Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays

Author:

Hallermayr Ariane123ORCID,Keßler Thomas13,Fujera Moritz1,Liesfeld Ben4,Bernstein Samuel4,von Ameln Simon5,Schanze Denny6,Steinke-Lange Verena123ORCID,Pickl Julia M. A.12,Neuhann Teresa M.1,Holinski-Feder Elke123ORCID

Affiliation:

1. MGZ—Medizinisch Genetisches Zentrum, 80335 Munich, Germany

2. Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität München, 80336 Munich, Germany

3. European Liquid Biopsy Society, 20246 Hamburg, Germany

4. Limbus Medical Technologies GmbH, 18055 Rostock, Germany

5. Immune-Oncological Centre Cologne (IOZK), 50674 Cologne, Germany

6. Institute of Human Genetics, University Hospital Magdeburg, Otto-von-Guericke University, 39120 Magdeburg, Germany

Abstract

Background: Liquid biopsy enables the non-invasive analysis of genetic tumor variants in circulating free DNA (cfDNA) in plasma. Accurate analytical validation of liquid biopsy NGS assays is required to detect variants with low variant allele frequencies (VAFs). Methods: Six types of commercial cfDNA reference materials and 42 patient samples were analyzed using a duplex-sequencing-based liquid biopsy NGS assay. Results: We comprehensively evaluated the similarity of commercial cfDNA reference materials to native cfDNA. We observed significant differences between the reference materials in terms of wet-lab and sequencing quality as well as background noise. No reference material resembled native cfDNA in all performance metrics investigated. Based on our results, we established guidelines for the selection of appropriate reference materials for the different steps in performance evaluation. The use of inappropriate materials and cutoffs could eventually lead to a lower sensitivity for variant detection. Conclusion: Careful consideration of commercial reference materials is required for performance evaluation of liquid biopsy NGS assays. While the similarity to native cfDNA aids in the development of experimental protocols, reference materials with well-defined variants are preferable for determining sensitivity and precision, which are essential for accurate clinical interpretation.

Publisher

MDPI AG

Subject

Cancer Research,Oncology

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