Validation of PNA-LNA PCR clamp assay for detection of EGFR exon 19 and 21 mutations in various types of clinical non-small cell lung cancer specimens

The aim of the study was to assess diagnostic reliability of PNA-LNA PCR clamp assay in EGFR mutations detection in different NSCLC samples. Evaluation was performed: (i) in reference NSCLC tissue FFPE samples (n=10), (ii) in comparison to direct sequencing in resected NSCLC tissue (n=199) and biopsy material specimens (n=179) characterized by different tumor cells content (TCC) and fixation [Table 1]. NSCLC samples characteristic. Resected tissue Biopsy material fresh-frozen FFPE FFPE Cytology smear 84 115 115 64 [Table 1] (i) PNA-LNA PCR clamp correctly detected all exon 19 deletions and L858R mutations in the reference FFPE materials, including those with meager TCC (5% and 10%). (ii) PNA-LNA PCR clamp method and direct sequencing presented high conformity (overall percent agreement, OPA=99%; Cohen’s Kappa score of 0.94 (95% CI=0.9, 0.99) in n=100 samples with >50% TCC analyzed. (iii) In total of 378 samples analyzed with PNA-LNA PCR clamp method, EGFR mutations were detected in 36 (9.5%). (iv) Only in 24 out of 36 (67%) mutation positive samples reevaluation with direct sequencing proved positive [Table 2]. PNA-LNA PCR clamp presented higher sensitivity in samples with TCC <50% (p=0.004). Comparison of PNA-LNA PCR clamp vs direct sequencing EGFR mutation detection sensitivity in materials with different TCC. PNA-LNA PCR clamp direct sequencing ≥50% 25/25 (100%) 22/25 (88%) 20<50% 6/6 (100%) 2/6 (33%) ≤20% 5/5 (100%) 0/5 (0%) total 36/36 (100%) 24/36 (67%) [Table 2] PNA-LNA PCR clamp method proved its diagnostic utility and high sensitivity of EGFR exon 19 and 21 mutations detection, especially in samples with TCC lower than 50%.