title: Development of pyrosequencing methods for the rapid detection of RAS mutations in clinical samples.
abstract: In advanced colorectal carcinoma (CRC) patients, extended RAS mutations testing (KRAS exons 2 to 4 and NRAS exons 2 to 4) is a prerequisite for patient stratification to anti-EGFr therapy. Accurately distinguishing mutant patients from potential responders has a clinically critical impact, and thus effective and low cost methods are needed for identification of the mutation status. We have developed quantitative pyrosequencing assays for sensitive and rapid detection of mutant RAS alleles in formalin-fixed, paraffin-embedded tissues. Exons 2 to 4 of KRAS and NRAS genes were PCR amplified and analyzed by pyrosequencing. For validation, PCR products were sequenced by conventional Sanger sequencing. Analytical sensitivity of these assays was determined by calculating the limit of detection. The results showed that low levels of mutant RAS alleles (2-13%) can be detected with pyrosequencing assays.
authors.affiliation: Department of Cancer Biology, Poitiers University Hospital, Poitiers, France. Electronic address: ulrich.cortes@chu-poitiers.fr.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Cortes
authors.affiliation: Department of Cancer Biology, Poitiers University Hospital, Poitiers, France.
authors.forename: Karline
authors.initials: K
authors.lastname: Guilloteau
authors.affiliation: Department of Cancer Biology, Poitiers University Hospital, Poitiers, France.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Rouvreau
authors.affiliation: Department of Cancer Biology, Poitiers University Hospital, Poitiers, France; Department of Pathology, Poitiers University Hospital, Poitiers, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Archaimbault
authors.affiliation: Department of Cancer Biology, Poitiers University Hospital, Poitiers, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Villalva
authors.affiliation: Department of Cancer Biology, Poitiers University Hospital, Poitiers, France; INSERMU1084, Poitiers, France; Poitiers University, Poitiers, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Karayan-Tapon
mesh_terms: D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D017063:Outcome Assessment, Health Care; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D018631:ras Proteins / Q000235:genetics*
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors; D018631:ras Proteins
keywords: Pyrosequencing; RAS