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Kim, Kim, Shin, Lee, Kim, Lee, Song, Kim, and Lee: Comparative Analysis of Peptide Nucleic Acid (PNA)-Mediated Real-Time PCR Clamping and DNA Direct Sequencing for EGFR Mutation Detection
Original Article
Tuberculosis and Respiratory Diseases

Tuberculosis and Respiratory Diseases 2011; 70(1): 21-27.

Published online: 31 January 2011

DOI: https://doi.org/10.4046/trd.2011.70.1.21

Comparative Analysis of Peptide Nucleic Acid (PNA)-Mediated Real-Time PCR Clamping and DNA Direct Sequencing for EGFR Mutation Detection

Hee Joung Kim, M.D.1, Wan Seop Kim, M.D.2, Kyeong-Cheol Shin, M.D.3, Gwan Ho Lee, M.D.3, Mi-Jin Kim, M.D.4, Jeong Eun Lee, M.D.5, Kyu Sang Song, M.D.6, Sun Young Kim, M.D.5, Kye Young Lee, M.D., Ph.D.1

1Department of Internal Medicine, Konkuk University School of Medicine, Seoul, Korea.

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.

Address for correspondence: Kye Young Lee, M.D., Ph.D. Department of Internal Medicine, Konkuk University School of Medicine, 4-12, Hwayang-dong, Gwangjin-gu, Seoul 143-729, Korea. Phone: 82-2-2030-7521, Fax: 82-2-2030-7748, kyleemd@kuh.ac.kr

Received 11 December 2010       Accepted 14 January 2011

Copyright © 2011. The Korean Academy of Tuberculosis and Respiratory Diseases. All rights reserved.

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.

Keywords: Peptide Nucleic Acids, Molecular Sequencing Data, Receptor, Epidermal Growth Factor, Epidermal Growth Factor

Figures and Tables

Table 1
Demographic characteristics of subjects
trd-70-21-i001

SD: standard deviation.

*Average±SD (median, range).

Table 2
EGFR mutation detected by PNA-mediated Real-Time PCR Clamping and DNA direct sequencing
trd-70-21-i002

Values are presented as number (%).

EGFR: epidermal growth factor receptor; PNA: peptide nucleic acid; PCR: polymerase chain reaction.

Table 3
Comparison of EGFR mutation results using PNA-mediated RT PCR Clamping and DNA direct sequencing
trd-70-21-i003

EGFR: epidermal growth factor receptor; PNA: peptide nucleic acid; PCR: polymerase chain reaction.

Table 4
Clinical response according to EGFR mutation status
trd-70-21-i004

Values are presented as number (%).

EGFR: epidermal growth factor receptor; PNA: peptide nucleic acid.

Acknowledgements

This study was supported by a grant from Korean Association for the Study of Lung Cancer (KASLC-1002).

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