TY - JOUR
T1 - Next-generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets
AU - Sahm, Felix
AU - Schrimpf, Daniel
AU - Jones, David T.W.
AU - Meyer, Jochen
AU - Kratz, Annekathrin
AU - Reuss, David
AU - Capper, David
AU - Koelsche, Christian
AU - Korshunov, Andrey
AU - Wiestler, Benedikt
AU - Buchhalter, Ivo
AU - Milde, Till
AU - Selt, Florian
AU - Sturm, Dominik
AU - Kool, Marcel
AU - Hummel, Manuela
AU - Bewerunge-Hudler, Melanie
AU - Mawrin, Christian
AU - Schüller, Ulrich
AU - Jungk, Christine
AU - Wick, Antje
AU - Witt, Olaf
AU - Platten, Michael
AU - Herold-Mende, Christel
AU - Unterberg, Andreas
AU - Pfister, Stefan M.
AU - Wick, Wolfgang
AU - von Deimling, Andreas
N1 - Publisher Copyright:
© 2015, Springer-Verlag Berlin Heidelberg.
PY - 2016/6/1
Y1 - 2016/6/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84949991007&partnerID=8YFLogxK
U2 - 10.1007/s00401-015-1519-8
DO - 10.1007/s00401-015-1519-8
M3 - Article
C2 - 26671409
AN - SCOPUS:84949991007
SN - 0001-6322
VL - 131
SP - 903
EP - 910
JO - Acta Neuropathologica
JF - Acta Neuropathologica
IS - 6
ER -