| Comparative Analysis of Peptide Nucleic Acid (PNA)-Mediated Real-Time PCR Clamping and DNA Direct Sequencing for EGFR Mutation Detection. |
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Hee Joung Kim, Wan Seop Kim, Kyeong Cheol Shin, Gwan Ho Lee, Mi Jin Kim, Jeong Eun Lee, Kyu Sang Song, Sun Young Kim, Kye Young Lee |
1Department of Internal Medicine, Konkuk University School of Medicine, Seoul, Korea. kyleemd@kuh.ac.kr
2Department of Pathology, Konkuk University School of Medicine, Seoul, Korea.
3Department of Internal Medicine, Yeungnam University College of Medicine, Daegu, Korea.
4Department of Pathology, Yeungnam University College of Medicine, Daegu, Korea.
5Department of Internal Medicine, Chungnam National University College of Medicine, Daejeon, Korea.
6Department of Pathology, Chungnam National University College of Medicine, Daejeon, Korea. |
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| Abstract |
BACKGROUND
Although the gold standard method for research trials on epidermal growth factor receptor (EGFR) mutations has been direct sequencing, this approach has the limitations of low sensitivity and of being time-consuming. Peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping is known to be a more sensitive detection tool. The aim of this study was to compare the detection rate of EGFR mutation and EGFR-tyrosine kinase inhibitor (TKI) responsiveness according to EGFR mutation status using both methodologies. METHODS: Clinical specimens from 112 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20, and 21. All clinical data and tumor specimens were obtained from 3 university hospitals in Korea. After genomic DNA was extracted from paraffin-embedded tissue specimens, both PNA-mediated PCR clamping and direct-sequencing were performed. The results and clinical response to EGFR-TKIs were compared. RESULTS: Sequencing revealed a total of 35 (22.9%) mutations: 8 missense mutations in exon 21 and 26 deletion mutations in exon 19. PNA-mediated PCR clamping showed the presence of genomic alterations in 45 (28.3%) samples, including the 32 identified by sequencing plus 13 additional samples (6 in exon 19 and 7 in exon 21). CONCLUSION: PNA-mediated PCR clamping is simple and rapid, as well as a more sensitive method for screening of genomic alterations in EGFR gene compared to direct sequencing. This data suggests that PNA-mediated PCR clamping should be implemented as a useful screening tool for detection of EGFR mutations in clinical setting. |
| Key Words:
Peptide Nucleic Acids, Molecular Sequencing Data, Receptor, Epidermal Growth Factor, Epidermal Growth Factor |
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