Verification of wild-type EGFR status in non-small cell lung carcinomas using a mutant-enriched PCR on selected cases

Yi Lin Chen, Cheng Chan Lu, Shu Ching Yang, Wen Pin Su, Ya Lan Lin, Wan Li Chen, Wenya Huang, Wu Chou Su, Nan Haw Chow, Chung Liang Ho

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14 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)486-494
Number of pages9
JournalJournal of Molecular Diagnostics
Issue number5
Publication statusPublished - 2014 Sept

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Molecular Medicine


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