Multi-Site Benchmarking Study Defines Analytical Performance Boundaries of Leading ctDNA Assays
This landmark cross-platform evaluation tested five industry-leading ctDNA sequencing assays using simulations, synthetic spike-ins and standardised reference samples across multiple sites. Above 0.5% variant allele frequency all assays performed well, but below this threshold detection became unreliable with wide inter-assay variation, particularly with limited input material. False negatives far outnumbered false positives, establishing that reliable sampling of rare ctDNA fragments -- not specificity -- is the primary analytical challenge for the field.
The original study
Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.
- Authors
- Deveson IW, Gong B, Lai K, LoCoco JS, Richmond TA, Schageman J, et al.
- Journal
- Nature biotechnology
- Type
- Journal Article, Research Support, N.I.H., Extramural, Research Support, N.I.H., Intramural, Research Support, Non-U.S. Gov't, Validation Study
- PMID
- 33846644
Original abstract
Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.