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Updated: Oct 24, 2025
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Liquid Biopsy Hotspot Variant Assays: Analytical Validation for Application in Residual Disease Detection and
Ariane Hallermayr1,2,3, Anna Benet-Pagès1,4, Verena Steinke-Lange1,5
1MGZ - Medizinisch Genetisches Zentrum, Munich, Germany.
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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