Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

Cristin Roma,Salvatore Pisconti,Alessandro Morabito,Anna Maria Rachiglio,Renato Franco,Raffaella Pasquale,Alessia Iannaccone,Gerardo Botti,Antonella De Luca,Nicoletta Chicchinelli,Claudia Esposito,Rita Mancini,Nicola Normanno

doi:10.1155/2013/385087

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.

Highlights

The discovery of driver mutations in key genes involved in regulating proliferation and survival of cancer cells and the development of drugs capable to block such oncogenic mechanisms are leading to remarkable successes in translational medicine [1, 2]
The aim of this study is to assess the feasibility of TaqMan Mutation Detection Assays (TMDA) based on Competitive Allele-Specific TaqMan PCR (castPCR) technology to detect Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) clinical specimens and to compare this method with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit
In order to identify the best threshold values for the EGFR TMDA, we first analysed with this method a training set of 30 NSCLC and 6 CRC samples, which were included as negative controls due to the rare presence of EGFR mutations in this tumor type

Summary

Introduction

The discovery of driver mutations in key genes involved in regulating proliferation and survival of cancer cells and the development of drugs capable to block such oncogenic mechanisms are leading to remarkable successes in translational medicine [1, 2]. The novel therapeutic approaches based on drugs directed against specific molecular agents are suitable only for molecularly selected populations of patients [3]. Molecular characterization is mandatory to identify patients which would most likely benefit from treatment with targeted therapies

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