title: Next-generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets.
abstract: With the number of prognostic and predictive genetic markers in neuro-oncology steadily growing, the need for comprehensive molecular analysis of neuropathology samples has vastly increased. We therefore developed a customized enrichment/hybrid-capture-based next-generation sequencing (NGS) gene panel comprising the entire coding and selected intronic and promoter regions of 130 genes recurrently altered in brain tumors, allowing for the detection of single nucleotide variations, fusions, and copy number aberrations. Optimization of probe design, library generation and sequencing conditions on 150 samples resulted in a 5-workday routine workflow from the formalin-fixed paraffin-embedded sample to neuropathological report. This protocol was applied to 79 retrospective cases with established molecular aberrations for validation and 71 prospective cases for discovery of potential therapeutic targets. Concordance of NGS compared to established, single biomarker methods was 98.0 %, with discrepancies resulting from one case where a TERT promoter mutation was not called by NGS and three ATRX mutations not being detected by Sanger sequencing. Importantly, in samples with low tumor cell content, NGS was able to identify mutant alleles that were not detectable by traditional methods. Information derived from NGS data identified potential targets for experimental therapy in 37/47 (79 %) glioblastomas, 9/10 (90 %) pilocytic astrocytomas, and 5/14 (36 %) medulloblastomas in the prospective target discovery cohort. In conclusion, we present the settings for high-throughput, adaptive next-generation sequencing in routine neuropathology diagnostics. Such an approach will likely become highly valuable in the near future for treatment decision making, as more therapeutic targets emerge and genetic information enters the classification of brain tumors.
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Sahm
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schrimpf
authors.affiliation: Division of Pediatric Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: David T W
authors.initials: DT
authors.lastname: Jones
authors.affiliation: Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Meyer
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Annekathrin
authors.initials: A
authors.lastname: Kratz
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Reuss
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Capper
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Koelsche
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Andrey
authors.initials: A
authors.lastname: Korshunov
authors.affiliation: Neurology Clinic, Heidelberg University Hospital, Heidelberg, Germany.|Clinical Cooperation Unit Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Wiestler
authors.affiliation: Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany.|Division of Applied Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Ivo
authors.initials: I
authors.lastname: Buchhalter
authors.affiliation: Department of Pediatric Oncology, Haematology and Immunology, Heidelberg University Hospital, and National Center for Tumor Diseases (NCT), Heidelberg, Germany.|Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Milde
authors.affiliation: Department of Pediatric Oncology, Haematology and Immunology, Heidelberg University Hospital, and National Center for Tumor Diseases (NCT), Heidelberg, Germany.|Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Selt
authors.affiliation: Division of Pediatric Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Pediatric Oncology, Haematology and Immunology, Heidelberg University Hospital, and National Center for Tumor Diseases (NCT), Heidelberg, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Sturm
authors.affiliation: Division of Pediatric Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Kool
authors.affiliation: Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Manuela
authors.initials: M
authors.lastname: Hummel
authors.affiliation: Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Bewerunge-Hudler
authors.affiliation: Department of Neuropathology, Otto von Guericke University Magdeburg, Magdeburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Mawrin
authors.affiliation: Center of Neuropathology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schüller
authors.affiliation: Department of Neurosurgery, Heidelberg University Hospital, Heidelberg, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: Jungk
authors.affiliation: Neurology Clinic, Heidelberg University Hospital, Heidelberg, Germany.|Clinical Cooperation Unit Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Wick
authors.affiliation: Department of Pediatric Oncology, Haematology and Immunology, Heidelberg University Hospital, and National Center for Tumor Diseases (NCT), Heidelberg, Germany.|Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ), German Consortium for Translational Cancer Research (DKTK), Heidelberg, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Witt
authors.affiliation: Neurology Clinic, Heidelberg University Hospital, Heidelberg, Germany.|Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Platten
authors.affiliation: Department of Neurosurgery, Heidelberg University Hospital, Heidelberg, Germany.
authors.forename: Christel
authors.initials: C
authors.lastname: Herold-Mende
authors.affiliation: Department of Neurosurgery, Heidelberg University Hospital, Heidelberg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Unterberg
authors.affiliation: Division of Pediatric Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.|Department of Pediatric Oncology, Haematology and Immunology, Heidelberg University Hospital, and National Center for Tumor Diseases (NCT), Heidelberg, Germany.
authors.forename: Stefan M
authors.initials: SM
authors.lastname: Pfister
authors.affiliation: Neurology Clinic, Heidelberg University Hospital, Heidelberg, Germany.|Clinical Cooperation Unit Neurooncology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wick
authors.affiliation: Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany. andreas.vondeimling@med.uni-heidelberg.de.|Clinical Cooperation Unit Neuropathology, German Consortium for Translational Cancer Research (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany. andreas.vondeimling@med.uni-heidelberg.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: von Deimling
mesh_terms: D001932:Brain Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D015336:Molecular Probe Techniques; D009154:Mutation / Q000235:genetics; D057089:Pathology, Molecular / Q000379:methods