title: The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.
abstract: Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.
authors.affiliation: Molecular Oncology and Genetics Unit, Department of Tumour Pathology and Pathomorphology, Franciszek Lukaszczyk Oncology Centre, Bydgoszcz, Poland; Department of Thoracic Surgery and Tumours, Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland.
authors.forename: Marzena Anna
authors.initials: MA
authors.lastname: Lewandowska
authors.affiliation: European Centre of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Poznan University of Technology, Pl. Marii Sklodowskiej-Curie 5, 60-965, Poznan, Poland.
authors.forename: Karol
authors.initials: K
authors.lastname: Czubak
authors.affiliation: European Centre of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Poznan University of Technology, Pl. Marii Sklodowskiej-Curie 5, 60-965, Poznan, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Klonowska
authors.affiliation: Department of Tumour Pathology and Pathomorphology, Franciszek Łukaszczyk Oncology Centre, Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Jozwicki
authors.affiliation: Department of Thoracic Surgery and Tumours, Franciszek Łukaszczyk Oncology Centre, Ludwik Rydygier Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz, Poland.
authors.forename: Janusz
authors.initials: J
authors.lastname: Kowalewski
authors.affiliation: European Centre of Bioinformatics and Genomics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Poznan University of Technology, Pl. Marii Sklodowskiej-Curie 5, 60-965, Poznan, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Kozlowski
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics*; D055088:Early Detection of Cancer / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005784:Gene Amplification; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D017354:Point Mutation
chemical_list: D014408:Biomarkers, Tumor; C512478:EGFR protein, human; D066246:ErbB Receptors