Liquid Biopsy Hotspot Variant Assays: Analytical Validation for Application in Residual Disease Detection and Treatment Monitoring. (1st November 2021)

• Record Type: Journal Article
• Authors: Hallermayr, Ariane; Benet-Pagès, Anna; Steinke-Lange, Verena; Mansmann, Ulrich; Rentsch, Markus; Holinski-Feder, Elke; Pickl, Julia M A
• Is Part Of: Clinical chemistry. Volume 67:Number 11(2021)
• Page Start: 1483
• Page End: 1491
• Publication Date: 2021-11-01
• Subjects: liquid biopsy -- circulating tumor DNA -- analytical validation -- droplet digital PCR -- residual disease -- tumor monitoring; Clinical chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Biochimie -- Périodiques; Diagnostics biologiques -- Périodiques; Biochemistry; Clinical chemistry; Pharmaceutical chemistry; Biochemistry; Laboratory Techniques and Procedures; Klinische chemie; Periodicals; 616.075605
• DOI: 10.1093/clinchem/hvab124
• Languages: English
• ISSNs: 0009-9147

Abstract

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.