Abstract 4925: Liquid biopsy using the ion AmpliSeq v2 cancer panel
Authors: Liu WeiHua , Yan Zhenyu , Horn Candice L. , Nunes Fabio , Bray Steven M. , Ebert Philip J. , Fang Peng , Biroschak Jennifer , Spittle Cindy , Galderisi Chad , Li Jin 
Published: August 2015

Abstract
Introduction:
Plasma cell-free circulating tumor DNA (ctDNA) has the potential to serve as a noninvasive surrogate to determine tumor genotype and monitor treatment response. Here we report the analytical validation of the Ion AmpliSeq v2 Cancer Panel (ACP) in plasma ctDNA samples.
Methods:
Mutations were assessed using the ACP, designed to survey 2800 mutations in 50 cancer-related genes. TruQ reference standards were used to determine LOD. Healthy donor and cancer patient plasma samples were used for other studies. Eight libraries were barcoded and sequenced with PGM sequencing 200 kit v2 on a 318 v2 chip. The sequencing data were analyzed with a combination of Torrent Suite 3.4.2, VarScan, our proprietary analysis pipeline and IGV. The background noise level of hotspot nt positions was calculated from multiple runs of known wild type DNA. In order to increase the sensitivity of the assay to detect low-level mutation, a custom program was developed to detect variants clearly different from background noise (95% confidence) and not reported by the two variant callers. PCR methods were used for concordance testing.
Results:
The performance characteristics of the AmpliSeq v2 Cancer Panel were demonstrated in 1) Minimum DNA input and LOD: With 10 ng DNA input, 43/43 and 42/43 expected mutations were detected at 2.5% or 1%, respectively. With 1 ng DNA input, 42/43 and 40/43 expected mutations were detected at 2.5% and 1%, respectively. 2) Coverage: The mean library coverage for 40 plasma libraries was 2912x with only 5 non-critical regions below 250x coverage. 3) Baseline error rate: 95% of 2290 hot-spots for SBS showed &lt;0.5% background error frequency assessed in 10 healthy control ctDNA samples. The mean background error frequency plus two folds of SD falls below 0.5% for 91% of SBS hot-spots. 4)Precision: All mutations expected for TruQ6 were detected in 4 independent runs at observed allele frequencies with 0.6% mean SD and 0.1% mean deviation from the true value. A 2% TP53 mutation R174W was reproducibly detected in a lung cancer plasma sample in 2 independent runs. 5) Concordance study: Consistent results were observed for 3 positive (Allele Freq 40%, 3% and 0.2%, respectively) and 3 negative melanoma plasma samples previously genotyped by AS-PCR for Braf V600E/K mutations. Additional cancer patient ctDNA samples are being assessed for concordance in the detection of other mutations.
Conclusions:
The ACP offers a useful tool for comprehensive somatic mutation profiling in ctDNA. The limit of detection is 1-2% mutation frequency for most mutations with slight variation across different nucleotide positions. The low concentration of ctDNA obtained from plasma samples can present a challenge. Less than 10 ng DNA input may result in allele drop out for low level mutations due to the inefficient amplification of multiplex PCR compared with PCR methods using a short length single amplicon. Higher DNA input to rescue allele drop-out is under evaluation.
Citation Format: WeiHua Liu, Zhenyu Yan, Candice L. Horn, Fabio Nunes, Steven M. Bray, Philip J. Ebert, Peng Fang, Jennifer Biroschak, Cindy Spittle, Chad Galderisi, Jin Li. Liquid biopsy using the ion AmpliSeq v2 cancer panel. [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 4925. doi:10.1158/1538-7445.AM2015-4925