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.

• MeSH
• Circulating Tumor DNA genetics   MeSH
• Precision Medicine * MeSH
• Medical Oncology * MeSH
• Humans MeSH
• Limit of Detection MeSH
• Neoplasms genetics   MeSH
• Reproducibility of Results MeSH
• Sequence Analysis, DNA standards   MeSH
• Practice Guidelines as Topic MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Validation Study MeSH

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.

• MeSH
• Vaccines, Acellular therapeutic use   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Histamine administration & dosage   MeSH
• Cricetinae MeSH
• Humans MeSH
• Mice MeSH
• Whooping Cough diagnosis   prevention & control   MeSH
• Pertussis Vaccine therapeutic use   MeSH
• Pertussis Toxin therapeutic use   MeSH
• Education methods   trends   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Congress MeSH
• Geographicals
• Czech Republic MeSH