title: Technical Validation of a Next-Generation Sequencing Assay for Detecting Actionable Mutations in Patients with Gastrointestinal Cancer.
abstract: Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- and tumor-specific genetic profiles as well as to optimally select targeted therapies. Here, we describe a custom-developed, next-generation sequencing test for detecting single-nucleotide variants (SNVs) and short insertions and deletions (indels) in 93 genes related to gastrointestinal cancer from routine formalin-fixed, paraffin-embedded clinical specimens. We implemented a validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures from cell lines with known genetic variants, which model a broad range of allele frequencies. Test sensitivity achieved >99% for both SNVs and indels, with allele frequencies >10%, with high specificity (97.4% for SNVs and 93.6% for indels). We further confirmed test accuracies using primary formalin-fixed, paraffin-embedded colorectal cancer specimens characterized by alternative and conventional clinical diagnostic technologies. Robust performance was observed on the formalin-fixed, paraffin-embedded specimens: sensitivity was 97.2% and specificity was 99.2%. We also observed high intrarun and inter-run reproducibility, as well as a low cross-contamination rate. Overall assessment using cell line samples and formalin-fixed, paraffin-embedded samples showed that our custom next-generation sequencing assay has consistent detection sensitivity down to 10% variant frequency.
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Sophie R
authors.initials: SR
authors.lastname: Wang
authors.affiliation: Cancer & Stem Cell Biology Program, Duke-National University of Singapore Graduate Medical School, Singapore.
authors.forename: Simeen
authors.initials: S
authors.lastname: Malik
authors.affiliation: Genome Institute of Singapore, Singapore; Cancer & Stem Cell Biology Program, Duke-National University of Singapore Graduate Medical School, Singapore; Department of Medical Oncology, National Cancer Centre Singapore, Singapore; Graduate School of Integrative Sciences and Engineering, National University of Singapore, Singapore.
authors.forename: Iain B
authors.initials: IB
authors.lastname: Tan
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Yang Sun
authors.initials: YS
authors.lastname: Chan
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Qiangze
authors.initials: Q
authors.lastname: Hoi
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Jack L
authors.initials: JL
authors.lastname: Ow
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Cassandra Z
authors.initials: CZ
authors.lastname: He
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Cindy E
authors.initials: CE
authors.lastname: Ching
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Dianne Y S
authors.initials: DYS
authors.lastname: Poh
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Hui Maan
authors.initials: HM
authors.lastname: Seah
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Katie H T
authors.initials: KHT
authors.lastname: Cheung
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Dharuman
authors.initials: D
authors.lastname: Perumal
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Arun G
authors.initials: AG
authors.lastname: Devasia
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Lu
authors.initials: L
authors.lastname: Pan
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Shimin
authors.initials: S
authors.lastname: Ang
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Seow Eng
authors.initials: SE
authors.lastname: Lee
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore.
authors.forename: Rachel
authors.initials: R
authors.lastname: Ten
authors.affiliation: Department of Medical Oncology, National Cancer Centre Singapore, Singapore.
authors.forename: Clarinda
authors.initials: C
authors.lastname: Chua
authors.affiliation: Genome Institute of Singapore, Singapore; Department of Medical Oncology, National Cancer Centre Singapore, Singapore; Division of Medical Sciences, National Cancer Centre Singapore, Singapore.
authors.forename: Daniel S W
authors.initials: DSW
authors.lastname: Tan
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: James Z Z
authors.initials: JZZ
authors.lastname: Qu
authors.affiliation: Genome Institute of Singapore, Singapore; Department of Pathology, Singapore General Hospital, Singapore, Singapore.
authors.forename: Yasmin M
authors.initials: YM
authors.lastname: Bylstra
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Lionel
authors.initials: L
authors.lastname: Lim
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Alexander
authors.initials: A
authors.lastname: Lezhava
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Pauline C
authors.initials: PC
authors.lastname: Ng
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Wong
authors.affiliation: Genome Institute of Singapore, Singapore; Department of Pathology, Singapore General Hospital, Singapore, Singapore. Electronic address: lim.kiat.hon@sgh.com.sg.
authors.forename: Tony
authors.initials: T
authors.lastname: Lim
authors.affiliation: Genome Institute of Singapore, Singapore; Cancer & Stem Cell Biology Program, Duke-National University of Singapore Graduate Medical School, Singapore; Cancer Science Institute Singapore, National University of Singapore, Singapore; Division of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore. Electronic address: tanbop@gis.a-star.edu.sg.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tan
mesh_terms: D014408:Biomarkers, Tumor; D004252:DNA Mutational Analysis / Q000379:methods / Q000592:standards; D005770:Gastrointestinal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054643:INDEL Mutation; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor