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.
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