Evelyn Dörner

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

GNA11 and N-RAS mutations: alternatives for MAPK pathway activating GNAQ mutations in primary melanocytic tumours of the central nervous system.

M. Gessi, J. Hammes, L. Lauriola, E. Dörner, J. Kirfel, G. Kristiansen, A. zur Muehlen, D. Denkhaus, A. Waha and T. Pietsch (2013) Neuropathology and Applied Neurobiology39, 417–425

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.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy.

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.

1q gain is a frequent finding in preoperatively treated Wilms tumors, but of limited prognostic value for risk stratification in the SIOP2001/GPOH trial

Segers et al. recently reported in this journal that gain of chromosome arm 1q is a marker of poor prognosis in Wilms tumors (Segers et al., 2013). They performed a cytogenetic analysis of 331 Wilms tumors treated in the British UKW2, UKW3, and the international SIOP 2001 nephroblastoma studies. About half of the tumors were primary nephrectomy specimens and the other half were tumor samples received after preoperative chemotherapy. Nineteen percent of Wilms tumors showed gain of 1q. There was no difference in age at diagnosis, stage, or anaplasia in tumors with and without 1q gain. Multivariate analysis showed an independent association of 1q gain with an adverse outcome for both event-free survival (EFS; hazard ration (HR) 5 2.45, P 5 0.02) and overall survival (OS; HR 5 4.28, P 5 0.004). Gratias et al. (2013) reported on behalf of the Children’s Oncology Group in a study on chromosome arm 1q gain in primary resected Wilms tumors with favorable histology that gain of 1q was associated with inferior EFS and OS. In 212 analyzed tumors using the multiplex ligationdependent probe amplification (MLPA) technique, they found a 1q gain in 58 specimens (27%). Eightyear EFS rate of 76% for tumors with 1q gain was significantly lower as compared to tumors without 1q gain (93%, P 5 0.0024). Risk stratification of patients in the SIOP2001/ GPOH study, as part of the SIOP2001 trial, is mainly based on stage and tumor classification after preoperative chemotherapy. Based on histopathologic classification three risk groups exist: high-risk tumors including blastemal-type (preoperatively treated) and nephroblastomas with diffuse anaplasia; low risk tumors consisting of completely necrotic tumors; all other types of Wilms tumors (mixed, epithelial, stromal, and regressive tumors as well as tumors with focal anaplasia) are considered as intermediate risk tumors according to this classification (Vujanić et al., 2002). The goal of our study was to determine if the prognostic significance of chromosome 1q gain is still existent in a homogenous preoperatively treated cohort of patients from the SIOP2001/ GPOH study using macrodissected tissue with more than 90% viable tumor cells. In a first step, we analyzed a cohort of 98 tumors (test set) with predominance of high-risk tumors to study the prognostic value of chromosome arm 1q gain (patient characteristics, see Table 1). Chromosome 1q gain was studied using MLPA. MLPA analysis was performed using the SALSA MLPA-probemix P303 with four primers located on 1q (1q21.1, 1q32.1, 1q42.12, 1q44) according to the manufacturer’s protocol (MRC Holland, Amsterdam, The Netherlands). In the test set, a gain of chromosome arm 1q was found in 45% (44/98) of tumors, predominantly in blastemal-type (n 5 32; 53%) tumors and in tumors with diffuse anaplasia (n 5 21, 67%). The 5-year EFS was 71% for those with one 1q gain and 81% for those without gain of 1q (P 5 0.214; Fig. 1A). Evaluation of EFS data of this high-risk enriched cohort using histopathologic risk grouping showed a significant difference in survival [high-risk tumors 63% EFS, intermediate risk tumors 92% (P 5 0.001), Fig. 1B]. In the second step, all patients (n 5 81) registered in the SIOP2001/GPOH study within 1 year (2007) were investigated (validation set) with the same technique to demonstrate the incidence of 1q gain in a nationwide unbiased study and to validate the prognostic value found in the test set (patients characteristics in Table 1). In the validation set, we found a gain of 1q in 59% of tumors. In Kaplan–Meier analysis there was no statistically significant difference in EFS [tumors with 1q gain 80% EFS, without 1q gain 91% EFS (P 5 0.188), Fig. 1C]. Evaluation of EFS data of the same patients using histopathologic risk grouping showed a significant difference in survival [high-risk tumors 70% EFS, intermediate risk tumors 88% (P 5 0.048), Fig. 1D]. In contrast to the test set, we did not find a higher incidence of gain of 1q in blastemal-type or diffusely anaplastic Wilms tumors as compared to other

Medulloblastoma with extensive nodularity: a tumour exclusively of infancy?

Medulloblastoma with extensive nodularity (MBEN) represents a rare medulloblastoma (MB) variant usually occurring in the first 3 years of life and associated with a good outcome [1]. It was previously designated as ‘cerebellar neuroblastoma’ and differs from the more common nodular/desmoplastic medulloblastoma by expanded lobular architecture and advanced neurocytic differentiation. MBENs show SHH pathway activation and can occur in patients with germline mutations of Patched1 (PTCH1) or Suppressor of fused (SUFU) [1–3]. It is believed that these tumours are derived from external granule cell progenitor cells, similar to desmoplastic/ nodular medulloblastomas [4]. Although adult medulloblastoma may show frequent SHH activation, MBEN has never been reported in adult patients. A 39-year-old male patient developed ataxia. The MRI showed a 2.8 cm contrast-enhancing cerebellar midline lesion (Figure 1a). After incomplete resection, the neuropathological examination revealed a tumour with a predominant (about 80% of the tumour tissue) grape-like lobular architecture and presence of reticulin-free islands with a neuropil-like matrix (Figure 1b–c), positive for synaptophysin (Dako, Hamburg, Germany). These nodules contained small cells with neurocytic cytology (Figure 1d) strongly positive for NeuN (Millipore, Darmstadt, Germany) (Figure 1e). Focally, the tumour cells displayed a streaming pattern in the nodular areas. The internodular regions with increased cellularity displayed a dense reticulin meshwork. Here, the tumour cells showed hyperchromatic nuclei and scant cytoplasm. The tumour cells of these internodular areas expressed the low affinity neurotrophin receptor p75-NTR (Thermo-Scientific, Waltham, MA, USA) (Figure 1f), a known SHH target [5] and Yap-1 (Cell Signaling, Danvers, MA, USA) indicative of SHH pathway activation. Neither nuclear positivity for ß-catenin (Roche-Ventana, Darmstadt, Germany) nor nuclear accumulation of p53 (Dako) was found. No expression of cytokeratins (Bachem, Weil am Rhein, Germany) and neurofilament protein (Dako) was observed. The proliferation marker MIB-1 (Ki67) (Dako) stained more than 20% of tumour cells in the internodular areas but was almost negative within the nodular areas. After the histological diagnosis, the patient received combined radio-chemotherapy. Radiotherapy was administered to the entire neuraxis (single dose 1.6 Gy, 5 fractions/week, total dose 35.2 Gy) with a boost to the posterior fossa (single dose 1.8 Gy, 5 fractions/ week, total dose 19.8 Gy). The total cumulative dose was 55.0 Gy. After irradiation, the residual tumour was no longer detectable in MRI. After radiotherapy, the patient received chemotherapy (CCNU/cisplatin, since the 5th cycle CCNU/carboplatin). Actually, 11 months after diagnosis, the patient completed the six of the eight planned cycles of chemotherapy. Clinically, the patient showed a treatment-related polyneuropathy and high-frequency hearing loss in one ear. For further molecular characterization, genomic DNA was extracted from FFPE tissue using standard methods (DNeasy Kit, Qiagen, D€ usseldorf, Germany). A molecular inversion probe (MIP) analysis (Oncoscan v3, Affymetrix, Santa Clara, CA, USA) revealed, besides chr. 7q gain, no significant cytogenetic alterations or allelic imbalances (Figure 1g). In particular, there was no allelic loss of 9q (PTCH1) or 10q (SUFU). MYC, MYCN and GLI2 were not amplified. The mutational probes included in the MIP assay revealed the presence of a PIK3CA E545K (c.1633 g>a) mutation, which was further confirmed by direct sequencing (Eurofins MWG Operon, Ebersberg, Germany) (not shown). Conversely, no TP53 allelic losses or mutations were evident in the MIP assay. The mutational analysis of exon 6 of Smoothened (SMOH) showed presence of an activating L412F mutation (Figure 1h). Medulloblastoma is the most common malignant intracranial tumour in childhood but can also be observed, more rarely, in adults [1]. Like MB in infants and small children, adult MBs show frequent activation of the SHH signaling pathway, usually caused by somatic mutations in PTCH1 or SMOH, [6]. Despite

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.