Functional Characterization and a Real-World Clinical Laboratory Pilot of the Foundation for the National Institutes of Health Circulating Tumor DNA Quality Control Materials

Published

November 6, 2025

Author(s)

Cloud Paweletz, Thomas Forbes, Laura M Yee, Grace Heavey, Hua-Jun He, Zhiyong He, Dana Connors, Daniel Stetson, Susan Keating, Kenneth Cole, Li Chen, Rini Pauly, Caroline Sigman, Liang-Chun Liu, Qiang Gan, Yves Konigshofer, Russell Garlick, Ephrem Chin, Gary Kelloff, Mickey Williams, Robert McCormack, Chris Karlovich

Abstract

Purpose We previously developed quality control materials (QCMs) to aid in the development of circulating tumor DNA (ctDNA) assays. In this study, we further characterize the performance of the QCMs relative to clinical samples. Methods QCMs were provided by three manufacturers. To functionally characterize the QCMs, we (1) evaluated EGFR L858R and ex19del (range, 0.5%-5.0% variant allele frequency [VAF]) in QCMs compared with clinical samples by droplet digital polymerase chain reaction (ddPCR), targeted-amplicon sequencing (Tag-seq), and hybrid capture next-generation sequencing (NGS); and (2) evaluated the QCMs and clinical samples near the Tag-seq limit of detection. A clinical pilot was also conducted in 11 clinical laboratories spanning four continents. Results For functional characterization, part 1, QCM VAFs for hybrid capture were similar to ddPCR for EGFR L858R but lower for ex19del. By contrast, hybrid capture results for EGFR L858R clinical samples showed a positive trend compared with ddPCR. For amplicon NGS, QCMs performed similarly to clinical samples for both variants. For part 2, observed hit rates approximated expected values. In the clinical pilot, median ex19del VAF was higher for Tag-seq than hybrid capture for both 1.0% and 0.5% QCM formulations. Median QCM L858R VAFs were similar for Tag-seq and hybrid capture, with greatest interlaboratory differences observed for Thermo Fisher Scientific QCMs. For non-EGFR variants, we observed assay and QCM-dependent trends, with no particular QCM or assay driving these trends. Conclusion This project revealed unexpected differences in performance of both assays and QCMs. These findings highlight the need for further validation across diverse alteration types and merit consideration by laboratories that rely on QCMs to develop and perform ctDNA assays for diagnostic applications.

Citation

Journal of Clinical Oncology-Precision Oncology

Volume

9

Pub Type

Journals

Download Paper

https://doi.org/10.1200/PO-25-00245

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Keywords

Cancer biomarker, ctDNA, liquid biopsy, quality control material, reference material, EGFR mutation

Citation

Paweletz, C. , Forbes, T. , Yee, L. , Heavey, G. , He, H. , He, Z. , Connors, D. , Stetson, D. , Keating, S. , Cole, K. , Chen, L. , Pauly, R. , Sigman, C. , Liu, L. , Gan, Q. , Konigshofer, Y. , Garlick, R. , Chin, E. , Kelloff, G. , Williams, M. , McCormack, R. and Karlovich, C. (2025), Functional Characterization and a Real-World Clinical Laboratory Pilot of the Foundation for the National Institutes of Health Circulating Tumor DNA Quality Control Materials, Journal of Clinical Oncology-Precision Oncology, [online], https://doi.org/10.1200/PO-25-00245, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=959733 (Accessed September 30, 2026)

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