Analytical Validation of a Hybrid Capture–Based Next-Generation Sequencing Clinical Assay for Genomic Profiling of Cell-Free Circulating Tumor DNA

Travis A. Clark, Jon H. Chung, Mark Kennedy, Jason D. Hughes, Niru Chennagiri, Daniel S. Lieber, Bernard Fendler, Lauren Young, Mandy Zhao, Michael Coyne, Virginia Breese, Geneva Young, Amy Donahue, Dean Pavlick, Alyssa Tsiros, Timothy Brennan, Shan Zhong, Tariq Mughal, Mark Bailey, Jie HeSteven Roels, Garrett M. Frampton, Jill M. Spoerke, Steven Gendreau, Mark Lackner, Erica Schleifman, Eric Peters, Jeffrey S. Ross, Siraj M. Ali, Vincent A. Miller, Jeffrey Gregg, Philip J. Stephens, Allison Welsh, Geoff A. Otto, Doron Lipson

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture–based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode–based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%–99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%–95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%–71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments–certified/College of American Pathologists–accredited/New York State–approved laboratory.

Original languageEnglish (US)
Pages (from-to)686-702
Number of pages17
JournalJournal of Molecular Diagnostics
Issue number5
StatePublished - Sep 1 2018

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine


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