Abstract A208: Mutation detection in FFPE and plasma circulating DNA with a focused ALK-EGFR-KRAS Next-Generation Sequencing panel.
Authors: Fang Peng , Darwanto Agus , Yan Zhenyu , Liu Weihua , Pelak Kimberly , Kristof Jessica , Mack Philip C. , Sankar Sabita , Galderisi Chad , Li Jin 
Published: August 2014

Abstract
Background: Mutations in ALK, EGFR and KRAS have well-established or anticipated clinical utility in predicting response to targeted therapies either approved or in development for patients with non-small cell lung cancer (NSCLC). Here we report results from a proof-of-concept study using a custom Ion AmpliSeq Next-Generation Sequencing (NGS) panel designed to sequence a range of clinically relevant exons in ALK, EGFR and KRAS genes in formalin fixed paraffin-embedded (FFPE) DNA and plasma circulating DNA.
Methods: The cumulative 2.1kb regions of interest (ROI) include exons 20-25 of ALK, exons 18-22 of EGFR, and exons 2-4 of KRAS, as well as the first 3 bp of the intron at the intron-exon boundaries. The custom panel was designed by Ion AmpliSeq Designer. Sequencing data were analyzed with Torrent Suite 3.4 and MolecularMD's proprietary analysis pipeline.
Results: The study demonstrated the robustness of the custom NGS assay with at least 90% of reads-on-target, an average coverage of above 5000x, and no less than 81% uniformity in the reads. In addition, the minimum coverage for each ROI in all the samples tested was no less than 1000x. The limit of detection (LOD) of the NGS assay, as determined using dilutions of cell line DNA standards, was 0.7% for single base substitution (SBS) mutations, and 1.2% for indels. In addition, the assay was able to quantify mutations with frequencies as low as ∼1% in plasma circulating DNA.
The assay detected 4 KRAS SBS mutations (G12C, G12D, G12V and Q61H), 2 EGFR SBS mutations (G719A, V769M) and 1 EGFR exon 19 deletion (G746-A750del) in 10 FFPE specimens tested from colon and lung cancer patients. These variants were each confirmed using Cancer Panels (Ion Torrent AmpliSeq and Illumina TrueSeq). The assay also detected T790M, L858R, V769M and the exon 19 deletion in EGFR in 7 plasma samples obtained from NSCLC patients. Each of these variants was confirmed by Sanger sequencing, Qiagen RGQ assay, or by MolecularMD's proprietary EGFR droplet digital PCR (ddPCR) assay. A low-frequency EGFR exon 19 deletion identified by the Qiagen RGQ assay was not seen in either the NGS assay or ddPCR assay. No other potential false negatives were identified in the custom NGS assay.
Conclusions: This study demonstrates the feasibility of creating a sensitive and specific ALK-EGFR-KRAS focused NGS assay that covers broader regions of the target genes than the hotspots represented in commercial panels. With a DNA input of merely 20ng of FFPE DNA, or 1-2ng of the plasma circulating DNA, the assay was able to detect both SBS and small indels in the targeted regions. Given the low DNA input requirements and the capability of plasma-based testing, this assay and other custom NGS panels may enable routine monitoring of mutation status for relevant genes in patients with various solid tumors, and may ultimately inform clinical decision-making.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A208.
Citation Format: Peng Fang, Agus Darwanto, Zhenyu Yan, Weihua Liu, Kimberly Pelak, Jessica Kristof, Philip C. Mack, Sabita Sankar, Chad Galderisi, Jin Li. Mutation detection in FFPE and plasma circulating DNA with a focused ALK-EGFR-KRAS Next-Generation Sequencing panel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A208.