Multiplex digital PCR assay to detect multiple KRAS and GNAS mutations associated with pancreatic carcinogenesis from minimal specimen amounts.

Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. Here we developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a 2-D plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, we determined the absolute quantification of different KRAS and GNAS variants. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 FNA samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized using template DNA at a concentration as low as 1-10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false positives using high-fidelity polymerase, allowing for the detection of various KRAS and GNAS mutations with high probability in small cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the FNA needle flush and microscopic lesions in resected specimens, have successfully been identified. The proposed method allows for low-cost and accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.