Cancer Genetics - (2015) -

BRIEF COMMUNICATION

ARID1A and TERT promoter mutations indedifferentiated meningiomaMalak S. Abedalthagafi a,1, Wenya Linda Bi b,1, Parker H. Merrill a,William J. Gibson c, Matthew F. Rose a, Ziming Du a, Joshua M. Francis d,e,Rose Du b, Ian F. Dunn b, Azra H. Ligon f, Rameen Beroukhim c,d,e,Sandro Santagata a,c,*aDivision of Neuropathology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA,USA; bDepartment of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA;cDepartment of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA; dDepartment of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; eBroad Institute of Massachusetts Institute of Technology and Harvard,Cambridge, MA, USA; fClinical Cytogenetics Laboratory, Center for Advanced Molecular Diagnostics, Department ofPathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Received Febru

2015; accepted Ma

* Corresponding

E-mail address:1 Both authors c

considered first aut

2210-7762/$ - see

http://dx.doi.org/10

Unlike patients with World Health Organization (WHO) grade I meningiomas, which are consid-

ered benign, patients with WHO grade III meningiomas have very high mortality rates. The

principles underlying tumor progression in meningioma are largely unknown, yet a detailed un-

derstanding of these mechanisms will be required for effective management of patients with

these high grade lethal tumors. We present a case of an intraventricular meningioma that at

first presentation displayed remarkable morphologic heterogeneitydcomposed of distinct re-

gions independently fulfilling histopathologic criteria for WHO grade I, II, and III designations.

The lowest grade regions had classic meningothelial features, while the highest grade regions

were markedly dedifferentiated. Whereas progression in meningiomas is generally observed

during recurrence following radiation and systemic medical therapies, the current case offers

us a snapshot of histologic progression and intratumoral heterogeneity in a native pretreatment

context. Using whole exome sequencing and high resolution array-based comparative

genomic hybridization, we observed marked genetic heterogeneity between the various areas.

Notably, in the higher grade regions we found increased aneuploidy with progressive loss of

heterozygosity, the emergence of mutations in the TERT promoter, and compromise of

ARID1A. These findings provide new insights into intratumoral heterogeneity in the evolution

of malignant phenotypes in anaplastic meningiomas and potential pathways of malignant

progression.

Keywords Meningioma, anaplastic, intratumoral heterogeneity, SWI/SNF complex, chromatin

remodeling

ª 2015 Elsevier Inc. All rights reserved.

Meningiomas are the most common primary tumors of thecentral nervous system, with 25e30% following an aggres-sive course with rapid growth, invasion into adjacent

ary 5, 2015; received in revised form March 5,

rch 6, 2015.

author.

ssantagata@partners.org

ontributed equally to this work and both are

hor.

front matter ª 2015 Elsevier Inc. All rights reserved.

.1016/j.cancergen.2015.03.005

structures, and rapid recurrence even after radical resectionand adjuvant treatment. These meningiomas are classifiedas World Health Organization (WHO) grade II or III and areassociated with premature mortality (1). Despite anemerging understanding of the genetic changes underlyingmeningioma formation (2e10), the factors that drive malig-nant progression in meningioma remain poorly understood(11e13). We encountered a case of a unique untreatedintraventricular meningioma, which allowed us to investigatemolecular factors associated with tumor progression anddedifferentiation.

2 M.S. Abedalthagafi et al.

Materials and methods

Pathologic examination

Histopathologic diagnosis and tumor purity of greater than80% was confirmed by review of the H&E stained sections bytwo neuropathologists (S.S., M.A.). This study was approvedby the human subject institutional review board of the Dana-Farber/Brigham and Women’s Cancer Center.

Immunohistochemistry and RNAscope

Immunohistochemical analysis was performed usingcommercially available antibodies following standard auto-mated and manual protocols on formalin-fixed, paraffin-embedded (FFPE) tissue. The slides were incubated withantibodies for MIB-1 (Ki-67, Dako, catalogue no. M7240)(Agilent Technologies, Santa Clara, CA), epithelial membraneantigen (EMA, Dako, catalogue no. M0613 clone: E29) (Agi-lent Technologies), and ARID1A (monoclonal ARID1A/BAF250a, catalogue no. sc-32761X) (Santa Cruz Biotech-nology, Santa Cruz, CA) and developed using 3,30-dia-minobenzidine (DAB) as the chromagen (Sigma-Aldrich, St.Louis, MO), before being counterstained with hematoxylin.Appropriate positive and negative controls were usedthroughout. In situ hybridization of TERT mRNA transcriptswas performed using the RNAscope assay with Probe-Hs-TERT (catalogue no. 605511) (Advanced Cell Diagnostics,Hayward, CA), following themanufacturer’s protocols (10,14).

Genomic analysis

Array-based comparative genomic hybridization (aCGH) wasperformed using a stock 1�1M Agilent SurePrint G3 humanCGH microarray chip in a Clinical Laboratory ImprovementAmendments (CLIA)ecertified laboratory. A minimum of1.3 mg DNA, corresponding to approximately 6�1-mmpunches from FFPE tissue blocks, was obtained for eachspecimen. Genomic DNA isolated from FFPE blocks washybridized with a commercial reference DNA sample repre-senting a pool of individuals with normal karyotypes (Prom-ega, Madison, WI). The array platform contains 963,029probes spaced with a 2.1-kb overall median probe spacingand a 1.8-kb probe spacing in RefSeq genes across thehuman genome. A genomic imbalance is reported when aminimum of eight consecutive probes, corresponding toapproximately 14e16 kb, show an average log2 ratio aboveþ0.25 or below �0.5.

Whole exome sequencing was performed on DNA iso-lated from each histopathologically distinct area and onmatched germline DNA to a mean depth of 80X, and analysisproceeded as previously described (8,15). Raw sequencingdata were processed using the Picard tools pipeline and theGenome Analysis Toolkit (GATK) (16,17). Mutation analysisfor single nucleotide variants (SNVs) was performed usingMuTect v1.1.4 (18); indel calling was performed using theGATK SomaticIndelDetector tool; and SNVs and indels wereannotated using Oncotator. To analyze somatic copy numberalterations from whole exome data, we used the ReCapSegalgorithm, which assesses homologue-specific copy ratios

from segmental estimates of multipoint allelic copy ratios atheterozygous loci incorporating the statistical phasing soft-ware (BEAGLE) and population haplotype panels (HAP-MAP3) (19e21).

Results

A 48-year-old woman presented with 5 days of headacheand gait instability, prompting imaging that revealed a6.9�4.7�5-cm heterogeneously contrast-enhancing mass,centered within the atrium of the right lateral ventricle,causing obstructive hydrocephalus (Figures 1A and 1B).A right parietal craniotomy was performed with gross totalresection of the tumor (Figure 1C). The patient’s symptomsimproved postoperatively and she remains alive 2 yearsfollowing the resection.

The histopathologic analysis was notable for the concur-rent presence of regions displaying classic features of lowgrade meningothelial meningioma (WHO grade I), others withfeatures of atypical WHO grade II meningioma, and areasharboring cells with a pronounced increase in the nucleocy-toplasmic ratio, frequent mitosis, and loss of discernablefeatures of meningothelial differentiationdhallmarks ofdedifferentiation in a WHO grade III anaplastic meningioma(Figure 1D). Consistent with this histologic progression, theMIB-1 proliferation index ranged from less than 1% to morethan 90% in corresponding grade I versus grade III regions(Figure 1D). Immunohistochemical results for epithelialmembrane antigen (EMA), a marker of meningothelial differ-entiation, also showed EMA expression in the low grade re-gions that was entirely lost in the highest grade regions,consistent with the morphologic dedifferentiation (Figure 1D).To better understand the molecular underpinnings of thehistologic heterogeneity within this tumor, we analyzed DNAextracted from core punches from each of these three regions,using high resolution aCGH, Sanger sequencing of the TERTpromoter, and whole exome sequencing (Figure 2).

The aCGH data showed partial single-copy loss of chro-mosomes 1p, 3q, 6q, 7p, 11q, 18q (monosomy 18), and 22q(monosomy 22) in all three areas (Figure 2A). The atypicaland anaplastic regions both harbored an expanded loss ofchromosome 11q; additional unique losses of 4 and 11p; andregions of 5, 15q, and 17q that were not detected in thegrade I areas. Expansion of 5p and 9q losses was distinct tothe grade III area, as were gains on chromosome 8 and 10q.Copy number analysis of the whole exome sequencing datashowed a similar pattern of losses and further revealedprogressive loss of heterozygosity across the three regionsand evidence for subclonal chromosomal losses in area 2that become clonal in area 3 (Figure 2B).

Because whole exome sequencing does not cover thepromoter region of the TERT gene, which has been reportedto be mutated in meningiomas with histologic progression(2), we performed Sanger sequencing of this region. Wefound wild-type sequences in the grade I area but the c.228C>T mutation in both the grade II and III areas. UsingRNAscope in situ hybridization (14), we found that theexpression of TERT mRNA is low in grade I areas andsharply increased in higher-grade areas (Figure 2C). Thewhole exome sequencing data revealed a low frequency ofother mutations in the three samples, with the notable

Figure 1 Intratumoral heterogeneity within a high grade meningioma. (A) T1-weighted gadolinium-enhanced axial MRI image

demonstrating a heterogeneously appearing contrast-avid tumor within the atrium of the right lateral ventricle, with (B) significant

peritumoral edema and associated hydrocephalus on the T2-weighted axial MRI image. (C) Postoperative T1-weighted axial MRI

image demonstrating complete resection. (D) A section of tumor, with three distinct-appearing histologic regions (designated areas

1e3). Immunohistochemical results for epithelial membrane antigen (EMA) and MIB-1 (Ki-67). Scale bar, 10 mm.

Meningioma intratumoral heterogeneity 3

presence of an ARID1A frameshift deletion (p.LHH2017fs) inthe grade II and III areas (Supplementary Table 1). Coupledwith single-copy loss of chromosome 1p, the frameshiftwould lead to a sharp decrease in ARID1A protein levels.Indeed, ARID1A levels are markedly reduced in the highgrade areas on IHC (Figure 2D).

Discussion

The mutation and copy number data reveal a concentric,rather than partially overlapping, pattern of alterations; nearlyall mutations and copy number alterations in the areas with

Figure 2 Genetic heterogeneity within a high grade meningioma. (A) High resolution aCGH was performed on DNA extracted from

core punches taken from area 1 (WHO grade (I), area 2 (WHO grade II), and area 3 (WHO grade III), using 1�1M Agilent SurePrint G3

human CGH microarray chip (3,31). In the results shown here, red indicates gain and blue indicates loss. (B) Results of copy number

analysis from exome sequencing data of the three areas are shown as allelic copy ratios. Red indicates gain and blue indicates loss.

(C) Results of in situ hybridization of TERT mRNA transcripts using a TERT-specific RNAscope probe in FFPE sections of the me-

ningioma samples (10,14). (D) Immunohistochemical results for ARID1A. (E) A schematic representation of clonal evolution leading to

dedifferentiation and intratumoral heterogeneity. Scale bar, 10 mm.

4 M.S. Abedalthagafi et al.

atypical histopathology were also observed in the samplewith higher grade anaplastic features. This finding suggests acommon clonal origin to the distinct areas. Notably, thistumor was newly diagnosed, without prior surgical or chemo-radiation treatment. It is interesting to speculate that the denovo molecular heterogeneity observed in our case reflects aset of clonal sweeps where each ensuing clone consists of asubclone of the previous dominant clone (Figure 2E). Onemight imagine that therapeutic interventions such aschemotherapy and radiation may precipitate a differentpattern in which multiple surviving subclones compete fordominance.

The shared pattern of single-copy loss, involving chro-mosomes 1p, 3q, 6q, 18q, and 22q, is consistent with pre-viously reported cytogenetic changes in high grademeningiomas (22). We found these alterations even in theareas with grade I histologic features, which had a low pro-liferative index and intact EMA expression, suggesting thatadditional alterations may be needed for histologic progres-sion. Such cytogenetic aberrations are uncommon in

meningioma with grade I histology, and the presence ofthese aberrations in our case suggests that the mechanismsof chromosomal instability that drive accumulation of therecurrent genomic aberrations in meningioma were presentin these well-differentiated areas as well. Noncontiguouslosses on 15q and 17q and the unique whole arm loss of 9qobserved only in the anaplastic region contrast with previ-ously observed gains of 9q, 15q, and 17q in some high grademeningiomas and reflect the current incomplete under-standing of the specific oncogenic drivers within these broadregions (13,22). No CDKN2A (9p21.3) deletions, a frequentfinding among aggressive meningiomas (13,23), wereobserved.

The differences in mutational profiles between the lowgrade (grade I) and high grade (grades II and III) areas areintriguing. The NF2 gene was intact in all three samples, withmore than 100X coverage at each site of all exons aftermanual review. Although it was reported in a prior study thatNF2-mutated meningiomas may also exhibit greater chro-mosomal instability than NF2 wild type counterparts, our

Meningioma intratumoral heterogeneity 5

case showed genomic progression with apparently intactNF2 (24). Mutations in the TERT promoter and in ARID1Awere most notable. Telomerase activation is well recognizedas a feature of malignant meningiomas (25), with a recentreport that TERT promoter mutations are specific to menin-giomas that recur displaying histologic progression (2). Theobservation of differential TERT expression within regions ofthe same tumor that had not been treated further supportsthe role of increased TERT expression in the malignantprogression of meningioma. Moreover, sequencing studieshave revealed that the genes encoding subunits ofmammalian SWI/SNF complexes are mutated in over 20% ofall human cancers (26e28). ARID1A is important in ensuringcorrect lineage progression and in limiting self-renewal (29).It is interesting to propose that the concomitant activation ofTERT and the compromise of the SWI/SNF complex or otherchromatin remodeling complexes through a range of geneticor epigenetic events (30) may synergize in allowing menin-gioma cells to develop high grade malignant phenotypes (12)and dedifferentiate.

Acknowledgments

We are grateful to Marian Slaney and Sebastian Valentinofor assistance with histology, Terri Woo for assistance withimmunohistochemistry, and Marc L. Listewnik for cytogeneticdata support for technical assistance. The work is supportedby 35-1041 grant from King Abdulaziz City for Science andTechnology (KACST) no. 110968, Saudi Arabia (M.A.A.),T32GM007753 from the National Institute of General MedicalSciences (W.J.G), the Brain Science Foundation (S.S.), andthe Jared Branfman Sunflowers for Life Fund for PediatricBrain and Spinal Cancer Research (S.S.).

Supplementary data

Supplementary data related to this article can be found onlineat http://dx.doi.org/10.1016/j.cancergen.2015.03.005.

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