Abstract 4902: Detecting low abundant mutations in circulating cell free DNA and FFPE samples
Authors: Laliberte Julie , Couture Catherine , Makarov Vladimir , Schumacher Cassie , Sandhu Sukhinder , Kurihara Laurie , Harkins Timothy 
Published: August 2015

Abstract
Detection of low abundant alterations in oncogenes from circulating cell free DNA (cfDNA) samples or formaldehyde fixed-paraffin embedded samples (FFPE) has been a challenge for the community. Additionally, cfDNA and FFPE samples are typically limited in the amount of material that can be obtained, and often the FFPE samples are highly degraded, compounding the ability to sequence them and to perform accurate variant calling. To overcome these challenges, we have developed a single tube, multiplexed amplicon sequencing technology that employ hundreds of primer pairs for amplification of targeted loci, producing ready-to-run libraries for Illumina sequencing platforms. The resulting amplicons are less than 150 bp in length, enabling amplification and sensitive detection of mutations from cfDNA-sized DNA fragments or highly damaged DNA. A 200+ amplicon panel across 55 genes was developed to target known clinically relevant oncological mutations. The panel design encompasses single exons (e.g. BRAF) as well as comprehensive exon coverage of entire genes (e.g. TP53).
DNA was extracted from FFPE, and cfDNA was extracted from fresh plasma. DNA was quantified by qPCR using 2 primer pairs targeting ALU repeat regions. 10ng of DNA input was used to perform the multiplexed PCR. Libraries were quantified by qPCR and sequenced with MiSEQ V2 reagents, paired end reads. Alignment was performed using Burrows-wheeler Aligner (BWA) and variant calling was performed with at least two validated publicly available tools and confirmed manually by IGV. A variety of tools were used to validate allele technology was used to validate the sequencing data.
We interrogated 80 FFPE samples using our amplicon technology.The percent of on-target bases was over 95% and the coverage uniformity was over 98%, where uniformity is the percent based covered over 20% of the mean coverage. Out of the 40 colorectal cancer samples tested, 17 showed mutations in KRAS. Out of the 20 melanoma samples (Cure line) also analyzed, 9 were positive for BRAF V600E. The presence of those mutations was confirmed using myTprimerTM KRAS and BRAF qPCR assay. The limit of detection of the assay was set at 3% due to the inherent noise caused by the damage from the FFPE samples. For cell lines with known mutations, the sensitivity was 0.5%. No false positive or false negative were reported. 20 FFPE samples from different cancer types were also assessed. Mutations were identified in ERBB2 (cervical cancer), ALK (lung cancer) and TP53 (colon cancer) genes, as well as MET amplification (lung cancer).
Cell free DNA provides a non-invasive tool to monitor cancer progression and treatment efficacy. Tumor DNA, matching blood DNA and cfDNA from 10 cancer patients will be subjected to our multiplex PCR oncology panel. Data of this study will be presented.
Our results show that this unique multiplex PCR panel is an excellent tool to assess multiple oncogenes in limiting clinical samples, enabling high throughput, cost effective NGS analysis.
Citation Format: Julie Laliberte, Catherine Couture, Vladimir Makarov, Cassie Schumacher, Sukhinder Sandhu, Laurie Kurihara, Timothy Harkins. Detecting low abundant mutations in circulating cell free DNA and FFPE samples. [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 4902. doi:10.1158/1538-7445.AM2015-4902