Real-time PCR and Targeted Next-generation Sequencing in the Detection of Low Level Mutations: Instructive Case Analyses

Background: Allele specific real-time PCR and next-generation sequencing (NGS) are widely used to detect somatic mutation in non-small cell lung cancer (NSCLC). Both methods commonly use formalin-fixed paraffin-embedded (FFPE) tissues as diagnostic materials. Real-time PCR has the advantage of being easy to use and more tolerant of variable DNA quality, but has limited multiplex capability. NGS, in contrast, allows simultaneous analysis of many genomic loci while revealing the exact sequence changes; it is, however, more technically demanding and more expensive to employed. A challenge for both platforms is the varied limit of detection (LoD) for target genomic loci, even within the same gene. The variability of detection sensitivity may be problematic if well-known actionable somatic mutations are missed.

Cases: We compared LoDs between real-time PCR and targeted NGS tests for some commonly observed mutations in NSCLC specimens.

Conclusions: The FDA-approved real-time PCR test was superior to the NGS in detecting low level exon 19 deletion (near 1% variant allele fraction (VAF)). The cancer hotspot NGS detects low level c.2369C > T, p.T790M (2-5% VAF) better than the FDA-approved real-time PCR method. We conclude that the real-time PCR and hotspot NGS methods have complementary strengths in accurately determining clinically important mutations in NSCLC.