title: Detection and comparison of peptide nucleic acid-mediated real-time polymerase chain reaction clamping and direct gene sequencing for epidermal growth factor receptor mutations in patients with non-small cell lung cancer.
abstract: EGFR tyrosine kinase inhibitors (EGFR-TKIs) are recommended as first-line therapy in patients with advanced, recurrent, or metastatic non-squamous non-small cell lung cancer (NSCLC) that have active EGFR mutations. The importance of rapid and sensitive methods for the detection of EGFR mutations is emphasized. The aim of this study is to examine the EGFR mutational status by both direct DNA sequencing and peptide nucleic acid (PNA)-mediated real-time PCR clamping and to evaluate the correlation between the EGFR mutational status and the clinical response to EGFR-tyrosine kinase inhibitors. Clinical specimens from 240 NSCLC patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21. All clinical data and tumor specimens were obtained from 8 centers of the Korean Molecular Lung Cancer Group (KMLCG). After genomic DNA was extracted from paraffin-embedded tissue specimens, we performed PNA-mediated real-time PCR clamping and direct DNA sequencing for the detection of EGFR mutations. Of 240 tumor samples, PNA-mediated PCR clamping was used to detect genomic alterations in 83 (34.6%) samples, including 61 identified by sequencing and 22 additional samples (10 in exon 19, 9 in exon 21, and 3 in both exons); direct DNA sequencing was used to identify a total of 63 (26.3%) mutations that contained 40 deletion mutations in exon 19 (63.5%) and 18 substitution mutations (28.6%) in exon 21. PNA-mediated PCR clamping was used to identify more mutations than clinical direct sequencing, whereas clinical outcomes were not significantly different between the groups harboring activating mutations detected by each method. These data suggest that PNA-mediated real-time PCR clamping exhibits high sensitivity and is a simple procedure relative to direct DNA sequencing that is a useful screening tool for the detection of EGFR mutations in clinical settings.
authors.affiliation: Department of Internal Medicine, Konkuk University School of Medicine, 4-12 Hwayang-dong, Gwangjin-gu, Seoul 143-729, Republic of Korea.
authors.forename: Hee Joung
authors.initials: HJ
authors.lastname: Kim
authors.forename: Kye Young
authors.initials: KY
authors.lastname: Lee
authors.forename: Young-Chul
authors.initials: YC
authors.lastname: Kim
authors.forename: Kyu-Sik
authors.initials: KS
authors.lastname: Kim
authors.forename: Sung Yong
authors.initials: SY
authors.lastname: Lee
authors.forename: Tae Won
authors.initials: TW
authors.lastname: Jang
authors.forename: Min Ki
authors.initials: MK
authors.lastname: Lee
authors.forename: Kyeong-Cheol
authors.initials: KC
authors.lastname: Shin
authors.forename: Gwan Ho
authors.initials: GH
authors.lastname: Lee
authors.forename: Jae Chol
authors.initials: JC
authors.lastname: Lee
authors.forename: Jeong Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Sun Young
authors.initials: SY
authors.lastname: Kim
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics; D000077192:Adenocarcinoma of Lung; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics*; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D020135:Peptide Nucleic Acids / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000970:Antineoplastic Agents; D020135:Peptide Nucleic Acids; D047428:Protein Kinase Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors