Pediatric low-grade gliomas (pLGG) show heterogeneous responses to MAPK inhibitors (MAPKi) in clinical trials. Thus, more complex stratification biomarkers are needed to identify patients likely to benefit from MAPKi therapy. Here, we identify MAPK-related genes enriched in MAPKi-sensitive cell lines using the GDSC dataset and apply them to calculate class-specific MAPKi sensitivity scores (MSSs) via single-sample gene set enrichment analysis. The MSSs discriminate MAPKi-sensitive and non-sensitive cells in the GDSC dataset and significantly correlate with response to MAPKi in an independent PDX dataset. The MSSs discern gliomas with varying MAPK alterations and are higher in pLGG compared to other pediatric CNS tumors. Heterogenous MSSs within pLGGs with the same MAPK alteration identify proportions of potentially sensitive patients. The MEKi MSS predicts treatment response in a small set of pLGG patients treated with trametinib. High MSSs correlate with a higher immune cell infiltration, with high expression in the microglia compartment in single-cell RNA sequencing data, while low MSSs correlate with low immune infiltration and increased neuronal score. The MSSs represent predictive tools for the stratification of pLGG patients and should be prospectively validated in clinical trials. Our data supports a role for microglia in the response to MAPKi.

Concurrent malignant brain tumors in patients with multiple sclerosis (MS) constitute a rare but paradigmatic phenomenon for studying neuroimmunological mechanisms from both molecular and clinical perspectives.

To explore the correlation between Nuclear Overhauser Enhancement (NOE)-mediated signals and tumor cellularity in glioblastoma utilizing the apparent diffusion coefficient (ADC) and cell density from histologic specimens. NOE is one type of chemical exchange saturation transfer (CEST) that originates from mobile macromolecules such as proteins and might be associated with tumor cellularity via altered protein synthesis in proliferating cells.

Influencing cancer metabolism by lifestyle changes is an attractive strategy as - if effective - exercise-induced problems may be less severe than those induced by classical anti-cancer therapies. Pursuing this idea, clinical trials evaluated the benefit of e.g. different diets such as the ketogenic diet, intermittent caloric restriction and physical exercise (PE) in the primary and secondary prevention of different cancer types. PE proved to be beneficial in the context of breast and colon cancer.Glioblastoma has a dismal prognosis, with an average overall survival of about one year despite maximal safe resection, concomitant radiochemotherapy with temozolomide followed by adjuvant temozolomide therapy. Here, we focused on the influence of PE as an isolated and adjuvant treatment in murine GB therapy.PE did not reduce toxic side effects of chemotherapy in mice administered in a dose escalating scheme as shown before for starvation. Although regular treadmill training on its own had no obvious beneficial effects, its combination with temozolomide was beneficial in the treatment of glioblastoma-bearing mice. As PE might partly act through the induction of reactive oxygen species, dihydroartemisinin - an approved anti-malarial drug which induces oxidative stress in glioma cells - was further evaluated in vitro and in vivo. Dihydroartemisinin showed anti-glioma activity by promoting autophagy, reduced the clonogenic survival and proliferation capacity of glioma cells, and prolonged the survival of tumor bearing mice. Using the reactive oxygen species scavenger n-acetyl-cysteine these effects were in part reversible, suggesting that dihydroartemisinin partly acts through the generation of reactive oxygen species.

Mutation-specific vaccines have become increasingly important in glioma immunotherapy; however, shared neoepitopes are rare. For diffuse gliomas, a driver mutation in the gene for isocitrate dehydrogenase type-1 has been shown to produce an immunogenic epitope currently targeted in clinical trials. For highly aggressive midline gliomas, a recurrent point mutation in the histone-3 gene (H3F3A) causes an amino acid change from lysine to methionine at position 27 (K27M). Here, we demonstrate that a peptide vaccine against K27M-mutant histone-3 is capable of inducing effective, mutation-specific, cytotoxic T-cell- and T-helper-1-cell-mediated immune responses in a major histocompatibility complex (MHC)-humanized mouse model. By proving an immunologically effective presentation of the driver mutation H3K27M on MHC class II in human H3K27M-mutant gliomas, our data provide a basis for the further clinical development of vaccine-based or cell-based immunotherapeutic approaches targeting H3K27M.

Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas (n = 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample (n = 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I n = 61, WHO°II n = 88, and WHO°III n = 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas.

Gliomas evade current therapies through primary and acquired resistance and the effect of temozolomide is mainly restricted to methylguanin-O6-methyltransferase promoter (MGMT) promoter hypermethylated tumors. Further resistance markers are largely unknown and would help for better stratification.

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.

Medulloblastomas (MB) molecularly designated as Group 3 (Grp 3) MB represent a more clinically aggressive tumor variant which, as a group, displays heterogeneous molecular characteristics and disease outcomes. Reliable risk stratification of Grp 3 MB would allow for appropriate assignment of patients to aggressive treatment protocols and, vice versa, for sparing adverse effects of high-dose radio-chemotherapy in patients with standard or low-risk tumors. Here we performed RNA-based analysis on an international cohort of 179 molecularly designated Grp 3 MB treated with HIT protocols. We analyzed the clinical significance of differentially expressed genes, thereby developing optimal prognostic subdivision of this MB molecular group. We compared the transcriptome profiles of two Grp 3 MB subsets with various outcomes (76 died within the first 60 months vs. 103 survived this period) and identified 224 differentially expressed genes (DEG) between these two clinical groups (Limma R algorithm, adjusted p-value < 0.05). We selected the top six DEG overexpressed in the unfavorable cohort for further survival analysis and found that expression of all six genes strongly correlated with poor outcomes. However, only high expression of KIRREL2 was identified as an independent molecular prognostic indicator of poor patients' survival. Based on clinical and molecular patterns, four risk categories were outlined for Grp 3 MB patients: i. low-risk: M0-1/MYC non-amplified/KIRREL2 low (n = 48; 5-year OS-95%); ii. standard-risk: M0-1/MYC non-amplified/KIRREL2 high or M2-3/MYC non-amplified/KIRREL2 low (n = 65; 5-year OS-70%); iii. high-risk: M2-3/MYC non-amplified/KIRREL2 high (n = 36; 5-year OS-30%); iv. very high risk-all MYC amplified tumors (n = 30; 5-year OS-0%). Cross-validated survival models incorporating KIRREL2 expression with clinical features allowed for the reclassification of up to 50% of Grp 3 MB patients into a more appropriate risk category. Finally, KIRREL2 immunopositivity was also identified as a predictive indicator of Grp 3 MB poor survival, thus suggesting its application as a possible prognostic marker in routine clinical settings. Our results indicate that integration of KIRREL2 expression in risk stratification models may improve Grp 3 MB outcome prediction. Therefore, simple gene and/or protein expression analyses for this molecular marker could be easily adopted for Grp 3 MB prognostication and may help in assigning patients to optimal therapeutic approaches in prospective clinical trials.

An obstacle to effective uniform treatment of glioblastoma, especially at recurrence, is genetic and cellular intertumoral heterogeneity. Hence, personalized strategies are necessary, as are means to stratify potential targeted therapies in a clinically relevant timeframe. Functional profiling of drug candidates against patient-derived glioblastoma organoids (PD-GBO) holds promise as an empirical method to preclinically discover potentially effective treatments of individual tumors. Here, we describe our establishment of a PD-GBO-based functional profiling platform and the results of its application to four patient tumors. We show that our PD-GBO model system preserves key features of individual patient glioblastomas in vivo. As proof of concept, we tested a panel of 41 FDA-approved drugs and were able to identify potential treatment options for three out of four patients; the turnaround from tumor resection to discovery of treatment option was 13, 14, and 15 days, respectively. These results demonstrate that this approach is a complement and, potentially, an alternative to current molecular profiling efforts in the pursuit of effective personalized treatment discovery in a clinically relevant time period. Furthermore, these results warrant the use of PD-GBO platforms for preclinical identification of new drugs against defined morphological glioblastoma features.

YAP1 is a transcriptional coactivator regulated by the Hippo signaling pathway, including NF2. Meningiomas are the most common primary brain tumors; a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 copy, implicating oncogenic YAP activity in these tumors. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2 wild-type meningiomas. Here, we show that human YAP1-MAML2-positive meningiomas resemble NF2 mutant meningiomas by global and YAP-related gene expression signatures. We then show that expression of YAP1-MAML2 in mice induces tumors that resemble human YAP1 fusion-positive and NF2 mutant meningiomas by gene expression. We demonstrate that YAP1-MAML2 primarily functions by exerting TEAD-dependent YAP activity that is resistant to Hippo signaling. Treatment with YAP-TEAD inhibitors is sufficient to inhibit the viability of YAP1-MAML2-driven mouse tumors ex vivo. Finally, we show that expression of constitutively active YAP1 (S127/397A-YAP1) is sufficient to induce similar tumors, suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a causal oncogenic driver and highlight TEAD-dependent YAP activity as an oncogenic driver in YAP1-MAML2 fusion meningioma as well as NF2 mutant meningioma in general.

There is an unmet need for the identification of biomarkers to aid in the diagnosis, clinical management, prognosis and follow-up of meningiomas. There is currently no consensus on the optimum management of WHO grade II meningiomas. In this study, we identified the calcium binding extracellular matrix glycoprotein, Fibulin-2, via mass-spectrometry-based proteomics, assessed its expression in grade I and II meningiomas and explored its potential as a grade II biomarker. A total of 87 grade I and 91 grade II different meningioma cells, tissue and plasma samples were used for the various experimental techniques employed to assess Fibulin-2 expression. The tumours were reviewed and classified according to the 2016 edition of the Classification of the Tumours of the central nervous system (CNS). Mass spectrometry proteomic analysis identified Fibulin-2 as a differentially expressed protein between grade I and II meningioma cell cultures. Fibulin-2 levels were further evaluated in meningioma cells using Western blotting and Real-time Quantitative Polymerase Chain Reaction (RT-qPCR); in meningioma tissues via immunohistochemistry and RT-qPCR; and in plasma via Enzyme-Linked Immunosorbent Assay (ELISA). Proteomic analyses (p < 0.05), Western blotting (p < 0.05) and RT-qPCR (p < 0.01) confirmed significantly higher Fibulin-2 (FBLN2) expression levels in grade II meningiomas compared to grade I. Fibulin-2 blood plasma levels were also significantly higher in grade II meningioma patients compared to grade I patients. This study suggests that elevated Fibulin-2 might be a novel grade II meningioma biomarker, when differentiating them from the grade I tumours. The trend of Fibulin-2 expression observed in plasma may serve as a useful non-invasive biomarker.

We and others have demonstrated that MYC-amplified medulloblastoma (MB) cells are susceptible to class I histone deacetylase inhibitor (HDACi) treatment. However, single drug treatment with HDACi has shown limited clinical efficacy. We hypothesized that addition of a second compound acting synergistically with HDACi may enhance efficacy.

DICER1 syndrome is a tumor predisposition syndrome that is associated with up to 30 different neoplastic lesions, usually affecting children and adolescents. Here we identify a group of mesenchymal tumors which is highly associated with DICER1 syndrome, and molecularly distinct from other DICER1-associated tumors. This group of DICER1-associated mesenchymal tumors encompasses multiple well-established clinicopathological tumor entities and can be further divided into three clinically meaningful classes designated "low-grade mesenchymal tumor with DICER1 alteration" (LGMT DICER1), "sarcoma with DICER1 alteration" (SARC DICER1), and primary intracranial sarcoma with DICER1 alteration (PIS DICER1). Our study not only provides a combined approach to classify DICER1-associated neoplasms for improved clinical management but also suggests a role for global hypomethylation and other recurrent molecular events in sarcomatous differentiation in mesenchymal tumors with DICER1 alteration. Our results will facilitate future investigations into prognostication and therapeutic approaches for affected patients.

Nowadays medulloblastoma (MB) tumors can be treated with risk-stratified approaches with up to 80% success rate. However, disease relapses occur in approximately 30% of patients and successful salvage treatment strategies at relapse remain scarce. Acquired copy number changes or TP53 mutations are known to occur frequently in relapses, while methylation profiles usually remain highly similar to those of the matching primary tumors, indicating that in general molecular subgrouping does not change during the course of the disease. In the current study, we have used RNA sequencing data to analyze the transcriptome profiles of 43 primary-relapse MB pairs in order to identify specific molecular features of relapses within various tumor groups. Gene variance analysis between primary and relapse samples demonstrated the impact of age in SHH-MB: the changes in gene expression relapse profiles were more pronounced in the younger patients (< 10 years old), which were also associated with increased DNA aberrations and somatic mutations at relapse probably driving this effect. For Group 3/4 MB transcriptome data analysis uncovered clear sets of genes either active or decreased at relapse that are significantly associated with survival, thus could be potential predictive markers. In addition, deconvolution analysis of bulk transcriptome data identified progression-associated differences in cell type enrichment. The proportion of undifferentiated progenitors increased in SHH-MB relapses with a concomitant decrease of differentiated neuron-like cells, while in Group 3/4 MB relapses cell cycle activity increases and differentiated neuron-like cells proportion decreases as well. Thus, our findings uncovered significant transcriptome changes in the molecular signatures of relapsed MB and could be potentially useful for further clinical purposes.

Homozygous deletion of CDKN2A/B was recently incorporated into the World Health Organization classification for grade 3 meningiomas. While this marker is overall rare in meningiomas, its relationship to other CDKN2A alterations on a transcriptomic, epigenomic, and copy number level has not yet been determined. We therefore utilized multidimensional molecular data of 1577 meningioma samples from 6 independent cohorts enriched for clinically aggressive meningiomas to comprehensively interrogate the spectrum of CDKN2A alterations through DNA methylation, copy number variation, transcriptomics, and proteomics using an integrated molecular approach. Homozygous CDKN2A/B deletions were identified in only 7.1% of cases but were associated with significantly poorer outcomes compared to tumors without these deletions. Heterozygous CDKN2A/B deletions were identified in 2.6% of cases and had similarly poor outcomes as those with homozygous deletions. Among tumors with intact CDKN2A/B (without a homozygous or heterozygous deletion), we found a distinct difference in outcome based on mRNA expression of CDKN2A, with meningiomas that had elevated mRNA expression (CDKN2Ahigh) having a significantly shorter time to recurrence. The expression of CDKN2A was independently prognostic after accounting for copy number loss and consistently increased with WHO grade and more aggressive molecular and methylation groups irrespective of cohort. Despite the discordant and mutually exclusive status of the CDKN2A gene in these groups, both CDKN2Ahigh meningiomas and meningiomas with CDKN2A deletions were enriched for similar cell cycle pathways but at different checkpoints. High mRNA expression of CDKN2A was also associated with gene hypermethylation, Rb-deficiency, and lack of response to CDK inhibition. p16 immunohistochemistry could not reliably differentiate between meningiomas with and without CDKN2A deletions but appeared to correlate better with mRNA expression. These findings support the role of CDKN2A mRNA expression as a biomarker of clinically aggressive meningiomas with potential therapeutic implications.

Pilocytic astrocytoma (PA) is the most common primary brain neoplasm in children and treated in curative intent with gross total resection (GTR). However, PA is rare in adults, resulting in limited knowledge on the natural clinical course. This study aimed to describe the clinical course and identify prognostic factors of adult patients with PA.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

Metastatic epithelioid sarcoma (EPS) remains a largely unmet clinical need in children, adolescents and young adults despite the advent of EZH2 inhibitor tazemetostat.

Molecular diagnostics including next generation gene sequencing are increasingly used to determine options for individualized therapies in brain tumor patients. We aimed to evaluate the decision-making process of molecular targeted therapies and analyze data on tolerability as well as signals for efficacy.