First insight into the somatic mutation burden of neurofibromatosis type 2-associated grade I and grade II meningiomas: a case report comprehensive genomic study of two cranial meningiomas with vastly different clinical presentation
- Ramita Dewan†1,
- Alexander Pemov†2,
- Amalia S. Dutra3,
- Evgenia D. Pak3,
- Nancy A. Edwards1,
- Abhik Ray-Chaudhury1,
- Nancy F. Hansen4,
- Settara C. Chandrasekharappa4,
- James C. Mullikin4, 5,
- Ashok R. Asthagiri6,
- NISC Comparative Sequencing Program,
- John D. Heiss1,
- Douglas R. Stewart2 and
- Anand V. Germanwala7, 8Email author
© The Author(s). 2017
Received: 28 March 2016
Accepted: 8 February 2017
Published: 13 February 2017
Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined.
Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansion
This is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.
KeywordsWhole exome sequencing Single nucleotide polymorphism Spectral karyotyping NF2 gene Somatic mutation Case report
Neurofibromatosis type 2 (NF2) is an autosomal dominant tumor syndrome characterized by the growth of multiple neoplasms within the central nervous system. Although bilateral vestibular schwannomas are the hallmark of NF2, meningiomas are the second most frequent intracranial tumor, and occur in about 52% of NF2 patients [1, 2]. Benign meningiomas (WHO grade I) feature a 5-year tumor recurrence rate of 5% as compared to 50–80% for anaplastic meningiomas (grade III), highlighting the importance of elucidating the molecular mechanisms which contribute to tumor progression .
The most common genetic mutation in meningiomas is NF2 inactivation, which is observed not only in NF2-associated tumors, but also in 47 to 72% of sporadic meningiomas, and is thus considered an integral step for meningioma tumor initiation [4–6]. Recent studies utilizing high throughput whole-exome and whole-genome sequencing have identified two distinct subtypes of sporadic meningiomas: tumors with or without an inactivated NF2 gene [7, 8]. Sporadic meningiomas with disrupted NF2 tend to display greater genomic instability (including several cases of chromothripsis) and higher grades than non-NF2 meningiomas. Non-NF2 tumors have been shown to contain recurrent oncogenic mutations in AKT1, KLF4, TRAF7 and SMO, indicating the alternate involvement of the PI3K-AKT and Hedgehog signaling pathways.
NF2-associated meningiomas are rarer than their sporadic counterparts and far fewer studies have investigated the genetics underlying their initiation and progression. Two case series evaluated meningiomas from NF2 patients only for the allelic imbalances most commonly observed in sporadic meningiomas, and confirmed frequent somatic inactivation of the NF2 gene, as well as losses of chromosome arms 1p, 6q, 9p, 10q, 14q and 18q [9, 10]. A more recent study used single nucleotide polymorphism array analysis to report increased chromosomal instability with increasing grade in NF2-associated meningiomas .
Here, we present an in-depth genomic study of grade I and grade II meningiomas that resided in close proximity in the brain of an NF2 patient. The tumors contained the same NF2 germline mutation and similar somatic hits affecting the normal remaining copy of the gene, yet differed drastically in genomic architecture and growth rate. The tumors were investigated using whole-exome sequencing complemented with SKY and SNP-array copy-number analysis.
Materials and methods
A 35-year-old woman was enrolled in the Institutional Review Board (IRB)-approved NF2 natural history study (NIH#08-N-0044) at the National Institute of Neurologic Disease and Stroke (NINDS). Prior MR imaging confirmed the NF2 Manchester diagnostic criteria of bilateral vestibular schwannoma in addition to numerous other significant findings: intracranial schwannomas involving cranial nerves V, VII, and VIII, intracranial meningiomas, cervical ependymomas, schwannomas along the cauda equina, and cervicothoracic meningiomas.
In preparation for surgery, the patient underwent frameless stereotactic navigation imaging on a 1.5 Tesla MRI scanner with and without gadolinium. 1-mm axial images were obtained with sagittal and coronal reconstruction. Image guidance registration was performed intraoperatively using facial registration.
A single image-guided right frontal craniotomy was used to resect an anterior grade II meningioma, in four discrete sections, and a posterior grade I meningioma. The two meningiomas were separated by an intervening section of normal brain, and were resected through a single image-guided right frontal craniotomy. The anterior grade II meningioma was noted to be soft and was removed in four anatomically discrete sections with alternating steps of circumferential dissection and suction. The posterior grade I meningioma was noted to be firm and was removed en bloc.
Tumor specimens were fixed in 10% buffered formalin immediately after removal, processed overnight, and subsequently embedded in paraffin. Five μm-thick sections were obtained from the paraffin blocks, and stained using the standard hematoxylin and eosin method.
Frozen tumor tissue was processed with Proteinase K, and DNA extraction was completed using the phenol:chloroform procedure. Frozen tumor tissue was minced with a scalpel, washed once in PBS, pH 7.4, and incubated in a solution containing 100 mM TrisHCl, pH 8.0, 5 mM EDTA, 0.5% SDS and 200 μg/mL Proteinase K (Invitrogen, Grand Island, NY) at 55 °C for 2–3 h or at 37 °C overnight. DNA was extracted by the phenol:chloroform procedure and precipitated with ice cold isopropanol. DNA pellets were air dried, re-suspended in 10 mM TrisHCl, pH 7.4 and 0.1 mM EDTA, aliquoted and stored at −20 °C.
Whole-exome sequencing (WES) of tumor and normal DNA
Capture of the coding portion (exome) of genomic DNA and library preparation for next generation sequencing was done using Roche NimbleGen (Madison, WI) SeqCap EZ Exome + UTR library (64 Mb of coding exons and miRNA regions plus 32 Mb
untranslated regions (UTR)) according to the manufacturer’s instructions. As an input, 1 μg of tumor and matching normal genomic DNA was used. Sequencing was completed on the Illumina HiSeq 2500 system (Illumina, San Diego, CA, USA). Among the six exomes sequenced, the average breadth of coverage was 89% (range 88–90%), and the average depth of coverage was 66X (range 54X-78X).
Raw sequencing data was further processed using an analytical pipeline that included ELAND (Illumina, Inc.) for initial alignment to the reference human genome (GRCh37); Novoalign, v.2.08.02  for local re-alignment; bam2mpg for genotype calling and calculation of the quality score Most Probable Genotype (MPG)  and ANNOVAR for functional annotation of genetic variants [14, 15]. The resulting data was formatted in VarSifter  format for further filtering.
Filtering consisted of removing all non-coding variants and nucleotides whose genotypes were identical in both the tumor and corresponding germline DNA, whose quality score (Most Probable Genotype, MPG) was less than 10 in either tumor or normal DNA, and whose ratio of quality score to depth of coverage was below 0.5 in germline DNA and below 0.4 in tumor DNA. All common variants (variants with minor allele frequency above 0.03 in ClinSeq and 1000 Genomes databases) were also removed. The resulting set was annotated with PolyPhen, SIFT and CADD tools to identify pathogenic mutations.
Sanger validation of mutations identified by WES
PCR primers were designed using Primer3 (v. 0.4.0) online software . PCR amplification was conducted using a 20 μL reaction mixture containing 20–50 ng of genomic DNA, 1x reaction buffer, 1.5 mM MgCl2, 4 dNTPs at 250 μM each, 10 pmole each of forward and reverse primers, and 2 units of ThermoFisher Scientific Taq DNA polymerase (Waltham, MA). PCR products were analyzed on Agilent 2100 BioAnalyzer (Santa Clara, CA) and sent for Sanger sequencing to ACGT, Inc. (Wheeling, IL). Sequencing was done on ABI 3730 DNA Analyzer, the data was processed with GeneMapper v.3.7 software (ThermoFisher Scientific), and the phred quality score for sequenced nucleotides was visualized using CodonCode Aligner v.6.0.2 (CodonCode Corp., Centerville, MA). Sequencing reaction tracks were visualized using FinchTV v.1.5.0 (Geospiza, Inc., Seattle, WA) and CodonCode Aligner software.
Sanger sequencing of NF2
Sanger sequencing of NF2 was conducted by Prevention Genetics (Marshfield, WI), a CLIA-certified DNA testing lab. PCR was used to amplify all NF2 coding exons, as well as ~20 bp flanking intronic or other non-coding sequence. Sequencing was performed separately in both the forward and reverse directions and all differences from the reference sequence were reported.
SNP genotyping was performed using HumanOmniExpressExome-8v1.2 Illumina BeadChip arrays (Illumina, San Diego, CA) per the manufacturer’s instructions. The arrays were read using the iScan platform (Illumina), and visualized with GenomeStudio v.2011.1 software (Illumina). The call rate for all the DNA samples was >99%. Genomic coordinates are per hg19.
Copy-number variation analysis
Copy-number variation (CNV) analysis of all tumor samples was performed using Nexus Copy Number software v.6.1 (BioDiscovery, Inc., Hawthorne, CA). “Allelic imbalance” refers to a locus with B-allele frequency classes other than 0, 0.5 or 1. Allele-Specific Copy number Analysis of Tumors (ASCAT) (v2.1) analysis of the data was performed as described by Van Loo and co-authors .
Spectral karyotyping (SKY)
Metaphase slide preparations were made from cultured meningioma primary cell cultures established from the grade II meningioma and hybridized with commercially available SKY probe set (Applied Spectral Imaging Inc., Carlsbad, CA) according to the manufacturer’s instructions. Mitotic arrest with colcemid (0.015 μg/mL, 2–4 h) (GIBCO, Gaithersburg, MD) was followed by hypotonic treatment (75 mM KCl, 20 min, 37 °C) and fixation in methanol–acetic acid mixture (3:1).
Histopathological analysis of the tumors
Germline and somatic mutations in the NF2 gene
Sequencing of exons and small flanking intronic regions of the NF2 gene from peripheral white blood cell DNA (germline) identified a constitutive mutation in the intronic region, two nucleotides upstream of the 5′-end of exon 13: c.1341-2A > C. This mutation likely disrupts the acceptor site of intron 12, thus affecting RNA splicing, and has been previously reported as pathogenic (Human Gene Mutation Database, CS115647) by Ellis and co-authors . It has not been previously annotated in the ExAC (mean coverage 15x), 1000 Genomes, or ESP datasets .
We noticed that the mutant signal (C) was weaker than that of the reference allele (A) (Fig. 3, see “Germline” panel). A similar A-to-C signal ratio was observed in both the plus and minus strand DNA sequences in both PG and our analyses (data in Fig. 3 is shown for the plus strand only). A-to-C substitution in the sequence 5′-GG[A]GGGCC-3′ converts it to a GC-rich 8 nucleotide-long stretch 5′-GG[C]GGGCC-3′. Such sequences can be more difficult to analyze due to the secondary DNA structure, which may explain the decreased mutant base C signal.
Copy-number SNP-array analysis
Grade II meningioma chromosomal aberrations identified by SNP-array analysis
p36.33 to p34.2
p33 to p31.3
p31.3 to p31.1
p31.1 to p22.3
p22.1 to p13.2
q21.2 to q21.3
q21.3 to q42.13
p15.5 to p15.4
p13.33 to p11.21
Spectral karyotyping (SKY) of grade II meningioma cells
Chromosomal translocations identified by SKY analysis in grade II meningioma fragments 2, 3 and 4
46, XX, t(10;18)
46, XX, t(1;8)
46, XX, t(5;14)
46, XX, t(11;8)
46, XX, t(8;8), t(11;12)
46, XX, t(1;2), t(2;4), t(7;8), t(9;21)
46, XX, t(1;3;22), t(11;21), t(2;18)
46, XX, t(3;13;14), t(17;21)
46, XX, t(3;13;14), t(17;21), inv(12)
Whole-exome sequencing (WES) of tumors and Sanger verification of mutations
After WES data processing and filtering, we identified two potentially damaging somatic mutations in the grade I meningioma, and nine somatic mutations in the four fragments of the grade II meningioma (Additional file 5). Of the nine mutations in the grade II meningioma, two were found in all four fragments.
Of the two mutations in the grade I meningioma, one (EPHB3) mutation was verified by Sanger sequencing, and of the nine mutations in the grade II tumor, two (CAPN5 and ADAMTSL3) were verified by Sanger sequencing. Importantly, the mutations in CAPN5 and ADAMTSL3 were detected by WES in all four fragments of grade II tumor and were verified by Sanger in all four fragments as well, suggesting that these mutations were likely present in the cell undergoing fast clonal expansion.
To our knowledge, this is the first whole-exome sequencing study of NF2-associated grade I and grade II meningiomas. Besides chromosome 22 loss, the genome of the grade I meningioma closely resembled that of a normal diploid cell, while the genome of the grade II tumor contained several chromosomal rearrangements previously observed in meningiomas, including losses in 1p, 2p, 2q, 3p, 3q, 6q, 12p, 14q, 18q, Xp, gain in 1q [21–25], and multiple translocations. Our observations confirm previous findings that inactivation of NF2 is likely to be the primary step in NF2-associated meningioma formation . In addition, we show that both benign and atypical tumors had a low somatic mutation burden. Although limited to a single patient, this data permits speculation that tumor progression to a higher grade likely occurs through multiple chromosomal gains, losses and translocations and to a lesser extent from the accumulation of point mutations and small indels.
Chromosomal translocations leading to the disruption of tumor suppressors or activation of proto-oncogenes are common in many neoplasms [27, 28]. Limited evidence suggests that chromosomal translocations may also be present in meningiomas  and systematic studies addressing this mechanism of tumorigenesis in meningiomas are emerging. We observed numerous chromosomal translocations (both balanced and unbalanced) as well as one case of highly irregular, shattered chromosomes. Interestingly, similar to the observation made by Brastianos et al. , close examination of SNP-array plots of chromosome 1 in the tumor revealed deletion of the 5′-half of the NEGR1 gene (not shown). These findings suggest that structural aberrations might be more frequent than previously believed in NF2-driven familial and sporadic meningiomas, and could represent one of the mechanisms of genetic instability and routes of tumor progression to higher grades.
By analyzing the genomic architecture and somatic mutations in multiple fragments of the grade II tumor, we gained insight into the clonal evolution of this fast growing neoplasm. We observed not only a remarkably uniform pattern of chromosomal gains and losses, but also the consistent presence of the only two potentially pathogenic mutations, in ADAMTSL3 and CAPN5, in all four fragments. These findings indicate that the aberrations were likely present in the initial cell undergoing fast clonal expansion, and that any of these aberrations/mutations could impact tumor progression and accelerate growth rate.
Germline mutations in CAPN5 (Calpain 5), which encodes a calcium-dependent endopeptidase, have been associated with neovascular inflammatory vitreoretinopathy . Though the role of the protein in neoplastic transformation is unclear, a recent study reported association of CAPN5 with promyelocytic leukemia nuclear bodies, which are involved in transcriptional regulation, cell differentiation, apoptosis, and cell senescence . The protein encoded by ADAMTSL3 (A Disintegrin And Metalloproteinase with TromboSpondin Like 3) is involved with extracellular matrix function and to cell–matrix interactions, and is frequently mutated and under-expressed in colorectal cancer . The gene belongs to a large family of proteins associated with microfibrils in the extracellular matrix, thus mediating sequestration of the TGFB superfamily of proteins and affecting wide array of cellular functions such as adhesion, migration, proliferation and angiogenesis [33, 34].
The majority of meningiomas are benign and asymptomatic tumors that require little or no treatment [35, 36]. However, a subset of tumors becomes more clinically aggressive as they evolve toward atypical and anaplastic stages, causing increased morbidity and mortality. Remarkably, the tumors we investigated had the same NF2 germline mutation, the same genetic background, similar chromosome 22 LOH and were residing within a few millimeters from one another in the patient’s brain, yet one remained as a slowly growing asymptomatic grade I meningioma and the other evolved into a fast growing grade II tumor. This observation underscores the importance of stochastic factors in meningioma progression, which are still poorly understood.
We performed an in-depth genomic study of NF2-associated benign and atypical meningiomas. Both tumors had inactivated second copies of NF2 and a low burden of somatic mutations. However, unlike the benign tumor, the atypical meningioma presented with widespread genomic aberrations, implying that chromosomal instability may be a key driving force in tumor progression. In addition, we identified two candidate driver genes, CAPN5 and ADAMTSL3, which could contribute to the elevated growth rate of the grade II meningioma. Future efforts should be focused on understanding the mechanistic links between NF2 deficiency and genomic instability.
- ADAMTSL3 :
A Disintegrin And Metalloproteinase with TromboSpondin Like 3
- AKT1 :
V-akt murine thymoma viral oncogene homolog 1
- BRD8 :
Bromodomain containing 8
- CAPN5 :
- EPHB3 :
EPH (ephrin) receptor B3
- KLF4 :
Kruppel-like factor 4 (gut)
Loss of heterozygosity
Neurofibromatosis type 2
- SMO :
Smoothened, frizzled class receptor
Single nucleotide polymorphism
Transforming growth factor beta
- TRAF7 :
TNF receptor associated factor 7
This study was supported by the Intramural Research Programs of the National Institute of Neurologic Disease and Stroke (NINDS), the Division of Cancer Epidemiology and Genetics of the National Cancer Institute (NCI), and the National Human Genome Research Institute (NHGRI).
This study was supported by funding from the Intramural Research Program of the National Institute of Neurologic Disease and Stroke (NINDS), the Division of Cancer Epidemiology and Genetics of the National Cancer Institute (NCI), and the National Human Genome Research Institute (NHGRI). The roles of each funding body were as follows: NINDS for study design, collection of data, and writing of the manuscript; NCI for study design, collection, analysis, and interpretation of data, and writing of the manuscript; and NHGRI for collection, analysis, and interpretation of data.
Availability of data and materials
RD carried out MRI volumetric image analysis, DNA samples preparation, genomic data analysis, assisted with clinical sample collection and co-drafted the manuscript (with AP); AP analyzed genomic data, participated in the study design (genomics and molecular biology) and co-drafted the manuscript (with RD); ASD analyzed SKY data and prepared the data for publication; EDP carried out the SKY experiments; NAE carried out histological sample preparation; AR-C performed pathological evaluation of tumors; NFH carried out WES data preparation; SCC carried out SNP-array experiments; JCM supervised WES sequencing and WES data preparation; ARA assisted with clinical sample collection; WES was carried out at NISC CSP; JDH supervised all clinical aspects of the study; DRS participated in the study design and critically evaluated the manuscript; AVG provided clinical care to the study’s patient, conceived of the study, carried out the surgery and tumor tissue collection, and critically evaluated the manuscript. All authors have read and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Written consent was obtained for publication of patient-related data in accordance with the NIH#08-N-0044 protocol for patient enrollment and informed consent, which is approved by the National Institute of Neurologic Disease and Stroke Institutional Review Board. A copy of the consent is available for review.
Ethics approval and consent to participate
Ethics approval was obtained in accordance with the NIH#08-N-0044 protocol which is approved by the National Institute of Neurologic Disease and Stroke Institutional Review Board. Written informed consent was obtained from the patient for study of her tissue in accordance with the NIH#08-N-0044 protocol for patient enrollment and informed consent, which is approved by the National Institute of Neurologic Disease and Stroke Institutional Review Board. A copy of the consent is available for review.
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