Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT Trial

Chih Jian Lih, David J. Sims, Robin D. Harrington, Eric Polley, Yingdong Zhao, Michele G. Mehaffey, Thomas D. Forbes, Biswajit Das, William D. Walsh, Vivekananda Datta, Kneshay N. Harper, Courtney H. Bouk, Lawrence V. Rubinstein, Richard M. Simon, Barbara A. Conley, Alice P. Chen, Shivaani Kummar, James H. Doroshow, Paul M. Williams

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

Robust and analytically validated assays are essential for clinical studies. We outline an analytical validation study of a targeted next-generation sequencing mutation-detection assay used for patient selection in the National Cancer Institute Molecular Profiling-Based Assignment of Cancer Therapy (NCI-MPACT) trial (NCT01827384). Using DNA samples from normal or tumor cell lines and xenografts with known variants, we assessed the sensitivity, specificity, and reproducibility of the NCI-MPACT assay in five variant types: single-nucleotide variants (SNVs), SNVs at homopolymeric (HP) regions (≥3 identical bases), small insertions/deletions (indels), large indels (gap ≥4 bp), and indels at HP regions. The assay achieved sensitivities of 100% for 64 SNVs, nine SNVs at HP regions, and 11 large indels, 83.33% for six indels, and 93.33% for 15 indels at HP regions. Zero false positives (100% specificity) were found in 380 actionable mutation loci in 96 runs of haplotype map cells. Reproducibility analysis showed 96.3% to 100% intraoperator and 98.1% to 100% interoperator mean concordance in detected variants and 100% reproducibility in treatment selection. To date, 38 tumors have been screened, 34 passed preanalytical quality control, and 18 had actionable mutations for treatment assignment. The NCI-MPACT assay is well suited for its intended investigational use and can serve as a template for developing next-generation sequencing assays for other cancer clinical trial applications.

Original languageEnglish (US)
Pages (from-to)51-67
Number of pages17
JournalJournal of Molecular Diagnostics
Volume18
Issue number1
DOIs
StatePublished - Jan 1 2016
Externally publishedYes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

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    Lih, C. J., Sims, D. J., Harrington, R. D., Polley, E., Zhao, Y., Mehaffey, M. G., Forbes, T. D., Das, B., Walsh, W. D., Datta, V., Harper, K. N., Bouk, C. H., Rubinstein, L. V., Simon, R. M., Conley, B. A., Chen, A. P., Kummar, S., Doroshow, J. H., & Williams, P. M. (2016). Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT Trial. Journal of Molecular Diagnostics, 18(1), 51-67. https://doi.org/10.1016/j.jmoldx.2015.07.006