Medulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.

Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

The large diversity of central nervous system (CNS) tumor types in children and adolescents results in disparate patient outcomes and renders accurate diagnosis challenging. In this study, we prospectively integrated DNA methylation profiling and targeted gene panel sequencing with blinded neuropathological reference diagnostics for a population-based cohort of more than 1,200 newly diagnosed pediatric patients with CNS tumors, to assess their utility in routine neuropathology. We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%). Discrepant results by neuropathological WHO-based and DNA methylation-based classification (30%) were enriched in histological high-grade gliomas, implicating relevance for current clinical patient management in 5% of all patients. Follow-up (median 2.5 years) suggests improved survival for patients with histological high-grade gliomas displaying lower-grade molecular profiles. These results provide preliminary evidence of the utility of integrating multi-omics in neuropathology for pediatric neuro-oncology.

Mutations in the TERT promoter represent the genetic underpinnings of tumor cell immortality. Beyond the two most common point mutations, which selectively recruit the ETS factor GABP to activate TERT, the significance of other variants is unknown. In seven cancer types, we identify duplications of wildtype sequence within the core promoter region of TERT that have strikingly similar features including an ETS motif, the duplication length and insertion site. The duplications recruit a GABP tetramer by virtue of the native ETS motif and its precisely spaced duplicated counterpart, activate the promoter and are clonal in a TERT expressing multifocal glioblastoma. We conclude that recurrent TERT promoter duplications are functionally and mechanistically equivalent to the hotspot mutations that confer tumor cell immortality. The shared mechanism of these divergent somatic genetic alterations suggests a strong selective pressure for recruitment of the GABP tetramer to activate TERT.

Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity given their stereotypic intracranial and paraspinal locations. Similar to many solid tumors, schwannomas and other nerve sheath tumors are primarily thought to arise due to aberrant hyperactivation of the RAS growth factor signaling pathway. Here, we sought to further define the molecular pathogenesis of schwannomas.

Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

CXCR4 is a chemokine receptor that recruits blood stem cells and increases tumor cell growth and invasiveness. We examined CXCR4 expression in vestibular schwannomas (VS) from patients with and without neurofibromatosis type 2 (NF2) and correlated the levels with the patients' clinical characteristics. The aim was to determine whether CXCR4 can be used as a prognostic marker and as a target for systemic therapy.

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood-cerebrospinal fluid barrier or the blood-brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell-cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.

A disintegrin and metalloproteinase 9 (ADAM9) is a member of the transmembrane ADAM family. It is expressed in different types of solid cancer and promotes tumor invasiveness. To the best of our knowledge, the present study was the first to examine ADAM9 expression in vestibular schwannomas (VS) from patients with and without neurofibromatosis type 2 (NF2) and to associate the data with clinical parameters of the patients. The aim of the present study was to evaluate if ADAM9 could be used as prognostic marker or therapeutic target. ADAM9 mRNA and protein levels were measured in VS samples (n=60). A total of 30 of them were from patients with neurofibromatosis. Healthy peripheral nerves from autopsies (n=10) served as controls. ADAM9 mRNA levels were measured by PCR, and protein levels were determined by immunohistochemistry (IHC) and western blotting (WB). The Hannover Classification was used to categorize tumor extension and hearing loss. ADAM9 mRNA levels were 8.8-fold higher in VS compared with in controls. The levels were 5.6-fold higher in patients with NF2 and 12-fold higher in patients with sporadic VS. WB revealed two mature isoforms of the protein, and according to IHC ADAM9 was mainly expressed by S100-positive Schwann cells. There was a strong correlation between ADAM9 mRNA expression and the level of functional impairment (r~1, p=0.01). Particularly, the secreted isoform of ADAM9 was expressed in patients with higher hearing impairment. ADAM9 mRNA was overexpressed in the tumor samples relative to healthy vestibular nerves, and there was an association between higher ADAM9 expression levels and greater hearing impairment. Therefore, ADAM9 may be a prognostic marker for VS, and ADAM9 inhibition might have the potential as a systemic approach for the treatment of VS.

Glioblastoma multiforme is the most aggressive primary brain tumor of adults, but lacks reliable and liquid biomarkers. We evaluated circulating plasma transcripts of metastasis-associated in colon cancer-1 (MACC1), a prognostic biomarker for solid cancer entities, for prediction of clinical outcome and therapy response in glioblastomas. MACC1 transcripts were significantly higher in patients compared to controls. Low MACC1 levels clustered together with other prognostically favorable markers. It was associated with patients' prognosis in conjunction with the isocitrate dehydrogenase (IDH) mutation status: IDH1 R132H mutation and low MACC1 was most favorable (median overall survival (OS) not yet reached), IDH1 wildtype and high MACC1 was worst (median OS 8.1 months), while IDH1 wildtype and low MACC1 was intermediate (median OS 9.1 months). No patients displayed IDH1 R132H mutation and high MACC1. Patients with low MACC1 levels receiving standard therapy survived longer (median OS 22.6 months) than patients with high MACC1 levels (median OS 8.1 months). Patients not receiving the standard regimen showed the worst prognosis, independent of MACC1 levels (low: 6.8 months, high: 4.4 months). Addition of circulating MACC1 transcript levels to the existing prognostic workup may improve the accuracy of outcome prediction and help define more precise risk categories of glioblastoma patients.

Recently, we described a disintegrin and metalloproteinase 9 (ADAM9) overexpression by Schwann cells of vestibular schwannoma (VS) and suggested that it might be a marker for VS tumor growth and invasiveness. This research note provides additional data utilizing a small cohort of VS primary cultures and tissue samples. We examined whether reconstitution of Merlin expression in VS cells regulates ADAM9 protein expression and performed lentiviral ADAM9 knock down to investigate possible effects on VS cells numbers. Moreover, the co-localization of ADAM9 and Integrins α6 and α2β1, respectively, was examined by immunofluorescence double staining.

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Inhibition of the protein kinase MPS1, a mitotic spindle-checkpoint regulator, reinforces the effects of multiple therapies against glioblastoma multiforme (GBM) in experimental settings. We analyzed MPS1 mRNA-expression in gliomas WHO grade II, III and in clinical subgroups of GBM. Data were obtained by qPCR analysis of tumor and healthy brain specimens and correlated with the patients' clinical data. MPS1 was overexpressed in all gliomas on an mRNA level (ANOVA, p < 0.01) and correlated with tumor aggressiveness. We explain previously published conflicting results on survival: high MPS1 was associated with poorer long term survival when all gliomas were analyzed combined in one group (Cox regression: t < 24 months, p = 0.009, Hazard ratio: 8.0, 95% CI: 1.7-38.4), with poorer survival solely in low-grade gliomas (LogRank: p = 0.02, Cox regression: p = 0.06, Hazard-Ratio: 8.0, 95% CI: 0.9-66.7), but not in GBM (LogRank: p > 0.05). This might be due to their lower tumor volume at the therapy start. GBM patients with high MPS1 mRNA-expression developed clinical symptoms at an earlier stage. This, however, did not benefit their overall survival, most likely due to the more aggressive tumor growth. Since MPS1 mRNA-expression in gliomas was enhanced with increasing tumor aggressiveness, patients with the worst outcome might benefit best from a treatment directed against MPS1.

Despite its significant overexpression in several malignant neoplasms, the expression of RPS27 in the central nervous system (CNS) is widely unknown. We identified the cell types expressing RPS27 in the CNS under normal and disease conditions. We acquired specimens of healthy brain (NB), adult pilocytic astrocytoma (PA) World Health Organization (WHO) grade I, anaplastic PA WHO grade III, gliomas WHO grade II/III with or without isocitrate dehydrogenase (IDH) mutation, and glioblastoma multiforme (GBM). RPS27 protein expression was examined by immunohistochemistry and double-fluorescence staining and its mRNA expression quantified by RT-PCR. Patients' clinical and tumor characteristics were collected retrospectively. RPS27 protein was specifically expressed in tumor cells and neurons, but not in healthy astrocytes. In tumor tissue, most macrophages were positive, while this was rarely the case in inflamed tissue. Compared to NB, RPS27 mRNA was in mean 6.2- and 8.8-fold enhanced in gliomas WHO grade II/III with (p < 0.01) and without IDH mutation (p = 0.01), respectively. GBM displayed a 4.6-fold increased mean expression (p = 0.02). Although RPS27 expression levels did not affect the patients' survival, their association with tumor cells and tumor-associated macrophages provides a rationale for a future investigation of a potential function during gliomagenesis and tumor immune response.

Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients' tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.

Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M+ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M+ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician's discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4+ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M+ diffuse midline glioma.