title: Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens.
abstract: Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington. Electronic address: cpritch@uw.edu.
authors.forename: Colin C
authors.initials: CC
authors.lastname: Pritchard
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington; Department of Genome Sciences, University of Washington, Seattle, Washington.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Salipante
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington.
authors.forename: Karen
authors.initials: K
authors.lastname: Koehler
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington.
authors.forename: Christina
authors.initials: C
authors.lastname: Smith
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington.
authors.forename: Sheena
authors.initials: S
authors.lastname: Scroggins
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington; Department of Pathology, University of Washington, Seattle, Washington.
authors.forename: Brent
authors.initials: B
authors.lastname: Wood
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington.
authors.forename: David
authors.initials: D
authors.lastname: Wu
authors.affiliation: Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Ming K
authors.initials: MK
authors.lastname: Lee
authors.affiliation: Department of Pathology, University of Washington, Seattle, Washington.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Dintzis
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington.
authors.forename: Andrew
authors.initials: A
authors.lastname: Adey
authors.affiliation: Department of Pathology, University of Washington, Seattle, Washington.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Division of Oncology, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Eaton
authors.affiliation: Division of Oncology, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Renato
authors.initials: R
authors.lastname: Martins
authors.affiliation: Division of Oncology, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Kari
authors.initials: K
authors.lastname: Stricker
authors.affiliation: Division of Oncology, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Margolin
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington.
authors.forename: Noah
authors.initials: N
authors.lastname: Hoffman
authors.affiliation: Section of Hematology and Oncology, Department of Medicine, University of Chicago, Chicago, Illinois.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Churpek
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington.
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Tait
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington; Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Mary-Claire
authors.initials: MC
authors.lastname: King
authors.affiliation: Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington.
authors.forename: Tom
authors.initials: T
authors.lastname: Walsh
mesh_terms: D056915:DNA Copy Number Variations / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D015321:Gene Rearrangement / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*