Detection of Epidermal Growth Factor Receptor Mutations in a Few Cancer Cells from Transbronchial Cytologic Specimens by Reverse Transcriptase-Polymerase Chain Reaction

Abstract

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. 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. 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. EGFR mutations were detected in a small number of cancer cells (ten cells), even in the presence of 1 × 10(6) WBCs, by the RNA-based methodology (either RT-PCR or PCR-RFLP) [sensitivity: <10(-5)]. However, the DNA-based method exhibited lower sensitivity (10(-1)). EGFR mutations were detected in 21 of 71 NSCLC samples (29.6%) and in 19 of 43 adenocarcinomas (44.2%) by the RNA-based methodology. The DNA-based methodology failed to detect EGFR mutations in several cases, while the RNA-based methodology was able to detect them. Rapid diagnosis during bronchoscopy, utilizing UFP staining, contributed to the selection of the best samples for genetic analysis. EGFR mutations could be detected in a small number of cancer cells by the RNA-based methodology, with higher sensitivity than the DNA-based methodology, even in samples where numerous normal cells were present. Our present strategy can be integrated into the clinical process without additional invasive examination of patients and provides information regarding the EGFR mutational status.