title: Verification of wild-type EGFR status in non-small cell lung carcinomas using a mutant-enriched PCR on selected cases.
abstract: EGFR genotyping is required for targeted therapy of lung adenocarcinoma. Because a false-negative result might prevent a patient from receiving appropriate targeted therapies, it is desirable to recheck equivocal results of EGFR genotyping. A cohort of 346 lung cancers was tested with a commercial kit for EGFR mutations; nine of the cases had upward real-time amplification curves at late cycles. They were also investigated using mutant-enriched PCR with peptide nucleic acid-locked nucleic acid (PNA-sequencing). Six of the nine equivocal cases harbored EGFR mutations. These cases likely had a small amount of mutant DNA near the detection limit of the commercial kit. Twenty nonequivocal, wild-type cases were reconfirmed using PNA-sequencing. We noticed a College of American Pathologists proficiency test material that showed a suspicious upward curve and eventually proved to have an H773_V774insPH in exon 20, for which a specific primer was not designed in the commercial kit. Further study using cloned DNA fragments showed that the upward curve most likely resulted from cross-reaction between similar, but nonidentical, sequences. It is desirable to keep the number of false-negative results as low as possible, but rechecking all wild-type cases is impractical. The late upward curves we observed helped identify suspicious cases for rechecking. A second method, such as PNA-sequencing, is recommended to verify wild-type cases.
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Associations of Medical Technologists, Tainan, Taiwan.
authors: Yi-Lin
authors: YL
authors: Chen
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; The Institute of Molecular Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Cheng-Chan
authors: CC
authors: Lu
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Shu-Ching
authors: SC
authors: Yang
authors: Department of Internal Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Wen-Pin
authors: WP
authors: Su
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Ya-Lan
authors: YL
authors: Lin
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Wan-Li
authors: WL
authors: Chen
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Wenya
authors: W
authors: Huang
authors: Department of Internal Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Wu-Chou
authors: WC
authors: Su
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; The Institute of Molecular Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan.
authors: Nan-Haw
authors: NH
authors: Chow
authors: Molecular Diagnosis Laboratory, Department of Pathology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan; Molecular Medicine Core Laboratory, Research Center of Clinical Medicine, College of Medicine, National Cheng Kung University Hospital, Tainan, Taiwan. Electronic address: clh9@mail.ncku.edu.tw.
authors: Chung-Liang
authors: CL
authors: Ho
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons; D005260:Female; D005820:Genetic Testing; D060005:Genotyping Techniques; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014057:Tomography, X-Ray Computed
chemical_list: D011933:Reagent Kits, Diagnostic; D066246:ErbB Receptors