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

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


Introduction: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / genetics*
  • Adenocarcinoma / pathology
  • Adenocarcinoma of Lung
  • Adult
  • Aged
  • Aged, 80 and over
  • Cell Count
  • Endoscopic Ultrasound-Guided Fine Needle Aspiration / methods*
  • ErbB Receptors / genetics*
  • Female
  • Humans
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / pathology
  • Male
  • Middle Aged
  • Mutation*
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • Sequence Analysis, DNA / methods*
  • ras Proteins / genetics*


  • KRAS protein, human
  • Proto-Oncogene Proteins
  • ErbB Receptors
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins