DETECTION OF CIRCULATING TUMOR DNA IN PATIENTS WITH PANCREATIC CANCER USING DIGITAL NEXT-GENERATION SEQUENCING.

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positives is a challenge. We evaluated digital next-generation sequencing (NGS) as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects including 67 with pancreatic ductal adenocarcinoma, and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pre-treated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two standard deviations above the mean in controls was considered positive. 37% of patients with pancreatic cancer, including 31% of patients with Stage I/II disease had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low normal-range digital NGS scores at mutation hot-spots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error, rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. The detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation cells in plasma DNA from controls.