title: Coamplification at lower denaturation temperature polymerase chain reaction enables selective identification of K-Ras mutations in formalin-fixed, paraffin-embedded tumor tissues without tumor-cell enrichment.
abstract: Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.
authors.affiliation: The Comprehensive Cancer Center of Drum Tower Hospital, Jangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Shaorong
authors.initials: S
authors.lastname: Yu
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.forename: Zhibo
authors.initials: Z
authors.lastname: Hou
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Qian
authors.forename: Lixia
authors.initials: L
authors.lastname: Yu
authors.forename: Jia
authors.initials: J
authors.lastname: Wei
authors.forename: Yitao
authors.initials: Y
authors.lastname: Ding
authors.forename: Baorui
authors.initials: B
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D005404:Fixatives; D005557:Formaldehyde; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D005404:Fixatives; D005557:Formaldehyde