Characteristics of the major plasma genotyping assays available for advanced nonsmall cell lung cancer
| Principle | Coverage | Diagnostic accuracy (tissue reference) | Advantages | Pitfalls | Preferred indications | |
| Targeted assays | Genotyping of pre-defined hotspots, exons or complete genes of interest | 1–7 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 |
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| NGS | ||||||
| Whole genome sequencing | Sequencing of the full genome | NA | Discovery 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 sequencing | Sequencing of the full exome (coding regions) | Discovery of new targets or mechanisms of resistance | Tumour mutation burden (but usually replaced by large gene panels) | |||
| Panels (hybrid capture) | Capture and hybridisation to probes of pre-determined regions of interest, then sequencing | Depend 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 variants | Initial 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 sequencing | PCR 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.