Analytic validation of a clinical circulating tumor DNA assay for patients with solid tumors

Background

Several circulating tumor DNA (ctDNA) profiling assays are commercially available, and physicians must be empowered to identify assays with the high level of accuracy required to meet the diagnostic needs of their patients. This study describes the analytic validation of a ctDNA assay optimized for clinical care.

Methods

Accuracy and reproducibility were validated using 117 reference samples with known alterations and 268 clinical ctDNA samples. A CLIA-validated NGS assay, droplet digital PCR, and break-point PCR were used to validate the alterations identified. A highly optimized, integrated workflow was developed that includes sample collection, storage and transport, ctDNA purification, library generation, and enrichment (solution hybridization capture), followed by high-depth sequencing (HiSeq2500). Computational methods were developed to enable sensitive and specific detection of all alteration classes, including base substitutions, small insertions and deletions (indels), copy number changes, and rearrangements/fusions.

Results

This ctDNA assay demonstrated unprecedented accuracy: >99% sensitivity and >99% positive predictive value (PPV) for base substitutions and indels, >99% sensitivity and 98% PPV for rearrangements/fusions with a limit-of-detection below 0.5%, plus robust detection of high-level, focal amplifications. The assay also accurately reports allele frequency. In 48 clinical ctDNA samples, 95 alterations of all classes were 100% confirmed by orthogonal testing. We also report results comparing alterations from patient-matched ctDNA and FFPE biopsies.

Conclusions

Developing a commercial ctDNA assay requires rigorous analytic validation and the availability of clinically relevant test metrics to ensure reliable interpretation and reporting to optimize targeted treatment options. This rigorous analytic validation study demonstrates high-accuracy detection in blood for all alteration classes, even when mutations are present at low allele frequency, thereby realizing the potential of ctDNA-based molecular profiling for the management of cancer. A large, ongoing prospective clinical trial will provide additional data on the appropriate clinical settings for use of this assay in patient care.

Clinical trial identification

Legal entity responsible for the study

Foundation Medicine, Inc.

Funding

Foundation Medicine, Inc.

Disclosure

P.J. Stephens, V.A. Miller: Employee of, stockholder in, and holds a leadership position for Foundation Medicine, Inc. T. Clark, M. Kennedy, J. He, G. Young, M. Zhao, M. Coyne, V. Breese, L. Young, S. Zhong, M. Bailey, B. Fendler, G. Otto, D. Lipson, J.S. Ross: Employee of and stockholder in Foundation Medicine, Inc. All other authors have declared no conflicts of interest.