Digital PCR-based plasma cell-free DNA mutation analysis for early-stage pancreatic tumor diagnosis and surveillance

Background Cell-free DNA (cfDNA) shed from tumors into the circulation offers a tool for cancer detection. Here, we evaluated the feasibility of cfDNA measurement and utility of digital PCR (dPCR)-based assays, which reduce subsampling error, for diagnosing pancreatic ductal adenocarcinoma (PDA) and surveillance of intraductal papillary mucinous neoplasm (IPMN). Methods We collected plasma from seven institutions for cfDNA measurements. Hot-spot mutations inKRASandGNASin the cfDNA from patients with PDA (n = 96), undergoing surveillance for IPMN (n = 112), and normal controls (n = 76) were evaluated using pre-amplification dPCR. Results Upon Qubit measurement and copy number assessment of hemoglobin-subunit (HBB) and mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) in plasma cfDNA,HBBoffered the best resolution between patients with PDA relative to healthy subjects [area under the curve (AUC) 0.862], whereasMT-ND1revealed significant differences between IPMN and controls (AUC 0.851). DPCR utilizing pre-amplification cfDNA afforded accurate tumor-derived mutantKRASdetection in plasma in resectable PDA (AUC 0.861-0.876) and improved post-resection recurrence prediction [hazard ratio (HR) 3.179, 95% confidence interval (CI) 1.025-9.859] over that for the marker CA19-9 (HR 1.464; 95% CI 0.674-3.181). CapturingKRASandGNAScould also provide genetic evidence in patients with IPMN-associated PDA and undergoing pancreatic surveillance. Conclusions Plasma cfDNA quantification by distinct measurements is useful to predict tumor burden. Through appropriate methods, dPCR-mediated mutation detection in patients with localized PDA and IPMN likely to progress to invasive carcinoma is feasible and complements conventional biomarkers.