Abstract 4826: Identification of low prevalence somatic mutations in heterogeneous tumor samples

Abstract

Abstract High throughput sequencing enables the digital measurement of each allele in a DNA sample. This provides an ideal method to interrogate mutations present in heterogeneous samples such as solid tumors in which clonal selection or contamination with stroma can hinder the identification of important somatic mutations. We developed an ultra-deep targeted sequencing (UDT-Seq) assay to screen 46 cancer genes via microdroplet PCR (RainDance Technologies) and direct sequencing of the amplicons on the Illumina GA. This UDT-Seq library interrogates ∼86 kb of DNA located in cancer mutational hotspots (87% of all COSMIC mutations) and ∼29 kb located in exons sequenced in HapMap samples for the assay calibration and performance evaluation. We devised a statistical filtering of the mutations by using both experimental estimation and statistical modeling of the sequencing error rate. We measured the performance of our assay by processing 4 blends of 4 HapMap samples, interrogating 160 SNPs with known prevalence in each blend. The sensitivity and specificity of our method is >90% and >99% respectively for variants present at 5% prevalence or greater. We next interrogated 4 cancer samples (xenografts, 2 of which with matching primary samples) from 4 different tissues. We were able to detect low-prevalence somatic mutations in all samples of which some are well-known driver mutations. Interestingly, the mutational profile from primary to xenograft is conserved for one sample but different for another. Finally, we analyzed the robustness of the detection and prevalence measurement after performing whole-genome amplification on DNA extracted from fresh frozen and FFPE tissue using Single Primer Isothermal Amplification technology (SPIA® -NuGEN Technologies). Featuring a streamlined sample preparation to interrogate a large number of bases, this assay is well suited for clinical applications to study clonal selection in cancer progression or treatment with sub-optimal heterogeneous cancer samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4826. doi:10.1158/1538-7445.AM2011-4826