Victoria E. Clark

Genomic Analysis of Non-NF2 Meningiomas Reveals Mutations in TRAF7, KLF4, AKT1, and SMO

Genetic Clues to Meningioma Meningiomas are the most common primary brain tumors in adults. Located within the layer of tissue covering the brain, these tumors are usually slow-growing and benign but can cause serious neurological complications. About half of these tumors have mutations in the neurofibromin 2 gene (NF2). To identify other genes that contribute to meningioma pathogenesis, Clark et al. (p. 1077, published online 24 January) performed genome sequence analysis on 300 tumors. Meningiomas fell into two general classes: benign tumors located at the skull base—which tend to harbor mutations in the TRAF7, KLF4, AKT1, and SMO genes—and higher-grade tumors located in the cerebral and cerebellar hemispheres harbor mutations in NF2. The mutational profiles of meningiomas, a common type of brain tumor, correlate with their anatomical location and clinical status. We report genomic analysis of 300 meningiomas, the most common primary brain tumors, leading to the discovery of mutations in TRAF7, a proapoptotic E3 ubiquitin ligase, in nearly one-fourth of all meningiomas. Mutations in TRAF7 commonly occurred with a recurrent mutation (K409Q) in KLF4, a transcription factor known for its role in inducing pluripotency, or with AKT1E17K, a mutation known to activate the PI3K pathway. SMO mutations, which activate Hedgehog signaling, were identified in ~5% of non-NF2 mutant meningiomas. These non-NF2 meningiomas were clinically distinctive—nearly always benign, with chromosomal stability, and originating from the medial skull base. In contrast, meningiomas with mutant NF2 and/or chromosome 22 loss were more likely to be atypical, showing genomic instability, and localizing to the cerebral and cerebellar hemispheres. Collectively, these findings identify distinct meningioma subtypes, suggesting avenues for targeted therapeutics.

Integrated genomic characterization of IDH1-mutant glioma malignant progression

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1), we studied paired tumor samples from 41 patients, comparing higher-grade, progressed samples to their lower-grade counterparts. Integrated genomic analyses, including whole-exome sequencing and copy number, gene expression and DNA methylation profiling, demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions, as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach.

Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene.

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole‐exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis‐related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well‐established cancer gene lysine (K)‐specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome‐sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D‐mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.

Optimal Surgical Management of Well-Differentiated Thyroid Cancer Arising in Struma Ovarii: A Series of 4 Patients and a Review of 53 Reported Cases

BACKGROUND Well-differentiated thyroid cancer arising in struma ovarii is rare. The optimal management of this entity remains undefined. Unilateral cystectomy, unilateral salpingo-oophorectomy (USO), or total abdominal hysterectomy with bilateral salpingo-oophorectomy (TAH/BSO), in addition to total thyroidectomy and radioactive iodine (RAI) ablation, have been employed by various groups. We hypothesized that in patients with thyroid cancer arising within struma ovarii, pelvic surgery alone would be sufficient, provided there is no evidence of gross extra-ovarian extension. METHODS We review a series of four patients from a single institution and 53 cases from the literature, comparing the extent of treatment and outcomes. Our literature review focused on low-risk patients with struma ovarii confined to the ovary, without evidence of gross extra-ovarian spread or distant metastases. Cumulative recurrence rate was determined by using the Kaplan-Meier method. RESULTS We report the treatment of four patients with well-differentiated thyroid cancer arising within struma ovarii. Patients underwent USO, BSO, or TAH/BSO. One patient underwent prophylactic total thyroidectomy in anticipation of RAI treatment, and was found to have a synchronous papillary thyroid carcinoma. All patients clinically remain without evidence of disease at a median follow-up of 9 (range 0.8-13) years. Treatment strategies in 53 cases from a review of the literature varied. The pooled cumulative recurrence rate of 57 cases with struma ovarii confined to the ovary was 7.5% at 25 years. CONCLUSIONS Thyroid cancer arising in struma ovarii is rare. Controversy exists regarding the extent of pelvic resection and management of the thyroid gland. In our series of four patients, all patients are alive without evidence of disease, and the 25-year recurrence rate of 57 cases was low (7.5%), despite a variety of approaches to surgical resection and adjuvant treatment. Extensive pelvic surgery and prophylactic total thyroidectomy to facilitate RAI therapy may be reserved for patients with gross extra-ovarian extension or distant metastases.

Integrated genomic analyses of de novo pathways underlying atypical meningiomas

Meningiomas are mostly benign brain tumours, with a potential for becoming atypical or malignant. On the basis of comprehensive genomic, transcriptomic and epigenomic analyses, we compared benign meningiomas to atypical ones. Here, we show that the majority of primary (de novo) atypical meningiomas display loss of NF2, which co-occurs either with genomic instability or recurrent SMARCB1 mutations. These tumours harbour increased H3K27me3 signal and a hypermethylated phenotype, mainly occupying the polycomb repressive complex 2 (PRC2) binding sites in human embryonic stem cells, thereby phenocopying a more primitive cellular state. Consistent with this observation, atypical meningiomas exhibit upregulation of EZH2, the catalytic subunit of the PRC2 complex, as well as the E2F2 and FOXM1 transcriptional networks. Importantly, these primary atypical meningiomas do not harbour TERT promoter mutations, which have been reported in atypical tumours that progressed from benign ones. Our results establish the genomic landscape of primary atypical meningiomas and potential therapeutic targets.

Temporal requirement of the alternative-splicing factor Sfrs1 for the survival of retinal neurons.

Alternative splicing is the primary mechanism by which a limited number of protein-coding genes can generate proteome diversity. We have investigated the role of the alternative-splicing factor Sfrs1, an arginine/serine-rich (SR) protein family member, during mouse retinal development. Loss of Sfrs1 function during embryonic retinal development had a profound effect, leading to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early to mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. By contrast, Sfrs1 was not required for the survival of the neurons generated later, including later-born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated alternative splicing for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated.

Absence of KMT2D/MLL2 mutations in abdominal paraganglioma

Dear Editors, Pheochromocytomas (PCCs) and abdominal paragangliomas (PGLs) are rare, chromaffin cell-derived tumours of the adrenal medulla and extra-adrenal paraganglia, respectively. Approximately 40% of these tumours carry a somatic or constitutional mutation in one or several susceptibility gene(s), and based on gene expression studies, mutations in these genes have been found to alter either the kinase signalling pathway or the pseudo-hypoxia response pathway. Although the majority of PCCs and PGLs are benign, a significant subset of PGLs is malignant. Recently, heterozygous missense lysine (K)-specific methyltransferase 2D (KMT2D) gene mutations were demonstrated in 14% (14/99) of investigated PCCs. KMT2D is also known as mixed-linage leukaemia 2 (MLL2), a gene containing 54 exons, encoding a histone methyltransferase that is known to convey methylation of histone-3 lysine-4 which in turn regulates DNA accessibility. As compared to normal adrenal medulla, PCCs expressed significantly higher levels of KMT2D which also was associated with increased levels of histone H3 tri-methyl K4 (H3K4me3). Moreover, functional analyses suggested an increase in cellular motility in KMT2D-transfected PC12 cells and were also found to exhibit a transcriptional profile enriched for genes within the extracellular matrix–receptor interaction pathways and the TGF-beta signalling network, suggesting a potential role for KMT2D regulation in mediating an enhanced migratory phenotype of the cells. Given these findings and the notion that PGLs are more often malignant and closely related to PCCs, we sought to investigate the potential role of KMT2D in PGLs. A total of 13 PGLs were collected at the Karolinska University Hospital, Stockholm, Sweden, and the patient sample acquisition and analysis were approved by the local ethical review board. Detailed genetic and clinical information is available in the Supplementary Table 1. The cohort comprises 9 malignant and 4 benign PGLs formerly screened for somatic and constitutional mutations in 14 known PCC/PGL predisposition genes, namely EGLN1, EPAS1, KIF1Bb, MAX, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, VHL and also MEN1 which has an disputed involvement. In our material, 8 of 13 cases displayed mutation in these genes (constitutional SDHB n = 4, somatic EPAS1 n = 3, somatic VHL n = 1). All cases were subject to mutational analysis using either whole-exome sequencing (3 cases) or KMT2D targeted next-generation sequencing using molecular inversion probes (MIP; 10 cases) as previously described. In short, targeted exome capture and whole-exome sequencing of genomic DNA from 3 PGLs and their constitutional tissues were performed using the NimbleGen 2.1M exome array followed by sequencing on the Illumina HiSeq 2000 platform. The mutations were called somatic based on the differences in reference and nonreference read proportions between tumours and their corresponding normal sample, and the somatic variant calls were calculated using Fisher’s exact test (Table S2). Known constitutional variants in the 1000 Genomes database were excluded from further analysis. In total, 13 nonsynonymous somatic mutations were identified in three PGLs, and the previously known mutations in PGL susceptibility genes were verified (one constitutional SDHB mutation in case 3, two somatic EPAS1 mutations in case 6 and one VHL mutation in case 8). All somatic variants are detailed in Supplementary Table 2 together with technical data regarding tumour and normal tissue genomic coverage. All 13 PGLs were found to exhibit wild-type KMT2D sequences. Eleven PGLs as well as 10 normal adrenal reference samples underwent KMT2D gene expression analysis using qRT-PCR with a KMT2D gene assay (FAM-MGB dyed, assay ID: Hs00231606_m1) as previously described in detail. As compared to the normal adrenals, PGLs expressed significantly higher levels of KMT2D (Mann–Whitney U-test, P = 0 022, Fig. 1a). There were no correlations seen between KMT2D expression and genotype, gender, biochemical profile or malignancy status. All PGLs were analysed for KMT2D gene copy number alterations as previously described. A majority was found diploid (n = 11; 85%), and two PGLs were found with copy number gain (3 gene copies). No losses at the KMT2D gene locus were found. The two cases with KMT2D gain demonstrated similar KMT2D mRNA levels as compared to copy number neutral tumours. Immunohistochemistry was performed as previously described. In short, goat polyclonal anti-MLL2 (I-18, sc-68671, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200 and rabbit polyclonal anti-histone H3 tri-methyl K4 (H3K4me3, ab8580, Abcam, Cambridge, UK) diluted 1:500 were applied on formalin-fixated paraffin-embedded material from 11 cases to detect KMT2D and H3K4me3 methylation, respectively. All cases stained either positive (>75% positive tumour nuclei, n = 7) or partially positive (50–75% positive tumour nuclei, n = 4) for nuclear KMT2D. For nuclear H3K4me3 levels, 8 cases were positive and 3 cases partially positive. These results are presented in Fig. 1b and detailed in Table S1. There was a significant positive correlation between nuclear KMT2D and nuclear H3K4me3 immunoreactivity (Fisher’s exact test, P < 0 0001). We conclude that KMT2D mutations are rare in PGLs. As PCCs display a KMT2D mutational frequency of 14%, the lack of mutations among the PGLs studied might possibly reflect the diverse genetic aetiologies of PCCs and PGLs. However, the relatively small sample size is a limitation of the current study. When studying preceding WES studies of PGL, Clinical Endocrinology (2016) 84, 632–637

Author Correction: Integrated genomic analyses of de novo pathways underlying atypical meningiomas

This corrects the article DOI: 10.1038/ncomms14433.

Genomic Landscape of Brain Tumors

The use of next-generation sequencing technologies has transformed our understanding of the molecular pathways driving the formation of central nervous system tumors. Tumor classification, once based primarily on observable histopathologic findings, is increasingly based on molecular characteristics, including driver somatic gene mutations, genomic stability, epigenetic changes, and gene expression profiles. These markers define clinically relevant entities that relate to anatomic location, response to therapy, and overall survival. The major categories of both adult and pediatric brain tumors are addressed here with a focus on the identifying genomic findings, their relationship to molecular pathways, and their implications for diagnosis, prognosis, and, ultimately, treatment.