Technical Validation of a Next-Generation Sequencing Assay for Detecting Actionable Mutations in Patients with Gastrointestinal Cancer

• Published in: The Journal of molecular diagnostics : JMD Vol. 18; no. 3; pp. 416 - 424
• Main Authors: Wang, Sophie R, Malik, Simeen, Tan, Iain B, Chan, Yang Sun, Hoi, Qiangze, Ow, Jack L, He, Cassandra Z, Ching, Cindy E, Poh, Dianne Y.S, Seah, Hui Maan, Cheung, Katie H.T, Perumal, Dharuman, Devasia, Arun G, Pan, Lu, Ang, Shimin, Lee, Seow Eng, Ten, Rachel, Chua, Clarinda, Tan, Daniel S.W, Qu, James Z.Z, Bylstra, Yasmin M, Lim, Lionel, Lezhava, Alexander, Ng, Pauline C, Wong, Christopher W, Lim, Tony, Tan, Patrick
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2016
• Subjects: Biomarkers, Tumor; DNA Mutational Analysis - methods; DNA Mutational Analysis - standards; Gastrointestinal Neoplasms - diagnosis; Gastrointestinal Neoplasms - genetics; High-Throughput Nucleotide Sequencing; Humans; INDEL Mutation; Mutation; Pathology; Polymorphism, Single Nucleotide; Reference Values; Reproducibility of Results; Sensitivity and Specificity
• ISSN: 1525-1578; 1943-7811
• DOI: 10.1016/j.jmoldx.2016.01.006

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.