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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). We collected plasma from seven institutions for cfDNA measurements. Hot-spot mutations in KRAS and GNAS in 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. 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, HBB offered the best resolution between patients with PDA relative to healthy subjects [area under the curve (AUC) 0.862], whereas MT-ND1 revealed significant differences between IPMN and controls (AUC 0.851). DPCR utilizing pre-amplification cfDNA afforded accurate tumor-derived mutant KRAS detection 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). Capturing KRAS and GNAS could also provide genetic evidence in patients with IPMN-associated PDA and undergoing pancreatic surveillance. 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.

Background & Aims Intraductal papillary mucinous neoplasms (IPMNs) are regarded as precursors of pancreatic ductal adenocarcinomas (PDAs), but little is known about the mechanism of progression. This makes it challenging to assess cancer risk in patients with IPMNs. We investigated associations of IPMNs with concurrent PDAs by genetic and histologic analyses. Methods We obtained 30 pancreatic tissues with concurrent PDAs and IPMNs, and 168 lesions, including incipient foci, were mapped, microdissected, and analyzed for mutations in 18 pancreatic cancer-associated genes and expression of tumor suppressors. Results We determined the clonal relatedness of lesions, based on driver mutations shared by PDAs and concurrent IPMNs, and classified the lesions into 3 subtypes. Twelve PDAs contained driver mutations shared by all concurrent IPMNs, which we called the sequential subtype. This subset was characterized by less diversity in incipient foci with frequent GNAS mutations. Eleven PDAs contained some driver mutations that were shared with concurrent IPMNs, which we called the branch-off subtype. In this subtype, PDAs and IPMNs had identical KRAS mutations but different GNAS mutations, although the lesions were adjacent. Whole-exome sequencing and methylation analysis of these lesions indicated clonal origin with later divergence. Ten PDAs had driver mutations not found in concurrent IPMNs, called the de novo subtype. Expression profiles of TP53 and SMAD4 increased our ability to differentiate these subtypes compared with sequencing data alone. The branch-off and de novo subtypes had substantial heterogeneity among early clones, such as differences in KRAS mutations. Patients with PDAs of the branch-off subtype had a longer times of disease-free survival than patients with PDAs of the de novo or the sequential subtypes. Conclusions Detailed histologic and genetic analysis of PDAs and concurrent IPMNs identified 3 different pathways by which IPMNs progress to PDAs—we call these the sequential, branch-off, and de novo subtypes. Subtypes might be associated with clinical and pathologic features and be used to select surveillance programs for patients with IPMNs.