Characteristics of the major plasma genotyping assays available for advanced nonsmall cell lung cancer

PrincipleCoverageDiagnostic accuracy (tissue reference)AdvantagesPitfallsPreferred indications
Targeted assaysGenotyping of pre-defined hotspots, exons or complete genes of interest1–7 hotspots of a gene (ddPCR)
7 hotspots (Therascreen)
42 mutations in four exons (Cobas)
Sensitivity 60–80% (EGFR)
Specificity (EGFR)
96% Cobas,
97% BEAMing
100% ddPCR
Highly sensitive
Highly specific
Quantitative (except Cobas)
Low turnaround time
Only targets pre-defined regions of interest:
Doesn't cover all targetable alterations
Doesn't capture a potentially subclonal resistance
Screening for pre-defined targetable mutations (i.e. EGFR activating mutation, EGFR resistance mutations (T790M, C797S))
Monitoring of response
  •  ddPCR




 Whole genome sequencingSequencing of the full genomeNADiscovery of new targets (fusion genes involving intronic areas)Risk of false positives (poor specificity)
Risk of identifying germline mutations
Heavy bioinformatics
Low sensitivity
None in routine, exploration of new targets in research (fusion genes involving intronic areas)
 Whole exome sequencingSequencing of the full exome (coding regions)Discovery of new targets or mechanisms of resistanceTumour mutation burden (but usually replaced by large gene panels)
 Panels (hybrid capture)Capture and hybridisation to probes of pre-determined regions of interest, then sequencingDepend on the gene panels (usually hotspots, exons or full genes in 30–400 genes)Sensitivity 70–90% for SNVs
Sensitivity 50–80% for fusions
Specificity 65% for hybrid capture
Specificity >99% for amplicon
Interrogates simultaneously pre-determined genes of interest
Comprehensive detection of known and unknown mutations
Detection of SNVs, CNVs, fusions
Lower cost and less bioinformatics data compared to whole genome sequencing or whole exome sequencing
Imperfect specificity and concordance with tissue, in particular for low AF variantsInitial and resistance genotyping (focusing on genes of therapeutic interest)
Tumour mutation burden (large, >300 genes panels)
 Panels (amplicon sequencing)PCR amplifications of hotspots/exons/genes of interest, then sequencingPCR amplification can bias CNVs and AFs
Unable to detect fusions without prior knowledge of partners

ddPCR: digital-droplet PCR; NGS: next-generation sequencing; NA: not available; SNV: single-nucleotide variation; CNV: copy-number variation; AF: allelic fraction.