Highly multiplexed digital PCR assay for simultaneous quantification of variant allele frequencies and copy number alterations of KRAS and GNAS in pancreatic cancer precursors.

Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasms (IPMNs) are pancreatic ductal adenocarcinoma (PDAC) precursor lesions. Detecting these precursors and monitoring their progression are crucial for early PDAC diagnosis. Digital PCR (dPCR) is a highly sensitive nucleic acid quantification technique and offers a cost-effective option for patient follow-up. However, the clinical utility of conventional dPCR is restricted by multiplexing constraints, particularly due to the challenge of simultaneously quantifying multiple mutations and amplifications. In this study, we applied highly multiplexed dPCR and melting curve analysis to simultaneously measure single nucleotide mutations and amplifications of KRAS and GNAS. The developed 14-plex assay included both wild-type and mutant KRAS, a common driver gene in both PanIN and IPMN, and GNAS, which is specifically mutated in IPMN, along with RPP30, a reference gene for copy number alterations (CNAs). This multiplex dPCR method detected all target mutations with a limit of detection below 0.2% while quantifying CNAs. Additionally, the assay accurately quantified variant allele frequencies in liquid biopsy and tissue samples from both pancreatic neoplasm precursor and PDAC patients, indicating its potential for use in comprehensive patient follow-up.