Abstract 5242: Utilization of next-generation sequencing to identify clinically-relevant mutations in cell-free circulating tumor DNA from patients with advanced pancreatic cancer
Authors: Tepper Clifford G. , Davis Ryan R. , Liu Stephenie Y. , Tsai Rebekah A. , Hutchins Irene M. , Mack Philip C. , Semrad Thomas J. 
Published: August 2015

Abstract
Pancreatic cancer is a uniquely lethal malignancy characterized by frequent mutations in KRAS, CDKN2A, SMAD4, TP53 and many other genes. Molecular characterization is complicated by relatively few patients presenting with surgically resectable disease, frequent absence of metastases in easily accessible sites, and short patient survival. We have shown that KRAS mutation can be detected in cell-free, circulating tumor DNA (ctDNA) isolated from the plasma in a subset of patients and is associated with poor prognosis. The ability to simultaneously detect multiple pancreatic cancer-specific mutations in ctDNA would open a new avenue for detection of clinically-relevant mutations. We collected blood from 28 patients with advanced pancreatic cancer prior to treatment on a phase II clinical trial of pharmacodynamically separated gemcitabine and erlotinib. DNA was extracted from both plasma and available matched FFPE tumor specimens and evaluated with the GeneRead DNA QuantiMIZE System (Qiagen). The mutation status of 160 commonly-mutated genes was determined using an amplicon sequencing assay. Sequencing libraries were prepared from DNA samples (40 ng input) by targeted enrichment of exonic regions using the Qiagen GeneRead Human Comprehensive Cancer Panel, which encompasses &gt;744 kb genomic content with ∼8,000 amplicons. Next-generation sequencing (NGS) was performed on the Illumina MiSeq for validation analyses of a subset of matched tumor-ctDNA samples, and subsequently on the Illumina HiSeq 2500 for ultra-deep sequencing (1,000X) of the entire sample set. Read mapping, variant discovery, and functional annotation were performed with the Qiagen GeneRead Variant Calling Pipeline. The technical success of this sequencing assay for ctDNA, as well as FFPE DNA, was demonstrated by the resulting data meeting the expected NGS quality standards, including a high percentage of mappable reads (∼90%). In addition, the integrity of the sequencing data obtained from ctDNA samples was supported by detection of 82-94% of the variants found in each matched tumor. Somatic, non-synonymous variants were identified in 29 different genes and at allele frequencies typically &lt;0.5. While 52-68% of the mutations identified in the tumor specimen could also be detected in the cell-free ctDNA, not all mutations of potential clinical relevance in tumor specimens were detected in the ctDNA specimens. Updated results from ultra-deep NGS analysis will be presented. The results demonstrate the feasibility of using a new targeted NGS assay for the simultaneous identification of mutations in 160 cancer-related genes in ctDNA. The sensitivity and specificity of variant identification achieved with this technique are potentially clinically relevant, and its optimization could provide a true liquid biopsy for molecular characterization of pancreatic cancer.
Citation Format: Clifford G. Tepper, Ryan R. Davis, Stephenie Y. Liu, Rebekah A. Tsai, Irene M. Hutchins, Philip C. Mack, Thomas J. Semrad. Utilization of next-generation sequencing to identify clinically-relevant mutations in cell-free circulating tumor DNA from patients with advanced pancreatic 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 5242. doi:10.1158/1538-7445.AM2015-5242