Analytical Validation of a Pan-Cancer Next-Generation Sequencing Assay for In-House Liquid Biopsy Testing

Liquid biopsy assays are transforming precision oncology by providing a less invasive alternative to tissue biopsies. These assays screen for tumor genetic alterations in circulating free DNA, which typically requires detecting variants at low allele frequencies and, therefore, a high sensitivity and specificity. This international, multicenter analytical performance study evaluated the Hedera Profiling 2 circulating tumor DNA test panel (HP2), a hybrid capture-based next-generation sequencing assay for the detection of somatic alterations in circulating free DNA. Covering 32 genes, HP2 enables the detection of single-nucleotide variants (SNVs), insertions and deletions (Indels), fusions, copy number variations, and microsatellite instability status from a single DNA-only workflow. The analytical performance was assessed using reference standards and a diverse cohort of 137 clinical samples precharacterized by orthogonal methods. In reference standards with variants spiked in at 0.5% allele frequency, sensitivity and specificity were 96.92% and 99.67%, respectively, for SNVs/Indels and 100% for fusions. In clinical samples, SNV/Indel detection showed high concordance (94% for European Society for Medical Oncology Scale of Clinical Actionability for Molecular Targets level I variants) with orthogonal methods. Evidence for solid sensitivity was also found for copy number variation detection and microsatellite instability status determination. Overall, the HP2 assay showed significant potential as a sensitive and efficient pan-cancer test for liquid biopsy testing in a decentralized molecular pathology laboratory setting.

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Publication Details

Journal
Journal of Molecular Diagnostics
Published
2025-09-25
DOI
https://doi.org/10.1016/j.jmoldx.2025.08.008
Citations
1
Primary Topic
Ovarian cancer diagnosis and treatment
Type
article
Field-Weighted Citation Impact
1.70

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article

Analytical Validation of a Pan-Cancer Next-Generation Sequencing Assay for In-House Liquid Biopsy Testing

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Journal of Molecular Diagnostics
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article

Analytical Validation of a Pan-Cancer Next-Generation Sequencing Assay for In-House Liquid Biopsy Testing

Jonas Behr, Hendrikus J. Dubbink, Ernst‐Jan M. Speel, Alexandre Harlé, Alexis Rapin, Clémence Michon, Aitor Rodríguez‐Casanova, Léa Payen, Pantelis Constantoulakis, Umberto Malapelle, Parth Shah, Deepak Pathak, Giancarlo Troncone, Imke Demers, Gianluca Russo, Francesco Pepe, Nicole Pfarr, Ángel Díaz‐Lagares, Laura Muinelo‐Romay, Ricarda Graf, Hrishikesh Kumar, Gaëlle Lescuyer, Marie Piecyk, Ellen Heitzer, Christophe Passot, Ronald van Marion, Eva Mayr, Thomas Vetterli, Sangamitra Boppudi, Mai-Lan Koppermann, Georgia Christopoulou, Violaine Bourdon, Marie Husson, Anne-Marie C Dingemans, Anes Hadjadj Aoul, Romain V Parillaud, Oskar Martínez de Ilarduya, Ramón Manuel Lago Lestón
article en
1 citations

Abstract

Liquid biopsy assays are transforming precision oncology by providing a less invasive alternative to tissue biopsies. These assays screen for tumor genetic alterations in circulating free DNA, which typically requires detecting variants at low allele frequencies and, therefore, a high sensitivity and specificity. This international, multicenter analytical performance study evaluated the Hedera Profiling 2 circulating tumor DNA test panel (HP2), a hybrid capture-based next-generation sequencing assay for the detection of somatic alterations in circulating free DNA. Covering 32 genes, HP2 enables the detection of single-nucleotide variants (SNVs), insertions and deletions (Indels), fusions, copy number variations, and microsatellite instability status from a single DNA-only workflow. The analytical performance was assessed using reference standards and a diverse cohort of 137 clinical samples precharacterized by orthogonal methods. In reference standards with variants spiked in at 0.5% allele frequency, sensitivity and specificity were 96.92% and 99.67%, respectively, for SNVs/Indels and 100% for fusions. In clinical samples, SNV/Indel detection showed high concordance (94% for European Society for Medical Oncology Scale of Clinical Actionability for Molecular Targets level I variants) with orthogonal methods. Evidence for solid sensitivity was also found for copy number variation detection and microsatellite instability status determination. Overall, the HP2 assay showed significant potential as a sensitive and efficient pan-cancer test for liquid biopsy testing in a decentralized molecular pathology laboratory setting.

Journal of Molecular DiagnosticsVol. 27(12)
Université Claude Bernard Lyon 1 (FR), Centre National de la Recherche Scientifique (FR), Medical University of Graz (AT), Maastricht University Medical Centre (NL), Maastricht University (NL), Institut de Cancérologie de Lorraine (FR), Hospices Civils de Lyon (FR), Institut de Cancérologie de l'Ouest (FR), Centre de Recherche en Automatique de Nancy (FR), Center for Research in Molecular Medicine and Chronic Diseases (ES), Frontier Science Foundation-Hellas (GR), Centre de Recherche en Cancérologie de Marseille (FR), Instituto de Investigación Sanitaria de Santiago (ES), Erasmus MC Cancer Institute (NL), Institut Paoli-Calmettes (FR), Center for Genomic Science (IT), Healing Institute (US), Maastro Clinic (NL), Technical University of Munich (DE), University of Naples Federico II (IT), Epigenomics (Germany) (DE)
AstraZeneca, Instituto de Salud Carlos III, Servicio Gallego de Salud
Openalex Percentile: Top 13%
Ovarian cancer diagnosis and treatment
1.70
DOIOpen Access PDFOpenAlex
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