Assessment of a High Sensitivity Method for Identification of R132x Mutations in Tumors and Plasma of Intrahepatic Cholangiocarcinoma Patients.

Hotspot codon 132 mutations (R132xm) are frequent in intrahepatic cholangiocarcinoma (ICC), are druggable by anti-m agents, and could represent a marker of disease progression. Developing an assay to identify R132xm would provide a useful tool to select patients benefitting from targeted treatments. We tested a quantitative real-time allele-specific polymerase chain reaction (qPCR)-based method to detect the main R132xm in an Italian ICC series ( = 61) of formalin-fixed paraffin-embedded (FFPE) samples, and on circulating-free DNA samples. The outcomes were compared with nested PCR/Sanger sequencing. Reconstitution experiments of plasmids harboring the different R132xm mixed with wild-type (WT) DNA demonstrated that qPCR is able to detect at least 2% of all mutated allele. High efficiency was also observed on patient-derived mutated DNA mixed with WT DNA (up to 10% and 0.3 ng of mutated template); qPCR detected 16.4% of mutated samples (one R132G, three R132C and six R132L) while nested PCR/Sanger sequencing only 8.2% (four R132L and one R132G). In a single patient with an R132C-mutated tumor, qPCR was also performed on plasma samples collected at four time-points, observing an increase correlating with disease progression. In conclusion, we developed a qPCR assay which could represent a fast, inexpensive and sensitive tool both for detection of R132xm in ICC samples and monitoring disease progression from liquid biopsy.