title: Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology.
abstract: Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.
authors.affiliation: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 83013 Mercogliano, Italy.
authors.forename: Cristin
authors.initials: C
authors.lastname: Roma
authors.affiliation: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 83013 Mercogliano, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Esposito
authors.affiliation: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 83013 Mercogliano, Italy.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Rachiglio
authors.affiliation: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 83013 Mercogliano, Italy.
authors.forename: Raffaella
authors.initials: R
authors.lastname: Pasquale
authors.affiliation: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 83013 Mercogliano, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Iannaccone
authors.affiliation: Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 80131 Naples, Italy.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Chicchinelli
authors.affiliation: Surgical Pathology Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 80131 Naples, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Franco
authors.affiliation: Department of Molecular and Clinical Medicine, Laboratory of Molecular and Cellular Biology, Università "La Sapienza", 00189 Rome, Italy ; Department of Surgery "A. Valdoni", Laboratory of Molecular and Cellular Biology, Università "La Sapienza", 00161 Rome, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Mancini
authors.affiliation: Medical Oncology Unit, SS Annunziata Hospital, 74123 Taranto, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Pisconti
authors.affiliation: Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 80131 Naples, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: De Luca
authors.affiliation: Surgical Pathology Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 80131 Naples, Italy.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Botti
authors.affiliation: Medical Oncology Unit, Department of Thoracic Surgical and Medical Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 80131 Naples, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Morabito
authors.affiliation: Laboratory of Pharmacogenomics, Centro di Ricerche Oncologiche di Mercogliano (CROM), Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 83013 Mercogliano, Italy ; Cell Biology and Biotherapy Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione Giovanni Pascale"(IRCCS), 80131 Naples, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Normanno
mesh_terms: D000483:Alleles; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D066246:ErbB Receptors / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: C512478:EGFR protein, human; D066246:ErbB Receptors