Abstract
Objective: Direct sequencing is widely accepted method for EGFR mutation identification, but has limited sensitivity. It often requires additional procedures, like microdissection, to enrich the sample in cancer cells, when their content in tissue specimen is less than 50%. PNA-LNA PCR clamping represents allele-specific approach to gene analysis and demonstrates potent accuracy and ability to detect mutant alleles even if present in low fraction of cells.
Method: 79 DNA samples isolated from fresh-frozen and FFPE tissues, which mutation status was formerly confirmed by sequencing, were analyzed by PNA-LNA PCR clamping for EGFR point mutation L858R in exon 21.
Results: L858R mutation was detected in 8/79 (10%) samples by direct sequencing, whereas in 12/79 (15%) samples by PNA-LNA PCR clamping. All mutant-positive samples by sequencing were correctly determined by PNA-LNA PCR clamp. The remaining 4 L858R mutant-positive samples were recognized as wild type by sequencing. Two of them contained only 5% and 20% of cancer cells, respectively. Surprisingly, in the other two samples PNA-LNA PCR clamping method detected only low levels of EGFR mutant allele, despite the cancer cell contents were high (100% and 80%).
Conclusions: PNA-LNA PCR clamp technique enables sensitive and reliable detection of EGFR mutant allele in specimens with cancer cell content insufficient for direct sequencing or genetically heterogenous. Regarding its extremely high sensitivity, PNA-LNA PCR clamping should be validated thoroughly prior implementation into EGFR diagnostic routine to prevent overdiagnosis.
- © 2011 ERS
