Direct EGFR Mutation Detection in FFPE Samples

A direct sequencing method was applied for detecting EGFR mutation without routine tumor enrichment. Retrieved Formalin-fixed, paraffin embedded (FFPE) tumor samples were used for genomic DNA extraction by the QIAmp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) amplification of EGFR exons 18 to 21, using intron-based primers was followed. Sequencing was performed in both the forward and reverse directions. Since September 2014, the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp method has been applied in almost all cases. Genomic DNA of EGFR mutation hot-spots were amplified by PCR with a PNA clamp primer synthesized from a PNA with a wild-type sequence and detected by a fluorescent primer that incorporates locked nucleic acids. This method for preferential amplification of the mutant sequence can detect EGFR mutation in specimens containing 100 to 1000 excess copies of wild-type EGFR sequence [39 ].