Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp

Circulating tumour DNA (ctDNA) is a potential proxy for tumour tissues. However, the analysis of mutations and mutational abundance using ctDNA remains challenging because ctDNA is present at low levels. In addition, the concordance between plasma and tumour tissues requires further investigation by high-sensitivity techniques. Here, we established an ultrasensitive, quantitative method for detecting rare mutations in plasma samples based on a dual PNA clamping-mediated LNA-PNA PCR clamp (LNA-dPNA PCR clamp). The novelty of our method is the coupling of PNA clamping with one-tube nested PCR to dually block wild-type DNA amplification and efficiently amplify mutant DNA. Then, four hotspot EGFR mutations (EGFR L858R, EGFR Exon 19 deletion, EGFR T790M, and EGFR C797S) were detected by our proposed method. Finally, we evaluated the concordance between plasma and tumour tissues by simultaneously detecting EGFR L858R by ddPCR and LNA-dPNA PCR clamp in 132 tissues and matched plasma samples from patients with NSCLC. For the four EGFR mutations, the amplification sensitivity of the LNA-dPNA PCR clamp was 100 copies per reaction, and the linearity was from 100 to 106-107 copies per reaction. The limit of detection for the LNA-dPNA PCR clamp was 0.01%-0.1%. The LNA-dPNA PCR clamp was similarly consistent with ddPCR in quantifying mutational abundance (R2 = 0.9568) and exhibited similar limit of detection (0.01%-0.1% vs. 0.01%), sensitivity (19.6 vs. 21.7), specificity (94.2 vs. 91.9), and concordance (68.2 vs. 67.4) to those of ddPCR for ctDNA detection. In conclusion, the LNA-dPNA PCR clamp will provide a labour-saving, cost-saving, ultrasensitive tool for detecting and quantifying plasma EGFR mutations.