title: Validation of a next-generation sequencing assay for clinical molecular oncology.
abstract: Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥ 1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥ 15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Cottrell
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri. Electronic address: halkateb@path.wustl.edu.
authors.forename: Hussam
authors.initials: H
authors.lastname: Al-Kateb
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bredemeyer
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Duncavage
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: David H
authors.initials: DH
authors.lastname: Spencer
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Haley J
authors.initials: HJ
authors.lastname: Abel
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Lockwood
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Ian S
authors.initials: IS
authors.lastname: Hagemann
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: O'Guin
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Lauren C
authors.initials: LC
authors.lastname: Burcea
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Sawyer
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Dayna M
authors.initials: DM
authors.lastname: Oschwald
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Stratman
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Dorie A
authors.initials: DA
authors.lastname: Sher
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Johnson
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Justin T
authors.initials: JT
authors.lastname: Brown
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Paul F
authors.initials: PF
authors.lastname: Cliften
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Bijoy
authors.initials: B
authors.lastname: George
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Leslie D
authors.initials: LD
authors.lastname: McIntosh
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Savita
authors.initials: S
authors.lastname: Shrivastava
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Tudung T
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Payton
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Watson
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Seth D
authors.initials: SD
authors.lastname: Crosby
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Head
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Robi D
authors.initials: RD
authors.lastname: Mitra
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Nagarajan
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri; Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Shashikant
authors.initials: S
authors.lastname: Kulkarni
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Karen
authors.initials: K
authors.lastname: Seibert
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Herbert W
authors.initials: HW
authors.lastname: Virgin
authors.affiliation: Department of Genetics, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Milbrandt
authors.affiliation: Department of Pathology and Immunology, Genomics and Pathology Services, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: John D
authors.initials: JD
authors.lastname: Pfeifer
mesh_terms: D004247:DNA / Q000032:analysis; D005820:Genetic Testing; D015894:Genome, Human; D006239:Haplotypes / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004247:DNA