title: Two allele-specific PCR assays for screening epidermal growth factor receptor gene hotspot mutations in lung adenocarcinoma.
abstract: In patients with non-small cell lung cancer, mutations in the EGFR tyrosine kinase domain have been associated with improved response to tyrosine kinase inhibitors such as gefitinib or erlotinib and prolonged survival. Two hotspot mutations located in exons 19 and 21 account for approximately 90% of EGFR mutations reported to date in lung adenocarcinoma. A Bi-PASA (bidirectional PCR amplification of specific alleles) assay for detecting the exon 19 deletion (codons 746-750) and an allele-specific PCR assay for the EGFR hotspot mutation L858R in exon 21 were designed. The assays were validated in normal control samples and in lung adenocarcinoma cell lines containing the mutation. The three-primer assay for the exon 21 point mutation and the four-primer assay for the exon 19 deletion were able to specifically discriminate wild-type and mutant DNA. The primer specificity was confirmed by genomic sequencing. The allele-specific PCR assays are fast and easy to perform in any routine PCR laboratory and no special equipment other than thermocyclers is required. They provide rapid, sensitive and cost-effective EGFR testing as part of standard lung cancer management for identifying patients who might clinically benefit from tyrosine kinase inhibitors. The Bi-PASA assay proved to be a suitable method to detect small deletions. Strategies in designing allele-specific primers described herein can be adapted to other screening assays for point mutations and small deletions.
authors.affiliation: HELIOS Klinikum Erfurt, Institute of Pathology, D-99089 Erfurt, Germany. rdahse@erfurt.helios-kliniken.de.
authors.forename: Regine
authors.initials: R
authors.lastname: Dahse
authors.forename: Alexander
authors.initials: A
authors.lastname: Berndt
authors.forename: Anne-Kristin
authors.initials: AK
authors.lastname: Dahse
authors.forename: Hartwig
authors.initials: H
authors.lastname: Kosmehl