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Background Analysis of circulating tumor DNA (ctDNA) in plasma is a powerful approach to guide decisions in personalized cancer treatment. Given the low concentration of ctDNA in plasma, highly sensitive methods are required to reliably identify clinically relevant variants. Methods We evaluated the suitability of 5 droplet digital PCR (ddPCR) assays targeting KRAS, BRAF, and EGFR variants for ctDNA analysis in clinical use. Results We investigated assay performance characteristics for very low amounts of variants, showing that the assays had very low limits of blank (0% to 0.11% variant allele frequency, VAF) and limits of quantification (0.41% to 0.7% VAF). Nevertheless, striking differences in detection and quantification of low mutant VAFs between the 5 tested assays were observed, highlighting the need for assay-specific analytical validation. Besides in-depth evaluation, a guide for clinical interpretation of obtained VAFs in plasma was developed, depending on the limits of blank and limits of quantification values. Conclusion It is possible to provide comprehensive clinical reports on actionable variants, allowing minimal residual disease detection and treatment monitoring in liquid biopsy.

Liquid Biopsy Hotspot Variant Assays: Analytical Validation for Application in Residual Disease Detection and Treatment Monitoring