Reliable detection of rare mutations in EGFR gene codon L858 by PNA-LNA PCR clamp in non-small cell lung cancer.

PNA-LNA PCR clamp real-time PCR method represents allele-specific approach to mutation analysis of EGFR gene in NSCLC. Due to its unique design, it is characterized by exceptionally high specificity and sensitivity but also allows detection of rare or not specifically-targeted EGFR mutations within examined exons, otherwise undetectable by other mutation-specific fluorescent probes. We herein present two cases of rare mutations revealed by PNA-LNA PCR clamping of NSCLC samples referred for routine EGFR gene molecular diagnostics. In one, the EGFR gene L858 codon mutation was detected by standard PNA-LNA PCR clamping, subsequently reconfirmed and characterized by direct sequencing of allele specific amplification products as the missense mutation c.2572C>A (p.L858M) paired with L861Q mutation on the same allele (in cis). In the second sample, low quality FFPE material from pleural biopsy, c.2573C>T missense mutation (p.L858P) was revealed. Still, repeated DNA analysis by PNA-LNA PCR clamp and direct sequencing demonstrated low level of mutant allele existing in a total allele pool suggesting rather artifactual c.2572C>T transition, a phenomenon quite frequent in low-volume FFPE samples upon fixation procedures. In conclusion, superior sensitivity and unique design of PNA-LNA PCR clamping are crucial for its excellent diagnostic effectiveness. As we demonstrated, the method allows detecting rare EGFR mutations, although it increases the risk of detection of a very low signal, e.g., generated by a small pool of mutated allele. Therefore, applicability of PNA-LNA PCR clamp product for the direct sequencing reevaluation is of key importance enabling reliable validation of results.