BACKGROUND: Recent studies have shown that tumor-specific DNA from multiple types of tumors can be detected circulating in plasma and this has raised the possibility of “liquid biopsies” using mutated tumor DNA as a potential diagnostic and prognostic biomarker. Detection of mutations with allele frequencies below 0.1% remains challenging however given that circulating cell-free DNA is highly degraded and in low abundance. Detection of multiple different mutations in the same sample presents an additional challenge particularly when the mutation panel may change from patient to patient. We have developed a novel approach, called SimSen-Seq, to introduce molecular barcodes into sequencing libraries with DNA inputs as low as 5ng. Barcodes enable differentiation of true mutants from background noise introduced by Taq polymerase errors and permits detection of variant alleles with frequencies below 0.1%. The barcodes are protected from mis-priming using a hairpin structure which permits a high degree of multiplexing and flexibility for detection of multiple mutations from one plasma sample. We are using this technology to test the utility of liquid biopsy as a biomarker for esophageal adenocarcinoma (EAC) diagnosis and disease monitoring.
METHODS: Blood samples were obtained at a single time point from patients with various stages of EAC and longitudinal blood samples were also collected from patients undergoing neoadjuvant therapy followed by surgery. Tumor samples were obtained from biopsy or resection specimens. Tumor DNA was sequenced using a targeted EAC panel to identify mutations in each tumor. SimSen-Seq assays were designed to identify these mutations in plasma, and hairpin barcodes were attached. Sequencing libraries were generated from circulating DNA, sequenced and analyzed using the barcodes to reduce background noise.
RESULTS: Mutations were identified in tumor samples from 37 patients. To date, 29 patients have had plasma analyzed; 5 stage I, 6 stage II, 13 stage III, and 5 stage IV. Of these 29, the same mutations have been identified in 15 plasma DNA samples (20% stage I, 50% stage II, 54% stage III, 80% stage IV. Six patients demonstrated multiple mutant alleles in plasma DNA. One patient with detectable pre-treatment ctDNA underwent serial blood draws during their treatment course, and post-operative detection of tumor-specific markers preceded physical evidence of disease recurrence.
DISCUSSION: SimSen-Seq shows promise as a novel ultra-sensitive, highly multiplexed sequencing method for identifying rare circulating mutations. Possible applications include prognostication in early stage patients and rapid monitoring of therapeutic response and recurrence. Further work is to evaluate this is ongoing.
Citation Format: Matthew Egyud, Jennifer Jackson, Emiko Yamada, Anders Ståhlberg, Paul Krzyzanowski, Virginia Litle, Lincoln Stein, Tony Godfrey. Detection of tumor-specific mutations in plasma DNA: A potential esophageal adenocarcinoma biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2746. doi:10.1158/1538-7445.AM2017-2746