Abstract 4276: T200: a high depth targeted exome sequencing platform to identify actionable alterations in FFPE solid tumor samples
Authors: Eterovic Karina , Chen Ken , Zhao Hao , Meric-Bernstam Funda , Luthra Raja , Kenneth Aldape , Routbort Mark , Kopetz Scott , Davies Michael , Groot John de , Moulder Stacy , Mao Yong , Horombe Chacha , Tang Lin-ya , Ping Song , Qingxiu Zhang , Nader Ezzeddine , Zhang Lan , Shaw Kenna M. , Mendelsohn John , Mills Gordon B. 
Published: May 2015

Abstract
Background: We developed and established a high depth targeted sequencing platform of 202 genes (all exons) to identify actionable DNA alterations in tumor samples. We aimed a minimum of 500x depth and defined actionable if the aberration had a direct link to an available clinical treatment or clinical trial. Our platform was optimized for FFPE specimens and low input DNA while the data analysis pipeline was designed to detect low frequency mutations and copy number alterations. Over 500 tumor samples and matched normal were analyzed using this platform.
Methods: Libraries were made from a minimum of 170 ng of FFPE DNA (tumors) or blood DNA (matched normal). Capture of 202 genes (over 5,000 exons) was performed using Nimblegen probes to a minimum of 50x fold enrichment. Captured libraries were sequenced using Illumina HiSeq2000. Duplicate reads were removed from the raw data and the reads were mapped hg19 reference genome. We used VarScan2 for calling somatic, germline and loss of heterozygosity SNVs and short indels. Copy number variation was also reported when significant gain/loss on an exon was detected. Additionally, we used other tolls to annotate the functional consequence of the SNVs, such as CanDrA to predict if an SNV is driver or passenger event.
Results: We sequenced over 500 tumor samples and their normal match (blood). The mean coverage for most of samples was over 1500x, which allowed us to detect low frequency mutations (5% or higher) with accuracy. The most common disease sites analyzed where we found genetic alterations were: breast, colon, brain, ovary, endometrial, skin, lung, prostate, head and neck, sarcoma, kidney and stomach. About 98% of all tumors analyzed had at least one somatic alteration (SNVs or copy number) and although the vast majority of the samples analyzed were derived from FFPE blocks, the number of samples that failed was less than 5%. Among the most common genes mutated are several “actionable genes” such as BRAF, EGFR and KRAS. The samples tested in this platform were also tested on ion torrent Ampliseq 46 gene panel (46 overlapping genes) in a CLIA certified laboratory at MD Anderson Cancer Center with a very high level of concordance.
Conclusion: With a very low level of failure and high depth provided, our results indicate that this platform can be used for FFPE tumor samples with high level of accuracy and sensitivity, showing that this platform is reliable to be implemented in clinical settings.
Citation Format: Karina Eterovic, Ken Chen, Hao Zhao, Funda Meric-Bernstam, Raja Luthra, Aldape Kenneth, Mark Routbort, Scott Kopetz, Michael Davies, John de Groot, Stacy Moulder, Yong Mao, Chacha Horombe, Lin-ya Tang, Song Ping, Zhang Qingxiu, Ezzeddine Nader, Lan Zhang, Kenna M. Shaw, John Mendelsohn, Gordon B. Mills. T200: a high depth targeted exome sequencing platform to identify actionable alterations in FFPE solid tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4276. doi:10.1158/1538-7445.AM2014-4276