Abstract 4813: High-depth sequencing of 800 cancer-associated genes in 48 matched tumor-normal tissues to identify somatic alterations including low-frequency variants in colorectal cancer

Abstract

Abstract Purpose: Colorectal cancer is the 3rd most common cancer globally accounting for 10% of the global cancer burden. Catalogues of somatic mutations in cancer (e.g. The Cancer Genome Atlas (TCGA)) are based upon exome sequencing, typically performed at depths of ∼ 80x, which will miss sub-clonal low-frequency somatic variants. We performed high depth sequencing of 800 cancer-associated genes across 48 tumor/normal pairs of CRC to catalogue somatic variants including those present at low variant allele frequencies. Method: We used the Agilent SureSelect Target Enrichment system to design a customized enrichment panel comprising all coding exons of ∼800 cancer-associated genes. From genomic DNA extracted from frozen tissue specimens (matched primary tumor and adjacent normal), we performed library construction, target enrichment and illumina next generation sequencing. We used 3 variant-calling algorithms - a Genome Analyzer Toolkit (GATK) based pipeline, LoFreq and MuTect to identify somatic variants including sub-clonal variants present at low variant allele frequencies. Results: Sequencing was performed to a median of 302-fold and 310-fold coverage across target regions in the tumor tissues and normal tissues respectively. The use of 3 bioinformatics variant-calling algorithm allowed sensitive and comprehensive identification of variants down to a variant allele frequency of 2.5%; selected variants were validated by an orthogonal sequencing platform (Ion Torrent). The validation rate was > 97%. Each patient had a variant in a median of 19 genes (range 8-100) with a different complement of clonal and sub-clonal variants. Recurrent alterations (e.g. APC, KRAS, TP53) tended to be clonal. Broad regions of allelic imbalance (based on heterozygous SNPs identified in each patient's normal tissue) could also be mapped in each patient. Conclusion: High Depth sequencing of 800 cancer-associated genes in 48 matched tumor normal tissues permits identification of somatic alterations including low-frequency variants and sub-clonal alterations in patients with colorectal cancer. Citation Format: Clarinda Chua, Dan Liang Ho, Yuka Suzuki, Simeen Malik, John McPherson, Anna Gan, Dennis Koh, Choon Leong Tang, Sarah Boon Hsi Ng, Patrick Tan, Steve Rozen, Iain Beehuat Tan. High-depth sequencing of 800 cancer-associated genes in 48 matched tumor-normal tissues to identify somatic alterations including low-frequency variants in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4813. doi:10.1158/1538-7445.AM2015-4813