gms | German Medical Science

60. Jahrestagung der Deutschen Gesellschaft für Neuropathologie und Neuroanatomie (DGNN)

Deutsche Gesellschaft für Neuropathologie und Neuroanatomie

26. - 28.08.2015, Berlin

Next-Generation-Sequencing in routine neuropathology diagnostics

Meeting Abstract

  • corresponding author presenting/speaker Felix Sahm - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany
  • Daniel Schrimpf - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany
  • Jochen Meyer - DKFZ, CCU Neuropathology, Heidelberg, Germany
  • David Jones - DKFZ, Division of Pediatric Neurooncology, Heidelberg, Germany
  • David Reuss - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany
  • David Capper - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany
  • Christian Koelsche - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany
  • Annekathrin Kratz - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany
  • Andrey Korshunov - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany
  • Mark Zapatka - DKFZ, Molecular Genetics, Heidelberg, Germany
  • Stefan Pfister - DKFZ, Division of Pediatric Neurooncology, Heidelberg, Germany; University Hospital Heidelberg, Department of Pediatric Oncology, Hematology & Immunology, Heidelberg, Germany
  • Andreas Unterberg - University Hospital Heidelberg, Dept. of Neurosurgery, Heidelberg, Germany
  • Wolfgang Wick - University Hospital Heidelberg, Neurology Clinic, Heidelberg, Germany; DKFZ, CCU Neurooncology, Heidelberg, Germany
  • Andreas von Deimling - University Hospital Heidelberg, Dept. of Neuropathology, Heidelberg, Germany; DKFZ, CCU Neuropathology, Heidelberg, Germany

Deutsche Gesellschaft für Neuropathologie und Neuroanatomie. 60th Annual Meeting of the German Society for Neuropathology and Neuroanatomy (DGNN). Berlin, 26.-28.08.2015. Düsseldorf: German Medical Science GMS Publishing House; 2015. Doc15dgnnNO3

doi: 10.3205/15dgnn11, urn:nbn:de:0183-15dgnn114

Veröffentlicht: 25. August 2015

© 2015 Sahm et al.
Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). Lizenz-Angaben siehe http://creativecommons.org/licenses/by/4.0/.


Gliederung

Text

With the numbers of prognostic and predictive genetic markers in neuro-oncology steadily growing, the need for comprehensive molecular work-up of neuropathology samples has tremendously increased. Although several aberrations can be detected by immunohistochemistry (mutant IDH1R132H and BRAFV600E protein, loss of ATRX expression), the traditional detection of mutations, intragenic deletions and gene fusions requires time consuming methods such as Sanger sequencing and fluorescence in-situ hybridization. Covering all potentially clinically relevant genes in diagnostic routine is virtually impossible by these means. Thus, reliable high-throughput methods allowing parallel analysis of multiple targets are required in diagnostic neuropathology.We developed an enrichment-based gene panel comprising the entire coding region and selected promoter regions of 130 genes recurrently mutated in brain tumors. Optimization of probe design, extraction, library generation and sequencing conditions on 150 samples yielded a 5-workday routine workflow from FFPE sample to neuropathological report: Library generation is based on the Agilent SureSelect enrichment technology and sequencing is performed on an Illumina NextSeq 500. Raw data are processed with BWA-MEM for alignment, SAMtools mpileup for single nucleotide variant calling, Platypus for indel calling and De-Fuse for fusion detection. Variants in the germline, where available, are subtracted from the variants in the tumor. Variants in the tumor are subsequently annotated with dbSNP, 1000-genome and COSMIC entries as well as with SIFT and PolyPhen2 scores to inform on their biological relevance.

An average coverage of 500x enables detection of mutations also in samples with low tumor cell content. Further, inclusion of intronic regions of genes involved in fusion events enabled the detection of 7 pivotal gene fusions in parenchymal and meningeal brain tumors. Finally, high density of the designed probes, combined with the high coverage, permits the detection of copy number variations within the target regions with high resolution.

In conclusion, we present first experience with high-throughput next-generation-sequencing in routine neuropathology. Such an approach will likely become indispensable as more therapeutic targets emerge and genetic information enters the classification of brain tumors.