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Article Abstract
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.
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