Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology

Nat Biotechnol. 2021 Sep;39(9):1115-1128. doi: 10.1038/s41587-021-00857-z. Epub 2021 Apr 12.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Circulating Tumor DNA / genetics*
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Limit of Detection
  • Medical Oncology*
  • Neoplasms / genetics*
  • Practice Guidelines as Topic
  • Precision Medicine*
  • Reproducibility of Results
  • Sequence Analysis, DNA / standards*

Substances

  • Circulating Tumor DNA