Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases


Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRMapproach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma(PA) cases as a novel and effective diagnostic technique. Materials and
Methods: We tested the sensitivity andspecificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNAsamples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirmmutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then usedtarget-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRASmutations and/or serum CA 19-9.
Results: It was found that the sensitivity of Sanger reached 0.5% with COLDPCR,whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probeHRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in thedisease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples.The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA19-9 increased to 0.934.
Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be asensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulatingDNA for diagnosing and treating PA.