Abstract 3967: Amplicon-based NGS detects targeted variants in paired tissue and ctDNA samples
Authors: Greer Adam H. , Mills Glenn , Yin Hong 
Published: July 2016

Abstract
Circulating tumor DNA (ctDNA) in blood stream has been recognized as an essential sample source for non-invasive biopsy of cancer patients. With next generation sequencing (NGS) technology it is possible to detect multiple cancer relevant mutation spots simultaneously. In order to understand the correlation of mutation spectrum between DNA from original solid tumor and DNA from plasma, we analyzed paired DNA samples with amplicon-based NGS. Paired frozen tissue and plasma samples were collected from 17 colon cancer patients under the IRB protocol. Tissue DNA was extracted with NucleoSpin Tissue kit of Clontech. The ctDNA was extracted from 0.5ml of plasma with Quick-cfDNA™ Serum & Plasma Kit from Zymo Research. The library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences, Inc. This panel generates 263 amplicons covering 56 clinically relevant oncology-related genes. Input ctDNA from plasma was 0.5 to 10 ng depending on the sample yield. The sequencing was conducted on Illumina's Miseq with MiSeq Reagent Kits v2 (300cycles). FastQ files were generated by Miseq Reporter. Non-synonymous variant calls by the tissue DNA sequencing were defined by coverage depth (30), frequency (&gt;5%), and forward/reverse balance (≠0). The defined non-synonymous variant calls were searched from variant calls by the paired ctDNA sequencing with the cutoff frequency at 0.1%. In 17 paired colon cancer samples, 86 non-synonymous variant calls were identified by tissue DNA sequencing. The variant calls of tissue DNA samples showed 56 of 86 variants matched variant calls in the paired ctDNA samples. Among 56 variant calls in ctDNA, 44 variant calls were assumed as germline variants according to the 1000 genome reference and similar frequency of the variant call between paired tissue DNA and ctDNA. There were 12 variant calls deemed somatic variant calls by the literature references and the differential frequency of the variant call between paired tissue DNA and ctDNA. The 12 detected somatic variant calls were clustered in seven cases. Twelve somatic variant calls with frequency (%) in tissue and plasma DNA samples are shown as APC S1456 frame shift (41.8 vs 6.17), KRAS G12D(37.0 vs 5.0),TP53 G105R(56.0 vs 11.3), HNF1A P291 frame shift(36.8 vs 2.41), HNF1A P291 frame shift(27.1 vs 1.2) KRAS G12D(23.1 vs 0.3), ERBB4 I377T(8.3 vs 0.5),PTEN L267 frame shift (12.4 vs 0.4), MAKP2K1 L57N(23.9 vs 0.7),TP53 R273C(25.8 vs 0.3), TP53 H178D(12.1 vs 1.1),TP53 T377 frame shift(11.2 vs 0.1). Remaining 30 somatic variant calls were detected only in tissue DNA samples. Our results indicated that the simultaneous detection of somatic variants of multiple genes in ctDNA samples with the amplicon-based DNA sequencing is applicable with variant frequency over 0.1%. The NGS analysis of paired tissue DNA and ctDNA is a reliable evaluation for cancer related mutations in ctDNA.
Citation Format: Adam H. Greer, Glenn Mills, Hong Yin. Amplicon-based NGS detects targeted variants in paired tissue and ctDNA samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3967.