that such diagnostics have the potential to improve patient outcome.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer

Monday, March 4, 2013 9:30 AM

Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 275, Monday Morning


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