Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations.
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.
Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.|Severance Biomedical Science, Yonsei University College of Medicine, Seoul, Korea.
Suki
S
Kang
Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.|Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea.
Baek Gil
BG
Kim
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea.
Hyun Ho
HH
Han
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea.
Joo Hyun
JH
Lee
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea.
Ji Eun
JE
Kim
Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.
Hyo Sup
HS
Shim
Department of Pathology, Yonsei University College of Medicine, Seoul, Korea.|Severance Biomedical Science, Yonsei University College of Medicine, Seoul, Korea.|Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea.
Nam Hoon
NH
Cho
D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D018773:Genes, erbB-1; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
C512478:EGFR protein, human; D066246:ErbB Receptors
EGFR mutation; diagnostic technique; enrichment ultra-deep pyrosequencing; lung cancer