Abstract 3152: CanSeq: prospective clinical whole-exome sequencing of FFPE tumor samples.
Authors: Wagle Nikhil , Van Allen Eliezer , Perrin Danielle , Friedrich Dennis , Fisher Sheila , Kryukov Gregory , Ambrogio Lauren , Auclair Daniel , Gray Stacy , Joffe Steven , Janne Pasi , Garber Judy , Macconaill Laura , Lindeman Neal , Rollins Barrett , Kantoff Phillip , Getz Gad , Gabriel Stacey , Garraway Levi A. 
Published: August 2014

Abstract
INTRODUCTION: Knowledge of tumor genomic alterations that may predict therapeutic responses represents a growing clinical need. Although there has been an increase in the use of hotspot genotyping and targeted sequencing panels of small numbers of genes, the use of clinical whole exome sequencing (WES) remains underdeveloped. Prospective WES in cancer patients presents several logistical and scientific challenges, including (i) generating robust sequencing data from small amounts of FFPE tumor tissue, (ii) establishing a clinically relevant turnaround time, (iii) achieving clinical interpretation of genomic alterations, and (iv) communicating results to the clinical team and patients. Here, we describe CanSeq, a clinical WES platform to detect genomic alterations in FFPE tumor samples and identify clinically actionable and biologically meaningful alterations in order to aid clinical decision-making and inform future research.
METHODS: We performed prospective WES on tumor and germline DNA from patients with advanced refractory cancer. Sequencing was performed at the Broad Institute using the Illumina HiSeq, starting with 100 ng of DNA or less from FFPE tumor tissue and matched normal blood. Data were analyzed using a novel algorithm to highlight clinically actionable mutations, indels, and copy number alterations. These were annotated and assigned levels of evidence, and an interactive web-based report was generated for review by clinicians.
RESULTS: The average turnaround time from sample receipt to data delivery was 16 days. Each tumor sample was sequenced at 90X or greater depth of coverage with more than 80% of exons with at least 30X coverage. Analysis of the first 15 patients revealed at least one plausibly actionable somatic mutation in 14 samples. These alterations include “standard of care” alterations (BRAF, EGFR), entry criteria for clinical trials (PIK3CA, KRAS, PTEN), potentially actionable alterations based on more limited evidence (STK11, ATM, CRKL, CTNNB1, PDGFRA, CDK4, CDKN2A, SMARCB1, TP53), and alterations that were theoretically targetable (JAK3, SYK). Additional biologically relevant somatic alterations and notable germline alterations were also identified. For several patients, actionable alterations were confirmed in a CLIA lab and impacted clinical decision-making, including enrollment in clinical trials.
CONCLUSION: We have developed a prospective clinical WES platform to robustly detect genomic alterations in archival FFPE tumor samples (using &lt;100 ng DNA) in a clinically appropriate time frame (&lt;3 wks). The platform enables rapid identification of clinically relevant information from vast numbers of somatic and germline events. The ability to use real world biopsy samples could facilitate the routine incorporation of WES into the care of cancer patients, speeding the advent of precision cancer medicine by identifying the actionable genetic changes in each patient's tumor.
Citation Format: Nikhil Wagle, Eliezer Van Allen, Danielle Perrin, Dennis Friedrich, Sheila Fisher, Gregory Kryukov, Lauren Ambrogio, Daniel Auclair, Stacy Gray, Steven Joffe, Pasi Janne, Judy Garber, Laura Macconaill, Neal Lindeman, Barrett Rollins, Phillip Kantoff, Gad Getz, Stacey Gabriel, Levi A. Garraway. CanSeq: prospective clinical whole-exome sequencing of FFPE tumor samples. [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 3152. doi:10.1158/1538-7445.AM2013-3152