ARID1A and TERT Promoter Mutations in Dedifferentiated Meningioma Malak S

Cancer Genetics - (2015) -

BRIEF COMMUNICATION ARID1A and TERT promoter mutations in dedifferentiated meningioma Malak 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,* a Division of Neuropathology, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; b Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; c Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA; d Department of Medical Oncology, Dana- Farber Cancer Institute, Boston, MA, USA; e Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA; f Clinical Cytogenetics Laboratory, Center for Advanced Molecular Diagnostics, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Unlike patients with World Health Organization (WHO) grade I meningiomas, which are considered 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 understanding 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 regions 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 the structures, and rapid recurrence even after radical resection central nervous system, with 25e30% following an aggres- and adjuvant treatment. These meningiomas are classiﬁed sive course with rapid growth, invasion into adjacent as World Health Organization (WHO) grade II or III and are associated with premature mortality (1). Despite an emerging understanding of the genetic changes underlying meningioma formation (2e10), the factors that drive malig- Received February 5, 2015; received in revised form March 5, nant progression in meningioma remain poorly understood 2015; accepted March 6, 2015. e * Corresponding author. (11 13). We encountered a case of a unique untreated E-mail address: [email protected] intraventricular meningioma, which allowed us to investigate 1 Both authors contributed equally to this work and both are molecular factors associated with tumor progression and considered ﬁrst author. dedifferentiation.

Materials and methods from segmental estimates of multipoint allelic copy ratios at heterozygous loci incorporating the statistical phasing soft- Pathologic examination ware (BEAGLE) and population haplotype panels (HAP- MAP3) (19e21). Histopathologic diagnosis and tumor purity of greater than 80% was confirmed by review of the H&E stained sections by Results two neuropathologists (S.S., M.A.). This study was approved by the human subject institutional review board of the Dana- A 48-year-old woman presented with 5 days of headache Farber/Brigham and Women’s Cancer Center. and gait instability, prompting imaging that revealed a 6.94.75-cm heterogeneously contrast-enhancing mass, Immunohistochemistry and RNAscope centered within the atrium of the right lateral ventricle, causing obstructive hydrocephalus (Figures 1A and 1B). Immunohistochemical analysis was performed using A right parietal craniotomy was performed with gross total commercially available antibodies following standard auto- resection of the tumor (Figure 1C). The patient’s symptoms mated and manual protocols on formalin-fixed, paraffin- improved postoperatively and she remains alive 2 years embedded (FFPE) tissue. The slides were incubated with following the resection. antibodies for MIB-1 (Ki-67, Dako, catalogue no. M7240) The histopathologic analysis was notable for the concur- (Agilent Technologies, Santa Clara, CA), epithelial membrane rent presence of regions displaying classic features of low antigen (EMA, Dako, catalogue no. M0613 clone: E29) (Agi- grade meningothelial meningioma (WHO grade I), others with lent Technologies), and ARID1A (monoclonal ARID1A/ features of atypical WHO grade II meningioma, and areas BAF250a, catalogue no. sc-32761X) (Santa Cruz Biotech- harboring cells with a pronounced increase in the nucleocy- nology, Santa Cruz, CA) and developed using 3,30-dia- toplasmic ratio, frequent mitosis, and loss of discernable d minobenzidine (DAB) as the chromagen (Sigma-Aldrich, St. features of meningothelial differentiation hallmarks of Louis, MO), before being counterstained with hematoxylin. dedifferentiation in a WHO grade III anaplastic meningioma Appropriate positive and negative controls were used (Figure 1D). Consistent with this histologic progression, the throughout. In situ hybridization of TERT mRNA transcripts MIB-1 proliferation index ranged from less than 1% to more was performed using the RNAscope assay with Probe-Hs- than 90% in corresponding grade I versus grade III regions TERT (catalogue no. 605511) (Advanced Cell Diagnostics, (Figure 1D). Immunohistochemical results for epithelial Hayward, CA), following the manufacturer’s protocols (10,14). membrane antigen (EMA), a marker of meningothelial differentiation, also showed EMA expression in the low grade regions that was entirely lost in the highest grade regions, Genomic analysis consistent with the morphologic dedifferentiation (Figure 1D). To better understand the molecular underpinnings of the Array-based comparative genomic hybridization (aCGH) was histologic heterogeneity within this tumor, we analyzed DNA performed using a stock 11M Agilent SurePrint G3 human extracted from core punches from each of these three regions, CGH microarray chip in a Clinical Laboratory Improvement using high resolution aCGH, Sanger sequencing of the TERT Amendments (CLIA)ecertified laboratory. A minimum of promoter, and whole exome sequencing (Figure 2). 1.3 mg DNA, corresponding to approximately 61-mm The aCGH data showed partial single-copy loss of chro- punches from FFPE tissue blocks, was obtained for each mosomes 1p, 3q, 6q, 7p, 11q, 18q (monosomy 18), and 22q specimen. Genomic DNA isolated from FFPE blocks was (monosomy 22) in all three areas (Figure 2A). The atypical hybridized with a commercial reference DNA sample repre- and anaplastic regions both harbored an expanded loss of senting a pool of individuals with normal karyotypes (Prom- chromosome 11q; additional unique losses of 4 and 11p; and ega, Madison, WI). The array platform contains 963,029 regions of 5, 15q, and 17q that were not detected in the probes spaced with a 2.1-kb overall median probe spacing grade I areas. Expansion of 5p and 9q losses was distinct to and a 1.8-kb probe spacing in RefSeq genes across the the grade III area, as were gains on chromosome 8 and 10q. human genome. A genomic imbalance is reported when a Copy number analysis of the whole exome sequencing data minimum of eight consecutive probes, corresponding to showed a similar pattern of losses and further revealed approximately 14e16 kb, show an average log2 ratio above progressive loss of heterozygosity across the three regions þ0.25 or below 0.5. and evidence for subclonal chromosomal losses in area 2 Whole exome sequencing was performed on DNA iso- that become clonal in area 3 (Figure 2B). lated from each histopathologically distinct area and on Because whole exome sequencing does not cover the matched germline DNA to a mean depth of 80X, and analysis promoter region of the TERT gene, which has been reported proceeded as previously described (8,15). Raw sequencing to be mutated in meningiomas with histologic progression data were processed using the Picard tools pipeline and the (2), we performed Sanger sequencing of this region. We Genome Analysis Toolkit (GATK) (16,17). Mutation analysis found wild-type sequences in the grade I area but the c.228 for single nucleotide variants (SNVs) was performed using C>T mutation in both the grade II and III areas. Using MuTect v1.1.4 (18); indel calling was performed using the RNAscope in situ hybridization (14), we found that the GATK SomaticIndelDetector tool; and SNVs and indels were expression of TERT mRNA is low in grade I areas and annotated using Oncotator. To analyze somatic copy number sharply increased in higher-grade areas (Figure 2C). The alterations from whole exome data, we used the ReCapSeg whole exome sequencing data revealed a low frequency of algorithm, which assesses homologue-specific copy ratios other mutations in the three samples, with the notable Meningioma intratumoral heterogeneity 3

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. presence of an ARID1A frameshift deletion (p.LHH2017fs) in Discussion the grade II and III areas (Supplementary Table 1). Coupled with single-copy loss of chromosome 1p, the frameshift The mutation and copy number data reveal a concentric, would lead to a sharp decrease in ARID1A protein levels. rather than partially overlapping, pattern of alterations; nearly Indeed, ARID1A levels are markedly reduced in the high all mutations and copy number alterations in the areas with grade areas on IHC (Figure 2D). 4 M.S. Abedalthagaﬁ et al.

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 11M 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 meningioma 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. atypical histopathology were also observed in the sample meningioma with grade I histology, and the presence of with higher grade anaplastic features. This finding suggests a these aberrations in our case suggests that the mechanisms common clonal origin to the distinct areas. Notably, this of chromosomal instability that drive accumulation of the tumor was newly diagnosed, without prior surgical or chemo- recurrent genomic aberrations in meningioma were present radiation treatment. It is interesting to speculate that the de in these well-differentiated areas as well. Noncontiguous novo molecular heterogeneity observed in our case reflects a losses on 15q and 17q and the unique whole arm loss of 9q set of clonal sweeps where each ensuing clone consists of a observed only in the anaplastic region contrast with previ- subclone of the previous dominant clone (Figure 2E). One ously observed gains of 9q, 15q, and 17q in some high grade might imagine that therapeutic interventions such as meningiomas and reflect the current incomplete under- chemotherapy and radiation may precipitate a different standing of the specific oncogenic drivers within these broad pattern in which multiple surviving subclones compete for regions (13,22).NoCDKN2A (9p21.3) deletions, a frequent dominance. finding among aggressive meningiomas (13,23), were The shared pattern of single-copy loss, involving chro- observed. mosomes 1p, 3q, 6q, 18q, and 22q, is consistent with pre- The differences in mutational profiles between the low viously reported cytogenetic changes in high grade grade (grade I) and high grade (grades II and III) areas are meningiomas (22). We found these alterations even in the intriguing. The NF2 gene was intact in all three samples, with areas with grade I histologic features, which had a low pro- more than 100X coverage at each site of all exons after liferative index and intact EMA expression, suggesting that manual review. Although it was reported in a prior study that additional alterations may be needed for histologic progres- NF2-mutated meningiomas may also exhibit greater chro- sion. Such cytogenetic aberrations are uncommon in mosomal instability than NF2 wild type counterparts, our Meningioma intratumoral heterogeneity 5 case showed genomic progression with apparently intact 7. Clark VE, Erson-Omay EZ, Serin A, et al. Genomic analysis of NF2 (24). Mutations in the TERT promoter and in ARID1A non-NF2 meningiomas reveals mutations in TRAF7, KLF4, were most notable. Telomerase activation is well recognized AKT1, and SMO. Science 2013;339:1077e1080. as a feature of malignant meningiomas (25), with a recent 8. Brastianos PK, Horowitz PM, Santagata S, et al. Genomic report that TERT promoter mutations are specific to menin- sequencing of meningiomas identifies oncogenic SMO and AKT1 mutations. Nat Genet 2013;45:285e289. giomas that recur displaying histologic progression (2). The 9. Aavikko M, Li SP, Saarinen S, et al. Loss of SUFU function in observation of differential TERT expression within regions of familial multiple meningioma. Am J Hum Genet 2012;91: the same tumor that had not been treated further supports 520e526. the role of increased TERT expression in the malignant 10. Du Z, Abedalthagafi M, Aizer AA, et al. Increased expression of progression of meningioma. Moreover, sequencing studies the immune modulatory molecule PD-L1 (CD274) in anaplastic have revealed that the genes encoding subunits of meningioma. Oncotarget 2015;6:4704e4716. mammalian SWI/SNF complexes are mutated in over 20% of 11. Yang HW, Kim TM, Song SS, et al. Alternative splicing of all human cancers (26e28). 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