Detection of Epidermal Growth Factor Receptor Mutations in a Few Cancer Cells from Transbronchial Cytologic Specimens by
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Detection of Epidermal Growth Factor Receptor Mutations in a Few Cancer Cells from Transbronchial Cytologic Specimens by Reverse Transcriptase-Polymerase Chain Reaction Nobuhiro Kanaji,1 Shuji Bandoh,1 Tomoya Ishii,1 Yoshio Kushida,2 Reiji Haba,2 Kohoji Kohno,2 Hiroaki Dobashi,1 Hiroaki Ohnishi1 and Takuya Matsunaga1 1 Department of Internal Medicine, Division of Endocrinology and Metabolism, Hematology, Rheumatology and Respiratory Medicine, Faculty of Medicine, Kagawa University, Kagawa, Japan 2 Department of Diagnostic Pathology, Faculty of Medicine, Kagawa University, Kagawa, Japan
Abstract
Background: Epidermal growth factor receptor (EGFR) mutational status has the potential to be useful for determining prospective therapies in patients with non-small cell lung cancer (NSCLC) when analyzed in transbronchial cell specimens. The efficacy of RNA-based methods for the detection of EGFR mutations in transbronchial cell specimens has not been studied. Ultrafast Papanicolaou (UFP) staining is a method used in the immediate assessment of cytology during bronchoscopic examination. Objectives: The aims of this study were (i) to compare the efficacy of RNA-based methodology for the detection of EGFR mutations with DNA-based methodology; and (ii) to assess the analysis of EGFR mutational status in transbronchial cell specimens, utilizing UFP staining. Methods: EGFR mutant PC9 and NCI-H1975 cells were combined with wild-type EGFR white blood cells (WBCs), and the RNA and DNA were extracted. The sensitivity for the detection of EGFR mutations was determined. Polymerase chain reaction (PCR)-based methods, including reverse transcriptase (RT)-PCR and PCR-restriction fragment length polymorphism (RFLP), and sequencing were performed to detect the EGFR mutations. Seventy-one cell samples from bronchoscopic examinations that utilized UFP staining in patients with NSCLC were also analyzed for EGFR mutations. Results: EGFR mutations were detected in a small number of cancer cells (ten cells), even in the presence of 1 · 106 WBCs, by the RNA-based methodology (either RT-PCR or PCR-RFLP) [sensitivity:
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