Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations
Metrics: PDF 1272 views | HTML 1810 views | ?
Suki Kang1,2, Baek Gil Kim1,3, Hyun Ho Han3, Joo Hyun Lee3, Ji Eun Kim3, Hyo Sup Shim1 and Nam Hoon Cho1,2,3
1 Department of Pathology, Yonsei University College of Medicine, Seoul, Korea
2 Severance Biomedical Science, Yonsei University College of Medicine, Seoul, Korea
3 Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea
Nam Hoon Cho, email:
Keywords: enrichment ultra-deep pyrosequencing, EGFR mutation, lung cancer, diagnostic technique
Received: February 15, 2015 Accepted: March 18, 2015 Published: April 12, 2015
Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01% and 0.05%, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692% in tissue samples. EGFR mutations with frequencies as low as 0.01% were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.