title: Analytical Validation and Application of a Targeted Next-Generation Sequencing Mutation-Detection Assay for Use in Treatment Assignment in the NCI-MPACT Trial.
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.
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland. Electronic address: jason.lih@nih.gov.
authors.forename: Chih-Jian
authors.initials: CJ
authors.lastname: Lih
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: David J
authors.initials: DJ
authors.lastname: Sims
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Robin D
authors.initials: RD
authors.lastname: Harrington
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Polley
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Yingdong
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Michele G
authors.initials: MG
authors.lastname: Mehaffey
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Forbes
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Biswajit
authors.initials: B
authors.lastname: Das
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: William D
authors.initials: WD
authors.lastname: Walsh
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Vivekananda
authors.initials: V
authors.lastname: Datta
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Kneshay N
authors.initials: KN
authors.lastname: Harper
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Courtney H
authors.initials: CH
authors.lastname: Bouk
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Lawrence V
authors.initials: LV
authors.lastname: Rubinstein
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Simon
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Conley
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Alice P
authors.initials: AP
authors.lastname: Chen
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Shivaani
authors.initials: S
authors.lastname: Kummar
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Leidos Biomedical Research Inc., and Frederick National Laboratory for Cancer Research, Frederick, Maryland; Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Williams
mesh_terms: D001483:Base Sequence; D062005:Biopsy, Large-Core Needle; D045744:Cell Line, Tumor; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D018579:Patient Selection; D010865:Pilot Projects; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA