title: Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies.
abstract: We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; Department of Pathology, Texas Children's Hospital, Houston, Texas; Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas. Electronic address: kevin.fisher@bcm.edu.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Fisher
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Linsheng
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; Department of Pathology, University of Texas Southwestern and Children's Medical Center, Dallas, Texas.
authors.forename: Jason
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Geoffrey H
authors.initials: GH
authors.lastname: Smith
authors.affiliation: Biostatistics and Bioinformatics Shared Resource, Emory University, Atlanta, Georgia.
authors.forename: Scott
authors.initials: S
authors.lastname: Newman
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Schneider
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Rathi N
authors.initials: RN
authors.lastname: Pillai
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Ragini R
authors.initials: RR
authors.lastname: Kudchadkar
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Taofeek K
authors.initials: TK
authors.lastname: Owonikoko
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Suresh S
authors.initials: SS
authors.lastname: Ramalingam
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: David H
authors.initials: DH
authors.lastname: Lawson
authors.affiliation: Winship Cancer Institute, Emory University, Atlanta, Georgia; Department of Surgery, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Delman
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Bassel F
authors.initials: BF
authors.lastname: El-Rayes
authors.affiliation: Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Malania M
authors.initials: MM
authors.lastname: Wilson
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: H Clifford
authors.initials: HC
authors.lastname: Sullivan
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Annie S
authors.initials: AS
authors.lastname: Morrison
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Serdar
authors.initials: S
authors.lastname: Balci
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: N Volkan
authors.initials: NV
authors.lastname: Adsay
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Gal
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Gabriel L
authors.initials: GL
authors.lastname: Sica
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Debra F
authors.initials: DF
authors.lastname: Saxe
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Karen P
authors.initials: KP
authors.lastname: Mann
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Hill
authors.affiliation: Department of Hematology and Oncology, Emory University School of Medicine, Atlanta, Georgia; Winship Cancer Institute, Emory University, Atlanta, Georgia.
authors.forename: Fadlo R
authors.initials: FR
authors.lastname: Khuri
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia; Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Rossi
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics; D005770:Gastrointestinal Neoplasms / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D057230:Limit of Detection; D008175:Lung Neoplasms / Q000235:genetics*; D008545:Melanoma / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D009154:Mutation; D016612:Paraffin Embedding; D011786:Quality Control; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D016159:Tumor Suppressor Protein p53; C495901:TP53 protein, human; C512478:EGFR protein, human; C508053:ERBB2 protein, human