Pyrosequencing Analysis of EGFR and KRAS Mutations in EUS and EBUS-Derived Cytologic Samples of Adenocarcinomas of the Lung

• Published in: Journal of thoracic oncology Vol. 8; no. 8; pp. 1012 - 1018
• Main Authors: Stigt, Jos A., ´t Hart, Nils A., Knol, Ageeth J., Uil, Steven M., Groen, Harry J.M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2013; Copyright by the European Lung Cancer Conference and the International Association for the Study of Lung Cancer
• Subjects: Adenocarcinoma - genetics; Adenocarcinoma - pathology; Adenocarcinoma of Lung; Adult; Aged; Aged, 80 and over; Cell Count; Cytology; Deoxyribonucleic acid mutational analysis; DNA sequencing; Endoscopic ultrasonography; Endoscopic Ultrasound-Guided Fine Needle Aspiration - methods; ErbB Receptors - genetics; Female; Humans; Lung Neoplasms - genetics; Lung Neoplasms - pathology; Male; Middle Aged; Mutation; Non–small-cell lung cancer; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins p21(ras); ras Proteins - genetics; Sequence Analysis, DNA - methods; Non–small-cell lung cancer; Deoxyribonucleic acid mutational analysis; Endoscopic ultrasonography; Cytology; DNA sequencing
• ISSN: 1556-0864; 1556-1380
• DOI: 10.1097/JTO.0b013e31829ce93e

Patients with stage IV non–small-cell lung cancer harboring an activating epidermal growth factor receptor (EGFR) mutation are eligible for treatment with EGFR tyrosine kinase inhibitors. With pyrosequencing, low-frequency mutations may be detected more easily even in small diagnostic samples like endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspirations (EBUS-TBNA). The diagnostic performance of pyrosequencing in analyzing cytological specimens is compared with the routinely used high-resolution melting (HRM) and Sanger sequencing. Patients diagnosed with adenocarcinoma of the lung were selected from a fine needle aspiration and transbronchial needle aspiration specimen database. If formalin-fixed paraffin-embedded tumor blocks were available, mutation analysis was performed for EGFR and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog genes using both pyrosequencing and HRM. When HRM showed abnormalities, Sanger sequencing was used. A total of 126 samples were available for mutation analysis. The analysis success rate for pyrosequencing and HRM were 97% and 93%, respectively. HRM failures were observed in fragmented DNA showing chains of 100 to 200 bp. A significant correlation between length of DNA fragments (100–300 bp versus 300–400 bp) and mean sample age (797 versus 317 days) was found (p < 0.0001), suggesting an influence of sample age on DNA quality. Pyrosequencing on cytological blocks, especially older tumor blocks, is feasible with a high diagnostic success rate. Failures in HRM were observed in DNA samples with short fragments related to longer storage times.