Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC)

Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Sequencing recognized 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 81131-70-6 supplier samples. 1. 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]. However, the novel therapeutic approaches based 81131-70-6 supplier on drugs directed against specific molecular agents are suitable only for molecularly selected populations of patients [3]. Therefore, molecular characterization is mandatory to identify patients which would most likely benefit from treatment with targeted therapies. Mutations in the epidermal growth factor receptor (EGFR) gene in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors (TKIs) [4, 81131-70-6 supplier 5]. These mutations are usually found in exons 18 through 21 of the TK domain of the EGFR and are either point mutations or in-frame small deletions or insertions [6]. Although more than 250 mutations of the EGFR have been described up to now, two variants, a single point mutation in exon 21, the L858R, and a series of small in-frame deletions in exon 19, account for approximately 90% of all EGFR mutations [6]. To be able to determine whether an EGFR TKI or chemotherapy is Rabbit polyclonal to AKAP5 the appropriate first-line therapy, guidelines recommend mutation testing 81131-70-6 supplier for all those patients with advanced NSCLC tumor and adenocarcinoma histology [7]. The sensitivity of assays for hot-spot mutation detection is usually a 81131-70-6 supplier key issue in molecular diagnostics due to several limitations of tumor samples: the poor quality of the DNA extracted from formalin fixed paraffin embedded (FFPE) tissues, the low quantity of DNA available, and the contamination of tumor sample by nonneoplastic cells carrying wild type alleles [3]. Direct sequencing of PCR products is still considered the gold standard for the identification of mutations, but it is usually laborious and requires at least 40% to 50% of tumor cells content to prevent false negative results [7, 8]. The limited sensitivity of direct sequencing has created a need for alternative techniques to detect common mutations, such as well real-time PCR based assays, pyrosequencing, high resolution melting, and PNA-PCR clamp [9]. These new methods are faster and more sensitive than sequencing. For example, the real-time PCR based EGFR Therascreen RGQ kit, employing Scorpion probes and the ARMS technology, allows for selective amplification of mutated sequences leading to a sensitivity of 1% (Table 1). Table 1 Sensitivity of methods for mutational analysis. Highly sensitive methods should be cautiously validated in routine diagnostic to ensure accuracy in tumor mutation testing. In this regard, Competitive Allele-Specific TaqMan PCR (castPCR) is usually a highly specific and sensitive technology, able to detect rare amounts of mutated DNA in a large background of normal, wild type genomic DNA [10]. An allele-specific primer and a FAM dye-labelled MGB (Minor Groove Binder) probe detect the mutant allele, while an MGB oligonucleotide blocker suppresses the wild type allele. Mutant allele assays are run with a gene reference assay that is designed to a mutation-free region of the gene. This process would work for identifying the existence or lack of a particular mutation in an example with a higher amount of specificity, allowing the recognition of less than 0.1% mutant allele in the current presence of a wild type allele background (Desk 1). Specifically, sensibility for TaqMan Mutation Recognition Assays (TMDA) continues to be described to become at least of 0.5% for some common EGFR mutations, like the L858R and exon 19 deletions [10]. Nevertheless, the awareness of diagnostic exams.