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

SIMPLE SUMMARY: Liquid biopsy is a promising tool for the detection of low-frequency variants present in cancer and mosaic disease and is more and more adopted in clinical practice. Still, accurate validation and standardization across laboratories is lacking. Here, we show that the reference materi...

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Autores principales: Hallermayr, Ariane, Keßler, Thomas, Fujera, Moritz, Liesfeld, Ben, Bernstein, Samuel, von Ameln, Simon, Schanze, Denny, Steinke-Lange, Verena, Pickl, Julia M. A., Neuhann, Teresa M., Holinski-Feder, Elke
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10605119/
https://www.ncbi.nlm.nih.gov/pubmed/37894392
http://dx.doi.org/10.3390/cancers15205024
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author Hallermayr, Ariane
Keßler, Thomas
Fujera, Moritz
Liesfeld, Ben
Bernstein, Samuel
von Ameln, Simon
Schanze, Denny
Steinke-Lange, Verena
Pickl, Julia M. A.
Neuhann, Teresa M.
Holinski-Feder, Elke
author_facet Hallermayr, Ariane
Keßler, Thomas
Fujera, Moritz
Liesfeld, Ben
Bernstein, Samuel
von Ameln, Simon
Schanze, Denny
Steinke-Lange, Verena
Pickl, Julia M. A.
Neuhann, Teresa M.
Holinski-Feder, Elke
author_sort Hallermayr, Ariane
collection PubMed
description SIMPLE SUMMARY: Liquid biopsy is a promising tool for the detection of low-frequency variants present in cancer and mosaic disease and is more and more adopted in clinical practice. Still, accurate validation and standardization across laboratories is lacking. Here, we show that the reference materials used during validation have a significant impact on a liquid biopsy next-generation-sequencing (NGS) assays analytical performance evaluation. With our findings, we developed a guide for the selection of suitable reference materials to eventually enable comparable results for patients across different laboratories. Highly sensitive and precise liquid biopsy NGS assays are critical to ensure accurate clinical interpretation. 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.
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spelling pubmed-106051192023-10-28 Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays Hallermayr, Ariane Keßler, Thomas Fujera, Moritz Liesfeld, Ben Bernstein, Samuel von Ameln, Simon Schanze, Denny Steinke-Lange, Verena Pickl, Julia M. A. Neuhann, Teresa M. Holinski-Feder, Elke Cancers (Basel) Article SIMPLE SUMMARY: Liquid biopsy is a promising tool for the detection of low-frequency variants present in cancer and mosaic disease and is more and more adopted in clinical practice. Still, accurate validation and standardization across laboratories is lacking. Here, we show that the reference materials used during validation have a significant impact on a liquid biopsy next-generation-sequencing (NGS) assays analytical performance evaluation. With our findings, we developed a guide for the selection of suitable reference materials to eventually enable comparable results for patients across different laboratories. Highly sensitive and precise liquid biopsy NGS assays are critical to ensure accurate clinical interpretation. 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. MDPI 2023-10-17 /pmc/articles/PMC10605119/ /pubmed/37894392 http://dx.doi.org/10.3390/cancers15205024 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hallermayr, Ariane
Keßler, Thomas
Fujera, Moritz
Liesfeld, Ben
Bernstein, Samuel
von Ameln, Simon
Schanze, Denny
Steinke-Lange, Verena
Pickl, Julia M. A.
Neuhann, Teresa M.
Holinski-Feder, Elke
Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
title Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
title_full Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
title_fullStr Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
title_full_unstemmed Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
title_short Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays
title_sort impact of cfdna reference materials on clinical performance of liquid biopsy ngs assays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10605119/
https://www.ncbi.nlm.nih.gov/pubmed/37894392
http://dx.doi.org/10.3390/cancers15205024
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