title: 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.
abstract: BACKGROUND
  The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique.
  
  
  METHODS
  We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9.
  
  
  RESULTS
  It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease 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 CA 19-9 increased to 0.934.
  
  
  CONCLUSIONS
  It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.
authors.affiliation: Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China E-mail : guo.wei@zs-hospital.sh.cn.
authors.forename: Jiong
authors.initials: J
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Zhang
authors.forename: Bin-Bin
authors.initials: BB
authors.lastname: Song
authors.forename: Bei-Li
authors.initials: BL
authors.lastname: Wang
authors.forename: Bai-Shen
authors.initials: BS
authors.lastname: Pan
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Lou
authors.forename: Wei
authors.initials: W
authors.lastname: Guo
mesh_terms: D000230:Adenocarcinoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D016022:Case-Control Studies; D003062:Codon / Q000235:genetics*; D003080:Cold Temperature; D004252:DNA Mutational Analysis; D005500:Follow-Up Studies; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D009691:Nucleic Acid Denaturation; D010190:Pancreatic Neoplasms / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012372:ROC Curve; D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins