Genetic Landscape of Sporadic Vestibular Schwannoma
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LABORATORY INVESTIGATION J Neurosurg 128:911–922, 2018 Genetic landscape of sporadic vestibular schwannoma *Aril Løge Håvik,1–3 Ove Bruland, MSc, PhD,2 Erling Myrseth, MD, PhD,4 Hrvoje Miletic, MD, PhD,5–7 Mads Aarhus, MD, PhD,8 Per-Morten Knappskog, MSc, PhD,2,3 and Morten Lund-Johansen, MD, PhD1,4,6 Departments of 1Clinical Medicine, 3Clinical Science, and 7Biomedicine, and 6K.G. Jebsen Brain Tumor Research Center, University of Bergen; 2Center for Medical Genetics and Molecular Medicine, and Departments of 4Neurosurgery and 5Pathology, Haukeland University Hospital, Bergen; and 8Department of Neurosurgery, Oslo University Hospitals, Ullevål Sykehus, Oslo, Norway OBJECTIVE Vestibular schwannoma (VS) is a benign tumor with associated morbidities and reduced quality of life. Except for mutations in NF2, the genetic landscape of VS remains to be elucidated. Little is known about the effect of Gamma Knife radiosurgery (GKRS) on the VS genome. The aim of this study was to characterize mutations occurring in this tumor to identify new genes and signaling pathways important for the development of VS. In addition, the authors sought to evaluate whether GKRS resulted in an increase in the number of mutations. METHODS Forty-six sporadic VSs, including 8 GKRS-treated tumors and corresponding blood samples, were sub- jected to whole-exome sequencing and tumor-specific DNA variants were called. Pathway analysis was performed using the Ingenuity Pathway Analysis software. In addition, multiplex ligation-dependent probe amplification was performed to characterize copy number variations in the NF2 gene, and microsatellite instability testing was done to investigate for DNA replication error. RESULTS With the exception of a single sample with an aggressive phenotype that harbored a large number of muta- tions, most samples showed a relatively low number of mutations. A median of 14 tumor-specific mutations in each sample were identified. The GKRS-treated tumors harbored no more mutations than the rest of the group. A clustering of mutations in the cancer-related axonal guidance pathway was identified (25 patients), as well as mutations in the CDC27 (5 patients) and USP8 (3 patients) genes. Thirty-five tumors harbored mutations in NF2 and 16 tumors had 2 mutational hits. The samples without detectable NF2 mutations harbored mutations in genes that could be linked to NF2 or to NF2- related functions. None of the tumors showed microsatellite instability. CONCLUSIONS The genetic landscape of VS seems to be quite heterogeneous; however, most samples had mutations in NF2 or in genes that could be linked to NF2. The results of this study do not link GKRS to an increased number of mutations. https://thejns.org/doi/abs/10.3171/2016.10.JNS161384 KEY WORDS vestibular schwannoma; NF2; axonal guidance; next-generation sequencing; molecular biology; USP8; CDC27 ESTIBULAR schwannomas (VSs) make up 8% of in- ing VS pathobiology is the linkage of both sporadic VS tracranial tumors, with an annual incidence rate (sVS) and familial VS to a loss of the tumor suppressor ranging from 11 to 22 per million.10,58 They lead to protein merlin, which is encoded by the NF2 gene.49,62 Va set of well-characterized complaints. Affected individu- Merlin mediates contact inhibition and suppresses tumor- als report reduced quality of life, but the life expectancy of igenesis (both at the cell membrane and in the nucleus) patients with VSs is similar to that of the average popula- through its interaction with integrins and receptor tyrosine tion.8,37,56 kinases, among other proteins.28,41,69 The proportion of To date, the most important contribution to understand- sVSs harboring NF2 mutations ranges from 15% to 84%.13 ABBREVIATIONS GKRS = Gamma Knife radiosurgery; IPA = Ingenuity Pathway Analysis; LOH = loss of heterozygosity; MLPA = multiplex ligation-dependent probe amplification; MSI = microsatellite instability; PCR = polymerase chain reaction; SNV = single-nucleotide variant; sVS = sporadic VS; VS = vestibular schwannoma; WES = whole-exome sequencing. SUBMITTED May 30, 2016. ACCEPTED October 31, 2016. INCLUDE WHEN CITING Published online April 14, 2017; DOI: 10.3171/2016.10.JNS161384. * Drs. Knappskog and Lund-Johansen contributed equally to this work. ©AANS 2018, except where prohibited by US copyright law J Neurosurg Volume 128 • March 2018 911 Unauthenticated | Downloaded 09/29/21 09:50 AM UTC A. L. Håvik et al. This might indicate that alternative mechanisms for de- Mini Kit (QIAGEN). DNA from blood was extracted regulating NF2-related functions in schwannoma cells ex- using QIAsymphony (QIAGEN). The DNA quality and ist, leading us to search for mutations in genes linked to quantity were evaluated with 1% SeaKem gel electropho- merlin. resis and NanoDrop spectroscopy (Thermo Fisher Scien- In addition, we directed our attention toward another tific), respectively. tumor suppressor gene candidate, CAV1, to see whether mutations in this gene or in genes controlling its expres- Whole-Exome Sequencing sion can provide an explanation for its universal down- 1 WES was performed as a custom service (HudsonAl- regulation in sVSs. Except for the hereditary disease pha Institute for Biotechnology). The capture kit was Nim- associated with VS, neurofibromatosis Type 2, little is bleGen SeqCap EZ Exome Library v3.0, and sequencing known about the development of VS. Numerous studies was paired-end sequencing (2 × 100 bp) with approxi- have been conducted in the search for an etiology, but with × 15,43,51,53 mately 85 coverage on Illumina HiSeq. The resulting inconclusive findings. Recently, Agnihotri et al. se- reads were aligned to the GRCh37/hg19 reference genome quenced the exomes of a limited number of VSs and spi- 27 3 with Burrows-Wheeler transform, and the resulting bam- nal schwannomas. To gain a better understanding of the files were delivered to us. underlying pathogenesis of sVS formation, we performed whole-exome sequencing (WES) of sVSs and normal cells Bioinformatic Mutation Analyses to identify tumor-specific mutations. We applied the Genome Analysis Toolkit (version The increasing use of radiosurgery in treatment of VS 33 has raised concerns in the neurosurgical community about 3.2) base quality score recalibration, indel realignment, radiation-induced mutational events leading to malignant duplicate removal, and we performed single-nucleotide transformation.17,31,35,39,48,54 So far, a few cases of postir- polymorphism and INDEL calling across all 92 samples radiation malignant VSs have been described. Our study simultaneously using variant quality score recalibration therefore aimed not only to study sVS, but also in particu- according to Genome Analysis Toolkit Best Practices lar to study the mutational status of cases of VS where the recommendations.14,63 For detection of somatic point tumor had undergone radiosurgery. substitutions with low allelic fraction, we used MuTect.12 ANNOVAR64 was used for functional annotation of the variants called from the 46 tumor-normal pairs. Methods Candidate variants were filtered according to the fol- Patients and Specimen Collection lowing criteria: 1) allelic frequency < 0.05 in 1000 g, Samples of VS tissue and EDTA-treated blood were esp6500 and exac03; 2) repeated alleles < 5, evaluated obtained from 46 patients who underwent first-time sub- manually in borderline cases; 3) allelic frequency in tu- occipital resection of a unilateral VS at the Department mor > 0.05, allelic frequency in germline < 0.05, sequenc- of Neurosurgery, Haukeland University Hospital, between ing depth > 7, alternative allele > 1, evaluated manually in August 2003 and October 2014 (Table 1). Five pieces of borderline cases. The allelic frequency threshold was set viable tumor tissue with diameters of 3–5 mm were col- to exclude normal variants unlikely to be pathogenic. We lected from the intracapsular part of the tumor. This was excluded areas prone to segmental duplication by setting a done in 1 session, and the tissue was collected from 1 tu- cutoff value for mapping quality to 30 (phred score). mor area only. Routine histology was done in each case. To increase the likelihood of detecting mutations im- Eight patients had been treated with Gamma Knife radio- portant to tumorigenesis, we chose to evaluate nonsyn- surgery (GKRS) for the same VS. GKRS was performed onymous single-nucleotide variants (SNVs); frameshift according to a standardized protocol of 12 Gy to the tumor indels; and stop-gain, stop-loss, and splice-site mutations periphery at minimum 95% coverage. Six patients exhib- as predicted by ANNOVAR. The missense SNVs also had ited cystic VS. Dexamethasone 4 mg, administered orally to be predicted as deleterious/probably damaging/disease 4 times for 1 day preoperatively, was routinely given, start- causing/functional (positive prediction) by one of the fol- ing the day before surgery. Written informed consent was lowing prediction algorithms used in ANNOVAR: SIFT, received from all patients before tissue harvesting, and the PolyPhen 2 HDIV and HVAR, LRT, Mutation Taster, study was approved by the regional ethical committee for Mutation Assessor, FATHMM, MetaSVM, and MetaLR. medical research in Western Norway. The samples were GERP++, PhyloP, and SiPhy annotations were used to as- either immediately snap-frozen or stored at -80° and then sess how conserved the mutated sites were across different transferred to liquid nitrogen in the Bergen Neurosurgical species. Tissue Bank at Haukeland University Hospital. One tu- After filtering of variants, the remaining candidates mor-normal pair was found to be an outlier, accounting for were validated visually using the Integrative Genomics a large number of called variants, and was hence investi- Viewer.45,60 The outlier was subjected to stricter filtering gated with microsatellite-based polymerase chain reaction criteria to reduce the number of possible passenger mu- (PCR) profiling to confirm that the samples were matched. tations: ≥ 2 of the aforementioned prediction algorithms for nonsynonymous SNVs had to be positive. The variants DNA Extraction passing these filtering criteria were used for subsequent For DNA extraction, tumor tissue was first disrupted analyses.