Subependymomas are benign tumors characteristically encountered in the posterior fossa of adults that show distinct epigenetic profiles assigned to the molecular group "subependymoma, posterior fossa" (PFSE) of the recently established DNA methylation-based classification of central nervous system tumors. In contrast, most posterior fossa ependymomas exhibit a more aggressive biological behavior and are allocated to the molecular subgroups PFA or PFB. A subset of ependymomas shows epigenetic similarities with subependymomas, but the precise biology of these tumors and their potential relationships remain unknown. We therefore set out to characterize epigenetic traits, mutational profiles, and clinical outcomes of 50 posterior fossa ependymal tumors of the PFSE group. On histo-morphology, these tumors comprised 12 ependymomas, 14 subependymomas and 24 tumors with mixed ependymoma-subependymoma morphology. Mixed ependymoma-subependymoma tumors varied in their extent of ependymoma differentiation (2-95%) but consistently exhibited global epigenetic profiles of the PFSE group. Selective methylome analysis of microdissected tumor components revealed CpG signatures in mixed tumors that coalesce with their pure counterparts. Loss of chr6 (20/50 cases), as well as TERT mutations (21/50 cases), were frequent events enriched in tumors with pure ependymoma morphology (p < 0.001) and confined to areas with ependymoma differentiation in mixed tumors. Clinically, pure ependymoma phenotype, chr6 loss, and TERT mutations were associated with shorter progression-free survival (each p < 0.001). In conclusion, our results suggest that subependymomas may acquire genetic and epigenetic changes throughout tumor evolution giving rise to subclones with ependymoma morphology (resulting in mixed tumors) that eventually overpopulate the subependymoma component (pure PFSE ependymomas).

Galunisertib, a Transforming growth factor-βRI (TGF-βRI) kinase inhibitor, blocks TGF-β-mediated tumor growth in glioblastoma. In a three-arm study of galunisertib (300 mg/day) monotherapy (intermittent dosing; each cycle =14 days on/14 days off), lomustine monotherapy, and galunisertib plus lomustine therapy, baseline tumor tissue was evaluated to identify markers associated with tumor stage (e.g., histopathology, Ki67, glial fibrillary acidic protein) and TGF-β-related signaling (e.g., pSMAD2). Other pharmacodynamic assessments included chemokine, cytokine, and T cell subsets alterations. 158 patients were randomized to galunisertib plus lomustine (n = 79), galunisertib (n = 39) and placebo+lomustine (n = 40). In 127 of these patients, tissue was adequate for central pathology review and biomarker work. Isocitrate dehydrogenase (IDH1) negative glioblastoma patients with baseline pSMAD2⁺ in cytoplasm had median overall survival (OS) 9.5 months vs. 6.9 months for patients with no tumor pSMAD2 expression (p = 0.4574). Eight patients were IDH1 R132H⁺ and had a median OS of 10.4 months compared to 6.9 months for patients with negative IDH1 R132H (p = 0.5452). IDH1 status was associated with numerically higher plasma macrophage-derived chemokine (MDC/CCL22), higher whole blood FOXP3, and reduced tumor CD3⁺ T cell counts. Compared to the baseline, treatment with galunisertib monotherapy preserved CD4⁺ T cell counts, eosinophils, lymphocytes, and the CD4/CD8 ratio. The T-regulatory cell compartment was associated with better OS with MDC/CCL22 as a prominent prognostic marker.

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and 'enhancer hijacking' events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

Biallelic inactivation of SMARCB1, encoding a member of the SWI/SNF chromatin remodeling complex, is the hallmark genetic aberration of atypical teratoid rhabdoid tumors (ATRT). Here, we report how loss of SMARCB1 affects the epigenome in these tumors. Using chromatin immunoprecipitation sequencing (ChIP-seq) on primary tumors for a series of active and repressive histone marks, we identified the chromatin states differentially represented in ATRTs compared with other brain tumors and non-neoplastic brain. Re-expression of SMARCB1 in ATRT cell lines enabled confirmation of our genome-wide findings for the chromatin states. Additional generation of ChIP-seq data for SWI/SNF and Polycomb group proteins and the transcriptional repressor protein REST determined differential dependencies of SWI/SNF and Polycomb complexes in regulation of diverse gene sets in ATRTs.

The introduction of the classification of brain tumours based on their DNA methylation profile has significantly changed the diagnostic approach for cases with ambiguous histology, non-informative or contradictory molecular profiles or for entities where methylation profiling provides useful information for patient risk stratification, for example in medulloblastoma and ependymoma. We present our experience that combines a conventional molecular diagnostic approach with the complementary use of a DNA methylation-based classification tool, for adult brain tumours originating from local as well as national referrals. We report the frequency of IDH mutations in a large cohort of nearly 1550 patients, EGFR amplifications in almost 1900 IDH-wildtype glioblastomas, and histone mutations in 70 adult gliomas. We demonstrate how additional methylation-based classification has changed and improved our diagnostic approach. Of the 325 cases referred for methylome testing, 179 (56%) had a calibrated score of 0.84 and higher and were included in the evaluation. In these 179 samples, the diagnosis was changed in 45 (25%), refined in 86 (48%) and confirmed in 44 cases (25%). In addition, the methylation arrays contain copy number information that usefully complements the methylation profile. For example, EGFR amplification which is 95% concordant with our Real-Time PCR-based copy number assays. We propose here a diagnostic algorithm that integrates histology, conventional molecular tests and methylation arrays.

EPNs comprise a heterogeneous group of neuroepithelial tumors, accounting for about 10% of all intracranial tumors in children and up to 30% of brain tumors in those younger than 3 years. Actually, the pattern therapy for low-grade EPNs includes complete surgical resection followed by radiation therapy. Total surgical excision is often not possible due to tumor location. The aim of this study was to evaluate, for the first time, the anti-tumor activity of Amblyomin-X in 4 primary cultures derived from pediatric anaplastic posterior fossa EPN, Group A (anaplastic, WHO grade III) and one primary culture of a high grade neuroepithelial tumor with MN1 alteration, which was initially misdiagnosed as EPN: i) by in vitro assays: comparisons of temozolomide and cisplatin; ii) by intracranial xenograft model. Amblyomin-X was able to induce cell death in EPN cells in a more significant percentage compared to cisplatin. The cytotoxic effects of Amblyomin-X were not detected on hFSCs used as control, as opposed to cisplatin-treatment, which promoted a substantial effect in the hAFSCs viability. TEM analysis showed ultrastructural alterations related to the process of cell death: mitochondrial degeneration, autophagosomes and aggregate-like structures. MRI and histopathological analyzes demonstrated significant tumor mass regression. Our results suggest that Amblyomin-X has a selective effect on tumor cells by inducing apoptotic cell death and may be a therapeutic option for Group AEPNs.

Oncolytic virotherapy may be a means of improving the dismal prognosis of malignant brain tumors. The rat H-1 parvovirus (H-1PV) suppresses tumors in preclinical glioma models, through both direct oncolysis and stimulation of anticancer immune responses. This was the basis of ParvOryx01, the first phase I/IIa clinical trial of an oncolytic parvovirus in recurrent glioblastoma patients. H-1PV (escalating dose) was administered via intratumoral or intravenous injection. Tumors were resected 9 days after treatment, and virus was re-administered around the resection cavity. Primary endpoints were safety and tolerability, virus distribution, and maximum tolerated dose (MTD). Progression-free and overall survival and levels of viral and immunological markers in the tumor and peripheral blood were also investigated. H-1PV treatment was safe and well tolerated, and no MTD was reached. The virus could cross the blood-brain/tumor barrier and spread widely through the tumor. It showed favorable pharmacokinetics, induced antibody formation in a dose-dependent manner, and triggered specific T cell responses. Markers of virus replication, microglia/macrophage activation, and cytotoxic T cell infiltration were detected in infected tumors, suggesting that H-1PV may trigger an immunogenic stimulus. Median survival was extended in comparison with recent meta-analyses. Altogether, ParvOryx01 results provide an impetus for further H-1PV clinical development.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

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.

Diagnosing bone and soft tissue neoplasms remains challenging because of the large number of subtypes, many of which lack diagnostic biomarkers. DNA methylation profiles have proven to be a reliable basis for the classification of brain tumours and, following this success, a DNA methylation-based sarcoma classification tool from the Deutsches Krebsforschungszentrum (DKFZ) in Heidelberg has been developed. In this study, we assessed the performance of their classifier on DNA methylation profiles of an independent data set of 986 bone and soft tissue tumours and controls. We found that the 'DKFZ Sarcoma Classifier' was able to produce a diagnostic prediction for 55% of the 986 samples, with 83% of these predictions concordant with the histological diagnosis. On limiting the validation to the 820 cases with histological diagnoses for which the DKFZ Classifier was trained, 61% of cases received a prediction, and the histological diagnosis was concordant with the predicted methylation class in 88% of these cases, findings comparable to those reported in the DKFZ Classifier paper. The classifier performed best when diagnosing mesenchymal chondrosarcomas (CHSs, 88% sensitivity), chordomas (85% sensitivity), and fibrous dysplasia (83% sensitivity). Amongst the subtypes least often classified correctly were clear cell CHSs (14% sensitivity), malignant peripheral nerve sheath tumours (27% sensitivity), and pleomorphic liposarcomas (29% sensitivity). The classifier predictions resulted in revision of the histological diagnosis in six of our cases. We observed that, although a higher tumour purity resulted in a greater likelihood of a prediction being made, it did not correlate with classifier accuracy. Our results show that the DKFZ Classifier represents a powerful research tool for exploring the pathogenesis of sarcoma; with refinement, it has the potential to be a valuable diagnostic tool.

Differentiating intrahepatic cholangiocarcinomas (iCCA) from hepatic metastases of pancreatic ductal adenocarcinoma (PAAD) is challenging. Both tumours have similar morphological and immunohistochemical pattern and share multiple driver mutations. We hypothesised that DNA methylation-based machine-learning algorithms may help perform this task.

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.

In this study, we provide a comprehensive clinical and molecular biological characterization of radiation-induced gliomas (RIG), including a risk assessment for developing gliomas. A cohort of 12 patients who developed RIG 9.5 years (3-31 years) after previous cranial radiotherapy for brain tumors or T-cell acute lymphoblastic leukemia was established. The derived risk of RIG development based on our consecutive cohort of 371 irradiated patients was 1.6% at 10 years and 3.02% at 15 years. Patients with RIG glioma had a dismal prognosis with a median survival of 7.3 months. We described radiology features that might indicate the suspicion of RIG rather than the primary tumor recurrence. Typical molecular features identified by molecular biology examination included the absence of Histon3 mutation, methylation profile of pedHGG-RTK1 and the presence of recurrent PDGFRA amplification and CDKN2A/B deletion. Of the two long-term surviving patients, one had gliomatosis cerebri, and the other had pleomorphic xanthoastrocytoma with BRAF V600E mutation. In summary, our experience highlights the need for tissue diagnostics to allow detailed molecular biological characterization of the tumor, differentiation of the secondary tumor from the recurrence of the primary disease and potentially finding a therapeutic target.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy diagnosed in women under age 40. We and others recently determined that germline and/or somatic deleterious mutations in SMARCA4 characterize SCCOHT. Alterations in this gene, or the related SWI/SNF chromatin remodeling gene SMARCB1, have been previously reported in atypical teratoid/rhabdoid tumors (ATRTs) and malignant rhabdoid tumors (MRTs). To further describe the somatic landscape of SCCOHT, we performed whole exome sequencing on 14 tumors and their matched normal tissues and compared their genomic alterations with those in ATRT and ovarian high grade serous carcinoma (HGSC). We confirmed that SMARCA4 is the only recurrently mutated gene in SCCOHT, and show that recurrent allelic imbalance is observed exclusively on chromosome 19p, where SMARCA4 resides. By comparing genomic alterations between SCCOHT, ATRT and HGSC, we demonstrate that SCCOHTs, like ATRTs, have a remarkably simple genome and harbor significantly fewer somatic protein-coding mutations and chromosomal alterations than HGSC. Furthermore, a comparison of global DNA methylation profiles of 45 SCCOHTs, 65 ATRTs, and 92 HGSCs demonstrates a strong epigenetic correlation between SCCOHT and ATRT. Our results further confirm that the genomic and epigenomic signatures of SCCOHT are more similar to those of ATRT than HGSC, supporting our previous hypothesis that SCCOHT is a rhabdoid tumor and should be renamed MRT of the ovary. Furthermore, we conclude that SMARCA4 inactivation is the main cause of SCCOHT, and that new distinct therapeutic approaches should be developed to specifically target this devastating tumor.

The spatial relationship of glioblastoma (GBM) to the subventricular zone (SVZ) is associated with inferior patient survival. However, the underlying molecular phenotype is largely unknown. We interrogated an SVZ-dependent transcriptome and potential location-specific prognostic markers.

Recently, we described a machine learning approach for classification of central nervous system tumors based on the analysis of genome-wide DNA methylation patterns [6]. Here, we report on DNA methylation-based central nervous system (CNS) tumor diagnostics conducted in our institution between the years 2015 and 2018. In this period, more than 1000 tumors from the neurosurgical departments in Heidelberg and Mannheim and more than 1000 tumors referred from external institutions were subjected to DNA methylation analysis for diagnostic purposes. We describe our current approach to the integrated diagnosis of CNS tumors with a focus on constellations with conflicts between morphological and molecular genetic findings. We further describe the benefit of integrating DNA copy-number alterations into diagnostic considerations and provide a catalog of copy-number changes for individual DNA methylation classes. We also point to several pitfalls accompanying the diagnostic implementation of DNA methylation profiling and give practical suggestions for recurring diagnostic scenarios.

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

To date, standardized strategies for the treatment of recurrent glioma are lacking. Chemotherapy with the alkylating agent BCNU (1,3-bis (2-chloroethyl)-1-nitroso-urea) is a therapeutic option even though its efficacy and safety, particularly the risk of pulmonary fibrosis, remains controversial. To address these issues, we performed a retrospective analysis on clinical outcome and side effects of BCNU-based chemotherapy in recurrent glioma.