Abstract

Detection of Tumor-specific Mutations in Circulating, Cell-free DNA: Potential for a Biomarker in Esophageal Adenocarcinoma

Tony Godfrey, Associate Chair of Research, Department of Surgery, Boston University Medical Center

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.