title: Targeted next-generation sequencing panel (GlioSeq) provides comprehensive genetic profiling of central nervous system tumors.
abstract: BACKGROUND
  Identification of genetic changes in CNS tumors is important for the appropriate clinical management of patients. Our objective was to develop a next-generation sequencing (NGS) assay for simultaneously detecting the various types of genetic alterations characteristic for adult and pediatric CNS tumors that can be applied to small brain biopsies.
  
  
  METHODS
  We report an amplification-based targeted NGS assay (GlioSeq) that analyzes 30 genes for single nucleotide variants (SNVs) and indels, 24 genes for copy number variations (CNVs), and 14 types of structural alterations in BRAF, EGFR, and FGFR3 genes in a single workflow. GlioSeq performance was evaluated in 54 adult and pediatric CNS tumors, and the results were compared with fluorescence in-situ hybridization, Sanger sequencing, and reverse transcription PCR.
  
  
  RESULTS
  GlioSeq correctly identified 71/71 (100%) genetic alterations known to be present by conventional techniques, including 56 SNVs/indels, 9 CNVs, 3 EGFRvIII, and 3 KIAA1549-BRAF fusions. Only 20 ng of DNA and 10 ng of RNA were required for successful sequencing of 100% frozen and 96% formalin-fixed, paraffin-embedded tissue specimens. The assay sensitivity was 3%-5% of mutant alleles for SNVs and 1%-5% for gene fusions. The most commonly detected alterations were IDH1, TP53, TERT, ATRX. CDKN2A, and PTEN in high-grade gliomas, followed by BRAF fusions in low-grade gliomas and H3F3A mutations in pediatric gliomas.
  
  
  CONCLUSIONS
  GlioSeq NGS assay offers accurate and sensitive detection of a wide range of genetic alterations in a single workflow. It allows rapid and cost-effective profiling of brain tumor specimens and thus provides valuable information for patient management.
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Marina N
authors.initials: MN
authors.lastname: Nikiforova
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Abigail I
authors.initials: AI
authors.lastname: Wald
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Melan
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Somak
authors.initials: S
authors.lastname: Roy
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Shan
authors.initials: S
authors.lastname: Zhong
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Hamilton
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Frank S
authors.initials: FS
authors.lastname: Lieberman
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Jan
authors.initials: J
authors.lastname: Drappatz
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Nduka M
authors.initials: NM
authors.lastname: Amankulor
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Ian F
authors.initials: IF
authors.lastname: Pollack
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Yuri E
authors.initials: YE
authors.lastname: Nikiforov
authors.affiliation: Department of Pathology, Division of Molecular & Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (M.N.N, A.I.W., M.A.M., S.R., S.Z., Y.E.N.); Department of Pathology, Division of Neuropathology, University of Pittsburgh Medical Center, Presbyterian Hospital, Pittsburgh, Pennsylvania (R.L.H.); Division of Hematology/Oncology, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (F.S.L., J.D.); Department of Neurological Surgery, Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (N.M.A., I.F.P.); Departments of Pathology and Neurosurgery, Northwestern University, Chicago, Illinois (C.H.).
authors.forename: Craig
authors.initials: C
authors.lastname: Horbinski
mesh_terms: D000293:Adolescent; D000328:Adult; D016543:Central Nervous System Neoplasms / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D056915:DNA Copy Number Variations / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D005910:Glioma / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics
chemical_list: D048493:Proto-Oncogene Proteins B-raf
keywords: CNS tumors; gene fusions; mutations; next generation sequencing; paraffin