title: Analytical Validation of the Next-Generation Sequencing Assay for a Nationwide Signal-Finding Clinical Trial: Molecular Analysis for Therapy Choice Clinical Trial.
abstract: The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Chih-Jian
authors.initials: CJ
authors.lastname: Lih
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Robin D
authors.initials: RD
authors.lastname: Harrington
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, 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, 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, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Courtney H
authors.initials: CH
authors.lastname: Bouk
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Vivekananda
authors.initials: V
authors.lastname: Datta
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Yau
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Rajesh R
authors.initials: RR
authors.lastname: Singh
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Routbort
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Rajyalakshmi
authors.initials: R
authors.lastname: Luthra
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Keyur P
authors.initials: KP
authors.lastname: Patel
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Geeta S
authors.initials: GS
authors.lastname: Mantha
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Savitri
authors.initials: S
authors.lastname: Krishnamurthy
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut.
authors.forename: Karyn
authors.initials: K
authors.lastname: Ronski
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut.
authors.forename: Zenta
authors.initials: Z
authors.lastname: Walther
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Karin E
authors.initials: KE
authors.lastname: Finberg
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Sandra
authors.initials: S
authors.lastname: Canosa
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Hayley
authors.initials: H
authors.lastname: Robinson
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Amelia
authors.initials: A
authors.lastname: Raymond
authors.affiliation: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Long P
authors.initials: LP
authors.lastname: Le
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: McShane
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: Barbara A
authors.initials: BA
authors.lastname: Conley
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: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: A John
authors.initials: AJ
authors.lastname: Iafrate
authors.affiliation: Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Sklar
authors.affiliation: Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
authors.forename: Stanley R
authors.initials: SR
authors.lastname: Hamilton
authors.affiliation: Molecular Characterization and Clinical Assay Development Laboratory, Frederick National Laboratory for Cancer Research, Frederick, Maryland. Electronic address: mickey.williams@nih.gov.
authors.forename: P Mickey
authors.initials: PM
authors.lastname: Williams
mesh_terms: D002986:Clinical Trials as Topic; D019295:Computational Biology / Q000379:methods; D014644:Genetic Variation; D023281:Genomics / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D011785:Quality Assurance, Health Care; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D057188:Workflow