Diagnosis of a glioblastoma (GBM) is triggered by the onset of symptoms and is based on cerebral imaging and histological examination. Serum-based biomarkers may support detection of GBM. Here, we explored serum protein concentrations of GBM patients and used data mining to explore profiles of biomarkers and determine whether these are associated with the clinical status of the patients. Gene and protein expression data for astrocytoma and GBM were used to identify secreted proteins differently expressed in tumors and in normal brain tissues. Tumor expression and serum concentrations of 14 candidate proteins were analyzed for 23 GBM patients and nine healthy subjects. Data-mining methods involving all 14 proteins were used as an initial evaluation step to find clinically informative profiles. Data mining identified a serum protein profile formed by BMP2, HSP70, and CXCL10 that enabled correct assignment to the GBM group with specificity and sensitivity of 89 and 96%, respectively (p < 0.0001, Fischer's exact test). Survival for more than 15 months after tumor resection was associated with a profile formed by TSP1, HSP70, and IGFBP3, enabling correct assignment in all cases (p < 0.0001, Fischer's exact test). No correlation was found with tumor size or age of the patient. This study shows that robust serum profiles for GBM may be identified by data mining on the basis of a relatively small study cohort. Profiles of more than one biomarker enable more specific assignment to the GBM and survival group than those based on single proteins, confirming earlier attempts to correlate single markers with cancer. These conceptual findings will be a basis for validation in a larger sample size.

Liquid biopsies come of age offering unexploited potential to monitor and react to tumor evolution. We developed a cost-effective assay to non-invasively determine the immune status of glioblastoma (GBM) patients. Employing newly developed printed peptide microarrays we assessed the B-cell response against tumor-associated antigens (TAAs) in 214 patients. Firstly, sera of long-term (36+ months, LTS, n=10) and short-term (6-10 months, STS, n=14) surviving patients were screened for prognostic antibodies against 1745 13-mer peptides covering known TAAs (TNC, EGFR, GLEA2, PHF3, FABP5, MAGEA3). Next, survival associations were investigated in two retrospective independent multicenter validation sets (n=61, n=129, all IDH1-wildtype). Reliability of measurements was tested using a second array technology (spotted arrays). LTS/STS screening analyses identified 106 differential antibody responses. Evaluating the Top30 peptides in validation set 1 revealed three prognostic peptides. Prediction of TNC peptide VCEDGFTGPDCAE was confirmed in a second set (p=0.043, HR=0.66 [0.44-0.99]) and was unrelated to TNC protein expression. Median signals of printed arrays correlated with pre-synthesized spotted microarrays (p<0.0002, R=0.33). Multiple survival analysis revealed independence of age, gender, KPI and MGMT status. We present a novel peptide microarray immune assay that identified increased anti-TNC VCEDGFTGPDCAE serum antibody titer as a promising non-invasive biomarker for prolonged survival.

Mutation is a fundamental process in tumorigenesis. However, the degree to which the rate of somatic mutation varies across the human genome and the mechanistic basis underlying this variation remain to be fully elucidated. Here, we performed a cross-cancer comparison of 402 whole genomes comprising a diverse set of childhood and adult tumors, including both solid and hematopoietic malignancies. Surprisingly, we found that the inactive X chromosome of many female cancer genomes accumulates on average twice and up to four times as many somatic mutations per megabase, as compared to the individual autosomes. Whole-genome sequencing of clonally expanded hematopoietic stem/progenitor cells (HSPCs) from healthy individuals and a premalignant myelodysplastic syndrome (MDS) sample revealed no X chromosome hypermutation. Our data suggest that hypermutation of the inactive X chromosome is an early and frequent feature of tumorigenesis resulting from DNA replication stress in aberrantly proliferating cells.

Recurrent medulloblastoma is a therapeutic challenge because it is almost always fatal. Studies have confirmed that medulloblastoma consists of at least four distinct subgroups. We sought to delineate subgroup-specific differences in medulloblastoma recurrence patterns.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.

Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ∼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.

Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

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.

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-repressive complex 2 (PRC2) that epigenetically silences gene transcription through histone H3 lysine trimethylation (H3K27me3). EZH2 has been implicated in stem cell maintenance and is overexpressed in hematological and solid malignancie`s including malignant glioma. EZH2 is thought to promote tumor progression by silencing tumor suppressor genes. Hence pharmacological disruption of the PRC2 is an attractive therapeutic strategy for cancer treatment. Here we show that EZH2 is expressed in human glioma and correlates with malignancy. Silencing of EZH2 reduced glioma cell proliferation and invasiveness. While we did not observe induction of cell cycle-associated tumor suppressor genes by silencing or pharmacological inhibition of EZH2, microarray analyses demonstrated a strong transcriptional reduction of the AXL receptor kinase. Neither histone nor DNA methylation appeared to be involved in the positive regulation of AXL by EZH2. Silencing AXL mimicked the antiinvasive effects of EZH2 knockdown. Finally, AXL expression is found in human gliomas with high EZH2 expression. Collectively these data suggest that EZH2 drives glioma invasiveness via transcriptional control of AXL independent of histone or DNA methylation.

Musashi1 (Msi1) is a highly conserved RNA-binding protein that is required during the development of the nervous system. Msi1 has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation, and has also been implicated in tumorigenesis, being highly expressed in multiple tumor types. We analyzed Msi1 expression in a large cohort of medulloblastoma samples and found that Msi1 is highly expressed in tumor tissue compared with normal cerebellum. Notably, high Msi1 expression levels proved to be a sign of poor prognosis. Msi1 expression was determined to be particularly high in molecular subgroups 3 and 4 of medulloblastoma. We determined that Msi1 is required for tumorigenesis because inhibition of Msi1 expression by small-interfering RNAs reduced the growth of Daoy medulloblastoma cells in xenografts. To characterize the participation of Msi1 in medulloblastoma, we conducted different high-throughput analyses. Ribonucleoprotein immunoprecipitation followed by microarray analysis (RIP-chip) was used to identify mRNA species preferentially associated with Msi1 protein in Daoy cells. We also used cluster analysis to identify genes with similar or opposite expression patterns to Msi1 in our medulloblastoma cohort. A network study identified RAC1, CTGF, SDCBP, SRC, PRL, and SHC1 as major nodes of an Msi1-associated network. Our results suggest that Msi1 functions as a regulator of multiple processes in medulloblastoma formation and could become an important therapeutic target.

Glioblastoma (GBM) is a typically lethal type of brain tumor with a median survival of 15 months postdiagnosis. This negative prognosis prompted the exploration of alternative treatment options. In particular, the reliance of GBM on angiogenesis triggered the development of anti-VEGF (vascular endothelial growth factor) blocking antibodies such as bevacizumab. Although its application in human GBM only increased progression-free periods but did not improve overall survival, physicians and researchers still utilize this treatment option due to the lack of adequate alternatives. In an attempt to improve the efficacy of anti-VEGF treatment, we explored the role of the egfl7 gene in malignant glioma. We found that the encoded extracellular matrix protein epidermal growth factor-like protein 7 (EGFL7) was secreted by glioma blood vessels but not glioma cells themselves, while no major role could be assigned to the parasitic miRNAs miR-126/126*. EGFL7 expression promoted glioma growth in experimental glioma models in vivo and stimulated tumor vascularization. Mechanistically, this was mediated by an upregulation of integrin α5β1 on the cellular surface of endothelial cells, which enhanced fibronectin-induced angiogenic sprouting. Glioma blood vessels that formed in vivo were more mature as determined by pericyte and smooth muscle cell coverage. Furthermore, these vessels were less leaky as measured by magnetic resonance imaging of extravasating contrast agent. EGFL7-inhibition using a specific blocking antibody reduced the vascularization of experimental gliomas and increased the life span of treated animals, in particular in combination with anti-VEGF and the chemotherapeutic agent temozolomide. Data allow for the conclusion that this combinatorial regimen may serve as a novel treatment option for GBM.

Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail.Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG.Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis (11.1 and 10.8 months median overall survival, respectively). Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization.These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones.H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms.

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

In adults, pilocytic astrocytomas (PA) account for less than 2% of gliomas, resulting in uncertainty regarding the clinical course and optimal treatment, particularly in cases where gross total resection (GTR) could not be achieved. Moreover, information on molecular markers and their prognostic impact is sparse. In order to improve risk stratification, we analyzed our institutional series of 58 patients aged 17 years and older with histology-proven intracranial PA World Health Organization grade I for clinical and molecular prognosticators. Anaplastic and NF1-associated tumors were excluded. O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was determined by pyrosequencing or 450k/850k DNA methylation array. A univariate log-rank test and multivariate StepAIC were applied to identify prognostic factors. The median age was 30 years (range 17-66). Tumors were located in the cerebral/cerebellar hemispheres, midline structures and cerebello-pontine angle in 53%, 38% and 9%. MGMT promoter methylation was present in eight patients (14%). GTR (39/58 patients) significantly reduced the likelihood of tumor recurrence (p = 0.0001). Tumor relapse occurred in 16 patients (28%) after a median progression-free survival (PFS) of 135 months (range 6-153 months); there was one tumor-related death. PFS at 5 and 10 years was 67% and 53%. In multivariate analysis, PFS was significantly prolonged in patients with GTR (HR 0.1; CI 0.03-0.37; p < 0.001), unmethylated MGMT promoter (HR 0.18; CI 0.05-0.64; p = 0.009) and midline tumors (HR 0.21; CI 0.06-0.78; p = 0.02). In conclusion, MGMT promoter methylation status and tumor location were identified as novel prognostic factors in adult PAs, pointing at distinct molecular subtypes and detecting patients in need of close observance and intensified treatment.

Spinal ependymal tumors form a histologically and molecularly heterogeneous group of tumors with generally good prognosis. However, their treatment can be challenging if infiltration of the spinal cord or dissemination throughout the central nervous system (CNS) occurs and, in these cases, clinical outcome remains poor. Here, we describe a new and relatively rare subgroup of spinal ependymal tumors identified using DNA methylation profiling that is distinct from other molecular subgroups of ependymoma. Copy number variation plots derived from DNA methylation arrays showed MYCN amplification as a characteristic genetic alteration in all cases of our cohort (n = 13), which was subsequently validated using fluorescence in situ hybridization. The histological diagnosis was anaplastic ependymoma (WHO Grade III) in ten cases and classic ependymoma (WHO Grade II) in three cases. Histological re-evaluation in five primary tumors and seven relapses showed characteristic histological features of ependymoma, namely pseudorosettes, GFAP- and EMA positivity. Electron microscopy revealed cilia, complex intercellular junctions and intermediate filaments in a representative sample. Taking these findings into account, we suggest to designate this molecular subgroup spinal ependymoma with MYCN amplification, SP-EPN-MYCN. SP-EPN-MYCN tumors showed distinct growth patterns with intradural, extramedullary localization mostly within the thoracic and cervical spine, diffuse leptomeningeal spread throughout the whole CNS and infiltrative invasion of the spinal cord. Dissemination was observed in 100% of cases. Despite high-intensity treatment, SP-EPN-MYCN showed significantly worse median progression free survival (PFS) (17 months) and median overall survival (OS) (87 months) than all other previously described molecular spinal ependymoma subgroups. OS and PFS were similar to supratentorial ependymoma with RELA-fusion (ST-EPN-RELA) and posterior fossa ependymoma A (PF-EPN-A), further highlighting the aggressiveness of this distinct new subgroup. We, therefore, propose to establish SP-EPN-MYCN as a new molecular subgroup in ependymoma and advocate for testing newly diagnosed spinal ependymal tumors for MYCN amplification.