title: Allele specific locked nucleic acid quantitative PCR (ASLNAqPCR): an accurate and cost-effective assay to diagnose and quantify KRAS and BRAF mutation.
abstract: The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.
authors.affiliation: Dipartimento di Ematologia e Scienze Oncologiche L e A Seragnoli, Sezione di Anatomia Istologia e Citologia Patologica M. Malpighi Università di Bologna-AUSL Ospedale Bellaria, Bologna, Italy. morandi@ausl.bologna.it
authors.forename: Luca
authors.initials: L
authors.lastname: Morandi
authors.forename: Dario
authors.initials: D
authors.lastname: de Biase
authors.forename: Michela
authors.initials: M
authors.lastname: Visani
authors.forename: Valentina
authors.initials: V
authors.lastname: Cesari
authors.forename: Giovanna
authors.initials: G
authors.lastname: De Maglio
authors.forename: Stefano
authors.initials: S
authors.lastname: Pizzolitto
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pession
authors.forename: Giovanni
authors.initials: G
authors.lastname: Tallini
mesh_terms: D000483:Alleles; D001681:Biological Assay / Q000191:economics / Q000379:methods*; D003110:Colonic Neoplasms / Q000235:genetics; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods* / Q000592:standards; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017186:Titrimetry; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; C477371:locked nucleic acid; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins