Verena Dreschmann

Supratentorial ependymomas of childhood carry C11orf95-RELA fusions leading to pathological activation of the NF-κB signaling pathway.

Methods). This fusion was not detectable in a large series of infratentorial and spinal ependymomas including myxopapillary ependymomas. It was also not detectable in ependymoblastomas, medulloblastomas or pineoblastomas. 14/19 samples from supratentorial ependymomas expressed this novel fusion transcript resulting in an N-terminal part of C11orf95 encoding 212aa of the hypothetical 678aa protein fused to relA that is thereby uncoupled from its normal upstream regulators. The c-terminal relA part of the putative fusion protein contains almost the full relA sequence; only the first three amino acids (encoded by exon 2) are deleted in-frame. The occurrence of the fusion did not seem to be correlated to a specific histology, although several but not all cases showed clear cell morphology, and the fusionpositive cases showed no predominant location within the supratentorium (supplementary Table 1). Interestingly, all five reLA fusion-negative samples were from female patients (two-tailed Fisher’s exact test, p = 0.03). The physiological function of C11orf95 is unclear. It has been described as a fusion partner of the gene coding the transcription factor Mkl2 in benign chondroid lipomas representing the molecular correlate of the t(11;16) (q13;p13) translocation found in these tumors [3, 5]. The breakpoint in chondroid lipomas is at exon 5, whereas it is at exon 2 in supratentorial ependymomas. RELA encodes relA (NF-κB3), a 65-kDa protein which interacts with IκB and p50 in the central signaling complex in the NF-κB pathway. After activation of cell surface receptors the signal is transmitted by the IKK complex that phosphorylates IκB and thereby controls the translocation of relA/p50 into the nucleus and transcription of specific target genes (reviewed in [4]). The key physiological function of NF-κB signaling is the orchestration of the inflammatory responses to both Clinical observations and studies on genetic alterations and gene expression indicated that supratentorial ependymomas differ from ependymomas of infratentorial or spinal location [6, 8]. To further elucidate the pathogenesis of supratentorial ependymomas, we performed paired-end rNA sequencing in 19 tumor samples from 18 patients (for clinical data, see supplementary Table 1). Mapping of the reads predicted a novel recurrent fusion mrNA between C11orf95, a gene with unknown function, and v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) encoding the relA p65 subunit of the central NF-κB complex. RELA is located approximately 1.9 Mbp telomeric from C11orf95 on the same chromosomal band 11q13. We confirmed the fusion by sanger sequencing of the cDNA (Fig. 1a; primer sequences, see supplementary

High frequency of H3F3A K27M mutations characterizes pediatric and adult high-grade gliomas of the spinal cord

We identified 16 HGG (11 glioblastomas, 3 anaplastic astrocytomas and 2 anaplastic gangliogliomas, 16/30, 53 %) and 1 diffuse astrocytoma harboring H3F3A mutations (Fig. 1a–e). The frequency of mutations among HGG affecting adults and children was similar (52 vs. 54 %). The mean age of adult patients with mutated tumors was lower (34 years) than that observed in patients with wild-type tumors (53 years) (Fig. 1f). All tumors affecting infants were H3F3A wild type. The distributions of mutated and wild-type HGG along the spinal cord appeared similar, but in cervical spine HGG were more frequently mutated (Fig. 1g). Neither H3F3A wild-type HGG nor DA showed immunoreactivity for mutant IDH1 protein (not shown). Histones are nuclear proteins which regulate DNA replication and transcription by modifying the nucleosome structure [9]. H3.3 histone proteins, encoded by H3.3A (H3F3A) and H3.3B, are deposited on chromatin in a replication-independent manner and are enriched at actively transcribed genes and heterochromatic regions [9]. H3F3A mutations occur in a hotspot region (K27 and G34) undergoing posttranslational modifications. Similar to IDH1 mutated gliomas in adults, reduced H3.3K27 trimethylation and deregulated DNA methylation are considered to play a major role in pediatric gliomagenesis [2]. Recently, several groups have highlighted the high frequency of H3F3A mutations in pediatric midline HGG as well as in DIPG [2, 3, 7, 8]. These mutations are not limited to pediatric patients, but can occur, although more rarely, in adults. H3F3A mutations were frequently observed in thalamic HGG affecting young adults [1] and adult DIPG, but rare in supratentorial adult HGG [6]. Notably, the patient’s age did not affect the pattern of methylation in H3F3A mutant tumors [1]. Diffuse high-grade gliomas (HGG) in the spinal cord are rare [5]. It is still undetermined whether they could share genetic alterations with their supratentorial counterparts: in particular, because the spinal cord has to be considered a midline structure, a high incidence of H3F3A mutations could be hypothesized, as reported in diffuse pontine gliomas (DIPG) and in pediatric midline HGG [1–3, 6–8]. In this study, we investigated the H3F3A status of 30 primary spinal HGG affecting pediatric and adult patients. Formalin-fixed paraffin-embedded specimens of 36 primary spinal diffusely infiltrating gliomas, including 30 HGG (18 glioblastomas, WHO IV; 10 anaplastic astrocytomas, WHO III; 2 anaplastic gangliogliomas, WHO III) and 6 diffuse astrocytomas (WHO II) [5] were retrieved from the archives of the Institute of Neuropathology, University of Bonn medical Center and of the DGNN Brain Tumor Reference Center, Bonn, Germany. No brainstem HGG were included. The clinical features are summarized in supplementary Table 1. The H3F3A status was evaluated by immunohistochemistry and/or by pyrosequencing, as described previously [4]. Antibodies against mutant H3.3K27M (Millipore, Temecula, USA), methylated H3.3K27me3 protein (Cell Signaling, Danvers, USA) as well as mutant IDH1 protein (Dianova, Hamburg, Germany) were used.

Genomic Alterations of Adamantinomatous and Papillary Craniopharyngioma.

Craniopharyngiomas are rare histologically benign but clinically challenging neoplasms. To obtain further information on the molecular genetics and biology of craniopharyngiomas, we analyzed a cohort of 121 adamantinomatous and 16 papillary craniopharyngiomas (ACP, PCP). We extracted DNA from formalin-fixed paraffin-embedded tissue and determined mutational status of CTNNB1, BRAF, and DDX3X by Sanger sequencing, next generation panel sequencing, and pyrosequencing. Sixteen craniopharyngiomas were further analyzed by molecular inversion profiling (MIP); 76.1% of the ACP were mutated in exon 3 of CTNNB1 encoding for &bgr;-catenin and there was a trend towards a worse event-free survival in cases mutated at Thr41. Next generation panel sequencing of 26 ACP did not detect any recurrent mutations other than CTNNB1 mutations. BRAF V600E mutations were found in 94% of the PCP, but not in ACP. GISTIC analysis of MIP data showed no significant larger chromosomal aberrations but a fraction of ACP showed recurrent focal gains of chromosomal material, other cases showed loss in the chromosomal region Xq28, and a third group and the PCP had stable genomes. In conclusion, the crucial pathogenetic event appears to be WNT activation in ACP, whereas it appears to be activation of the Ras/Raf/MEK/ERK pathway by BRAF V600E mutations in PCP.

Intramedullary gangliogliomas: histopathologic and molecular features of 25 cases ☆

Gangliogliomas are uncommon glioneuronal tumors, which usually arise in the cerebral hemispheres and occasionally in the brain stem. Gangliogliomas occurring in the spinal cord are extremely rare. In this study, we analyzed the clinical, histopathologic, and molecular features of 25 spinal gangliogliomas. The cases included in our series affected mostly children and young adults (15 males and 10 females; mean age, 20 years; median age, 14 years; age range, 1-72 years) and were predominantly localized in the cervical and thoracic spine. From the clinical point of view (detailed follow-up available for 9 pediatric cases; mean follow-up: 2 years 10 months; range, 3 months to 5 years 10 months), most patients showed stable disease after subtotal resection. Radiotherapy was rarely used as adjuvant treatment. Histologically, gangliogliomas (WHO grade I) (21 cases) showed features largely similar to their supratentorial counterparts. Anaplastic gangliogliomas (World Health Organization grade III) (4 cases) showed features of anaplasia (including high cellularity and increased mitotic and proliferation activity). From a molecular point of view, only 2 tumors (2/19, 11%) harbored a BRAF(V600E) mutation. In conclusion, although spinal gangliogliomas display histologic and clinical features similar to their supratentorial counterparts, they show a relatively low frequency of BRAF(V600E) mutations, alteration otherwise common in hemispheric and brain stem gangliogliomas.

Dysembryoplastic Neuroepithelial Tumor of the Septum Pellucidum and the Supratentorial Midline: Histopathologic, Neuroradiologic, and Molecular Features of 7 Cases.

Dysembryoplastic neuroepithelial tumors (DNTs) are one of the most common epilepsy-associated low-grade glioneuronal tumors of the central nervous system. Although most DNTs occur in the cerebral cortex, DNT-like tumors with unusual intraventricular or periventricular localizations have been reported. Most of them involve the septum pellucidum and the foramen of Monro. In this study, we have described the neuroradiologic, histopathologic, and molecular features of 7 cases (4 female and 3 male; patient age range, 3 to 34 y; mean age, 16.7 y). The tumors, all localized near the supratentorial midline structures in proximity to the foramen of Monro and septum pellucidum, appeared in magnetic resonance imaging as well-delimited cystic lesions with cerebrospinal fluid-like signal on T1-weighted and T2-weighted images, some of them with typical fluid-attenuated inversion recovery ring sign. Histologically, they shared features with classic cortical DNTs but did not display aspects of multinodularity. From a molecular point of view the cases investigated did not show KIAA1549-BRAF fusions or FGFR1 mutations, alterations otherwise observed in pilocytic astrocytomas, or MYB and MYBL1 alterations that have been identified in a large group of pediatric low-grade gliomas. Moreover, BRAFV600E mutations, which so far represent the most common molecular alteration found in cortical DNTs, were absent in this group of rare periventricular tumors.

High-Resolution Genomic Analysis of Cribriform Neuroepithelial Tumors of the Central Nervous System

Abstract Cribriform neuroepithelial tumors (CRINET) are one of several recently characterized entities in the broad spectrum of solid tumors with SMARCB1-INI1 loss. This neoplasm seems to be exceedingly rare and displays unique neuropathologic and clinical features. To date, only a few cases of CRINET have been characterized from a molecular point of view. In this study, we investigated the molecular features of 3 cases of CRINET using multiplex ligation-dependent probe amplification and molecular inversion profiling approaches. Along with mutations and deletions of SMARCB1-INI1, molecular inversion profiling analysis revealed a stable genomic profile without significant large chromosomal changes. Focal alterations (gains) were observed in individual cases at chromosomes 4q12 (PDGFRA), 12q15 (MDM2), 7p15.1 (NPY), and 18q11.2 (CDH2). Genomic Identification of Significant Targets in Cancer analysis highlighted focal alterations, including gains at chromosomes 16q23.2 (MAF), 17q23 (AXIN2), and 8p12 (ADAM3A). No cases showed BRAFV600E or CTNNB1 mutations. These data indicate that CRINET present stable genetic features and lack alterations commonly identified in other pediatric brain tumors. Further studies are required to determine whether specific alterations and specific signaling pathways, in addition to SMARCB1-INI1, may be implicated in the biology of this rare tumor and whether there are additional molecular similarities between CRINET and atypical teratoid/rhabdoid tumors.

Type, Frequency, and Spatial Distribution of Immune Cell Infiltrates in CNS Germinomas: Evidence for Inflammatory and Immunosuppressive Mechanisms

Central nervous system germinomas are characterized by a massive immune cell infiltrate. We systematically characterized these immune cells in 28 germinomas by immunophenotyping and image analysis. mRNA expression was analyzed by Nanostring technology and in situ RNA hybridization. Tumor infiltrating lymphocytes (TILs) were composed of 61.8% ± 3.1% (mean ± SE) CD3-positive T cells, including 45.2% ± 3.5% of CD4-positive T-helper cells, 23.4% ± 1.5% of CD8-positive cytotoxic T cells, 5.5% ± 0.9% of FoxP3-positive regulatory T cells, and 11.9% ±1.3% PD-1-positive TILs. B cells accounted for 35.8% ± 2.9% of TILs and plasma cells for 9.3% ± 1.6%. Tumor-associated macrophages consisted of clusters of activated PD-L1-positive macrophages and interspersed anti-inflammatory macrophages expressing CD163. Germinoma cells did not express PD-L1. Expression of genes encoding immune cell markers and cytokines was high and comparable to mRNA levels in lymph node tissue. IFNG and IL10 mRNA was detected in subfractions of TILs and in PD-L1-positive macrophages. Taken together, the strong immune reaction observed in germinomas involves inflammatory as well as various suppressive mechanisms. Expression of PD-1 and PD-L1 and infiltration of cytotoxic T cells are biomarkers predictive of response to anti-PD-1/PD-L1 therapies, constituting a rationale for possible novel treatment approaches.

Childhood supratentorial ependymomas with YAP1-MAMLD1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features

Ependymoma with YAP1‐MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1‐MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1‐MAMLD1 fusion was documented by RT‐PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo‐rosettes, small to medium‐sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot‐like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1‐MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1–11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow‐up, 4.84 years). In this to date, largest series of ependymomas with YAP1‐MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings.