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J Thorac Oncol. 2013 Aug;8(8):1012-8. doi: 10.1097/JTO.0b013e31829ce93e.

Pyrosequencing analysis of EGFR and KRAS mutations in EUS and EBUS-derived cytologic samples of adenocarcinomas of the lung.

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Department of Pulmonology, Isala Klinieken, Zwolle, Amsterdam, The Netherlands.



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.

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