Abstract
Molecular markers have become pivotal in brain tumor diagnostics. Mutational analyses by targeted next-generation sequencing of DNA and array-based DNA methylation assessment with copy number analyses are increasingly being used in routine diagnostics. However, the broad variety of gene fusions occurring in brain tumors is marginally covered by these technologies and often only assessed by targeted assays. Here, we assessed the feasibility and clinical value of investigating gene fusions in formalin-fixed paraffin-embedded (FFPE) tumor tissues by next-generation mRNA sequencing in a routine diagnostic setting. After establishment and optimization of a workflow applicable in a routine setting, prospective diagnostic application in a neuropathology department for 26 months yielded relevant fusions in 66 out of 101 (65%) analyzed cases. In 43 (43%) cases, the fusions were of decisive diagnostic relevance and in 40 (40%) cases the fusion genes rendered a druggable target. A major strength of this approach was its ability to detect fusions beyond the canonical alterations for a given entity, and the unbiased search for any fusion event in cases with uncertain diagnosis and, thus, uncertain spectrum of expected fusions. This included both rare variants of established fusions which had evaded prior targeted analyses as well as the detection of previously unreported fusion events. While the impact of fusion detection on diagnostics is highly relevant, it is especially the detection of “druggable” fusions which will most likely provide direct benefit to the patients. The wider application of this approach for unbiased fusion identification therefore promises to be a major advance in identifying alterations with immediate impact on patient care.
Similar content being viewed by others
References
Bandopadhayay P, Ramkissoon LA, Jain P, Bergthold G, Wala J, Zeid R et al (2016) MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism. Nat Genet 48:273–282. https://doi.org/10.1038/ng.3500
Bridge JA, Liu XQ, Sumegi J, Nelson M, Reyes C, Bruch LA et al (2013) Identification of a novel, recurrent SLC44A1-PRKCA fusion in papillary glioneuronal tumor. Brain Pathol 23:121–128. https://doi.org/10.1111/j.1750-3639.2012.00612.x
Capper D, Jones DTW, Sill M, Hovestadt V, Schrimpf D, Sturm D et al (2018) DNA methylation-based classification of central nervous system tumours. Nature 555:469–474. https://doi.org/10.1038/nature26000
Chan E, Bollen AW, Sirohi D, Van Ziffle J, Grenert JP, Kline CN et al (2017) Angiocentric glioma with MYB-QKI fusion located in the brainstem, rather than cerebral cortex. Acta Neuropathol 134:671–673. https://doi.org/10.1007/s00401-017-1759-x
Hou Y, Pinheiro J, Sahm F, Reuss DE, Schrimpf D, Stichel D et al (2019) Papillary glioneuronal tumor (PGNT) exhibits a characteristic methylation profile and fusions involving PRKCA. Acta Neuropathol. https://doi.org/10.1007/s00401-019-01969-2
Hovestadt V, Remke M, Kool M, Pietsch T, Northcott PA, Fischer R et al (2013) Robust molecular subgrouping and copy-number profiling of medulloblastoma from small amounts of archival tumour material using high-density DNA methylation arrays. Acta Neuropathol 125:913–916. https://doi.org/10.1007/s00401-013-1126-5
Jones DT, Hutter B, Jager N, Korshunov A, Kool M, Warnatz HJ et al (2013) Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma. Nat Genet 45:927–932. https://doi.org/10.1038/ng.2682
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:R36. https://doi.org/10.1186/gb-2013-14-4-r36
Kline CN, Joseph NM, Grenert JP, van Ziffle J, Talevich E, Onodera C et al (2017) Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy. Neuro Oncol 19:699–709. https://doi.org/10.1093/neuonc/now254
Korshunov A, Ryzhova M, Hovestadt V, Bender S, Sturm D, Capper D et al (2015) Integrated analysis of pediatric glioblastoma reveals a subset of biologically favorable tumors with associated molecular prognostic markers. Acta Neuropathol 129:669–678. https://doi.org/10.1007/s00401-015-1405-4
Lin A, Rodriguez FJ, Karajannis MA, Williams SC, Legault G, Zagzag D et al (2012) BRAF alterations in primary glial and glioneuronal neoplasms of the central nervous system with identification of 2 novel KIAA1549:BRAF fusion variants. J Neuropathol Exp Neurol 71:66–72. https://doi.org/10.1097/NEN.0b013e31823f2cb0
Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK et al (2016) The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131:803–820. https://doi.org/10.1007/s00401-016-1545-1
McPherson A, Hormozdiari F, Zayed A, Giuliany R, Ha G, Sun MG et al (2011) deFuse: an algorithm for gene fusion discovery in tumor RNA-Seq data. PLoS Comput Biol 7:e1001138. https://doi.org/10.1371/journal.pcbi.1001138
Nagaishi M, Nobusawa S, Matsumura N, Kono F, Ishiuchi S, Abe T et al (2016) SLC44A1-PRKCA fusion in papillary and rosette-forming glioneuronal tumors. J Clin Neurosci 23:73–75. https://doi.org/10.1016/j.jocn.2015.04.021
Nikiforova MN, Wald AI, Melan MA, Roy S, Zhong S, Hamilton RL et al (2016) Targeted next-generation sequencing panel (GlioSeq) provides comprehensive genetic profiling of central nervous system tumors. Neuro Oncol 18:379–387. https://doi.org/10.1093/neuonc/nov289
Pages M, Lacroix L, Tauziede-Espariat A, Castel D, Daudigeos-Dubus E, Ridola V et al (2015) Papillary glioneuronal tumors: histological and molecular characteristics and diagnostic value of SLC44A1-PRKCA fusion. Acta Neuropathol Commun 3:85. https://doi.org/10.1186/s40478-015-0264-5
Panagopoulos I, Gorunova L, Bjerkehagen B, Heim S (2014) The “grep” command but not FusionMap, FusionFinder or ChimeraScan captures the CIC-DUX4 fusion gene from whole transcriptome sequencing data on a small round cell tumor with t(4;19)(q35;q13). PLoS One 9:e99439. https://doi.org/10.1371/journal.pone.0099439
Qaddoumi I, Orisme W, Wen J, Santiago T, Gupta K, Dalton JD et al (2016) Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology. Acta Neuropathol 131:833–845. https://doi.org/10.1007/s00401-016-1539-z
Sahm F, Schrimpf D, Jones DT, Meyer J, Kratz A, Reuss D et al (2016) Next-generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets. Acta Neuropathol 131:903–910. https://doi.org/10.1007/s00401-015-1519-8
Schweizer L, Koelsche C, Sahm F, Piro RM, Capper D, Reuss DE et al (2013) Meningeal hemangiopericytoma and solitary fibrous tumors carry the NAB2-STAT6 fusion and can be diagnosed by nuclear expression of STAT6 protein. Acta Neuropathol 125:651–658. https://doi.org/10.1007/s00401-013-1117-6
Sturm D, Orr BA, Toprak UH, Hovestadt V, Jones DT, Capper D et al (2016) New brain tumor entities emerge from molecular classification of CNS-PNETs. Cell 164:1060–1072. https://doi.org/10.1016/j.cell.2016.01.015
Tomic TT, Olausson J, Wilzen A, Sabel M, Truve K, Sjogren H et al (2017) A new GTF2I-BRAF fusion mediating MAPK pathway activation in pilocytic astrocytoma. PLoS One 12:e0175638. https://doi.org/10.1371/journal.pone.0175638
Acknowledgements
We thank Hai Yen Nguyen, Laura Dörner, and Lisa Kreinbihl for skillful technical assistance. This study was supported by the Else Kröner-Fresenius Stiftung (EKFS, 2017_EKES.24) and the Pediatric Low Grade Astrocytoma Fund (PLGA Fund) at the Pediatric Brain Tumor Foundation (PBTF).
Author information
Authors and Affiliations
Corresponding authors
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Stichel, D., Schrimpf, D., Casalini, B. et al. Routine RNA sequencing of formalin-fixed paraffin-embedded specimens in neuropathology diagnostics identifies diagnostically and therapeutically relevant gene fusions. Acta Neuropathol 138, 827–835 (2019). https://doi.org/10.1007/s00401-019-02039-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00401-019-02039-3