Abstract 928: Bias-corrected next generation sequencing of cell free DNA for detection of actionable mutations and rearrangements in advanced non-small cell lung cancer
Authors: Paweletz Cloud P. , Raymond Chris K. , Sacher Adrian G. , Kuang Yanan , O'Connell Allison , Lim Lee , Li Mark , Armour Chris , English Jessie M. , Kirschmeier Paul T. , Jänne Pasi A. , Oxnard Geoffrey 
Published: August 2015

Abstract
Genotype-directed targeted therapies are transforming the care of advanced cancer. However, having adequate tumor tissue for comprehensive genotyping remains a challenge for many patients. We recently reported on a new assay for noninvasive plasma genotyping using droplet digital PCR (ddPCR) of cell free DNA (cfDNA) (Oxnard et al., CCR, 2014). While appealing in its simplicity, ddPCR assays cannot detect complex genomic alterations such as chromosomal rearrangements and cannot be massively multiplexed; a key requirement for efficiently testing for the increasing number of rare but targetable genomic alterations in non-small cell lung cancer (NSCLC).
Here we report on a novel, targeted, bias-corrected, and quantitative Next Generation Sequencing (NGS) solution for analyses of cfDNA. Our plasma NGS assay is focused on targetable driver mutations and rearrangements in lung cancer targeting 23 exons, 9 introns covering ∼17kb for 10 driver genes. Rather than using traditional hybrid capture, our assay integrates efficient fragment cloning, guided target retrieval, and molecular annotation of library sequences, which reduces off-target reads and improves signal-to-noise ratio. Performance of this assay panel was optimized in two iterations of probe design using targeted sequencing results of fifteen annotated NSCLC-derived cell lines. We were able to detect hotspot base changes and indels with 100% sensitivity and specificity at mutant allele frequencies of ∼1% and with 92.3% specificity at allele frequencies less than ≤0.1%. An automated pipeline for mutation calling correctly identified rare alleles with a sensitivity and specificity for point mutations and indels of &gt;90% at allele frequencies of ≤0.2%. We then piloted the technology using cfDNA from a patient with newly diagnosed advanced ALK-rearranged NSCLC and two NSCLC patients with micro-duplication/insertions in Her2neu. Blinded to the true tumor sequences, our NGS platform detected the correct ALK rearrangement (EML4-ALK v2) in 2 separate reads as well as the correct Her2neu insertions (2311-2322dup and 2332-2340 dup) in circulating DNA. We further show clinical translation to plasma from a cohort of 20 lung cancer patients that harbored rare driver genetic events, including ALK, ROS1 and RET rearrangements by tissue sequencing.
We are the first to sequence chromosomal rearrangements in plasma without prior knowledge of tissue genotyping results. The potential for this NGS-based plasma genotyping assay to allow for the noninvasive, multiplexed detection of complex, targetable genomic alterations including rearrangements and rare mutations represents a significant improvement over existing methods.
Citation Format: Cloud P. Paweletz, Chris K. Raymond, Adrian G. Sacher, Yanan Kuang, Allison O'Connell, Lee Lim, Mark Li, Chris Armour, Jessie M. English, Paul T. Kirschmeier, Pasi A. Jänne, Geoffrey Oxnard. Bias-corrected next generation sequencing of cell free DNA for detection of actionable mutations and rearrangements in advanced non-small cell lung 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 928. doi:10.1158/1538-7445.AM2015-928