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.

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.

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.

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.

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.

Multi-drug resistance (MDR) is the main cause of low effectiveness of cancer chemotherapy. P-glycoprotein (P-gp) is one of the main factors determining MDR. Some studies indicate the potential role of melatonin (MLT) in MDR. In this study, we examined the effect of MLT on colon cancer cell's resistance to doxorubicin (DOX). Using the sulforhodamine B (SRB), method the effect of tested substances on the survival of LoVo (colon cancer cells sensitive to DOX) and LoVoDX (colon cancer cells resistant to DOX) was rated. Using immunocytochemistry (ICC), the expression of P-gp in the LoVo and LoVoDX was determined. With the real-time PCR (RT-PCR) technique, the ABCB1 expression in LoVoDX was evaluated. Based on the results, it was found that MLT in some concentrations intensified the cytotoxicity effect of DOX in the LoVoDX cells. In the ICC studies, it was demonstrated that certain concentrations of MLT and DOX cause an increase in the percentage of cells expressing P-gp, which correlates positively with ABCB1 expression (RT-PCR). The mechanism of overcoming resistance by MLT is probably not only associated with the expression of P-gp. It seems appropriate to carry out further research on the use of MLT as the substance supporting cancer chemotherapy.

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Retinoblastoma is a malignancy of the eye in children characterized by biallelic inactivation of the retinoblastoma 1 gene (RB1), located at chromosome 13q14.2. Children with interstitial chromosome 13q deletions that include the RB1 gene show a predisposition to develop retinoblastoma and variable other features. Large 13q deletions with severe clinical phenotype are nearly always the result of a de novo mutation, i.e. the pathogenic alteration is not detected in parents. This results in a low risk for siblings to develop 13q deletion syndrome.

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Molecular groups of medulloblastoma (MB) are well established. Novel risk stratification parameters include Group 3/4 (non-WNT/non-SHH) methylation subgroups I-VIII or whole-chromosomal aberration (WCA) phenotypes. This study investigates the integration of clinical and molecular parameters to improve risk stratification of non-WNT/non-SHH MB. Non-WNT/non-SHH MB from the HIT2000 study and the HIT-MED registries were selected based on availability of DNA-methylation profiling data. MYC or MYCN amplification and WCA of chromosomes 7, 8, and 11 were inferred from methylation array-based copy number profiles. In total, 403 non-WNT/non-SHH MB were identified, 346/403 (86%) had a methylation class family Group 3/4 methylation score (classifier v11b6) ≥ 0.9, and 294/346 (73%) were included in the risk stratification modeling based on Group 3 or 4 score (v11b6) ≥ 0.8 and subgroup I-VIII score (mb_g34) ≥ 0.8. Group 3 MB (5y-PFS, survival estimation ± standard deviation: 41.4 ± 4.6%; 5y-OS: 48.8 ± 5.0%) showed poorer survival compared to Group 4 (5y-PFS: 68.2 ± 3.7%; 5y-OS: 84.8 ± 2.8%). Subgroups II (5y-PFS: 27.6 ± 8.2%) and III (5y-PFS: 37.5 ± 7.9%) showed the poorest and subgroup VI (5y-PFS: 76.6 ± 7.9%), VII (5y-PFS: 75.9 ± 7.2%), and VIII (5y-PFS: 66.6 ± 5.8%) the best survival. Multivariate analysis revealed subgroup in combination with WCA phenotype to best predict risk of progression and death. The integration of clinical (age, M and R status) and molecular (MYC/N, subgroup, WCA phenotype) variables identified a low-risk stratum with a 5y-PFS of 94 ± 5.7 and a very high-risk stratum with a 5y-PFS of 29 ± 6.1%. Validation in an international MB cohort confirmed the combined stratification scheme with 82.1 ± 6.0% 5y-PFS in the low and 47.5 ± 4.1% in very high-risk groups, and outperformed the clinical model. These newly identified clinico-molecular low-risk and very high-risk strata, accounting for 6%, and 21% of non-WNT/non-SHH MB patients, respectively, may improve future treatment stratification.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.

Two distinct genetically defined entities of ependymoma arising in the supratentorial compartment are characterized by the presence of either a C11orf95-RELA or a YAP-MAMLD1 fusion, respectively. There is growing evidence that supratentorial ependymomas without these genetic features exist. In this study, we report on 18 pediatric non-RELA/non-YAP supratentorial ependymomas that were systematically characterized by means of their histology, immunophenotype, genetics, and epigenomics. Comprehensive molecular analyses included high-resolution copy number analysis, methylation profiling, analysis of fusion transcripts by Nanostring technology, and RNA sequencing. Based upon histological and immunohistochemical features two main patterns were identified-RELA-like (n = 9) and tanycytic ependymomas (n = 6). In the RELA-like group histologically assigned to WHO grade III and resembling RELA-fused ependymomas, tumors lacked nuclear expression of p65-RelA as a surrogate marker for a pathological activation of the NF-κB pathway. Three tumors showed alternative C11orf95 fusions to MAML2 or NCOA1. A methylation-based brain tumor classifier assigned two RELA-like tumors to the methylation class "EP, RELA-fusion"; the others demonstrated no significant similarity score. Of the tanycytic group, 5/6 tumors were assigned a WHO grade II. No gene fusions were detected. Methylation profiling did not show any association with an established methylation class. We additionally identified two astroblastoma-like tumors that both presented with chromothripsis of chromosome 22 but lacked MN1 breaks according to FISH analysis. They revealed novel fusion events involving genes in chromosome 22. One further tumor with polyploid cytogenetics was interpreted as PFB ependymoma by the brain tumor methylation classifier but had no relation to the posterior fossa. Clinical follow-up was available for 16/18 patients. Patients with tanycytic and astroblastoma-like tumors had no relapse, while 2 patients with RELA-like ependymomas died. Our data indicate that in addition to ependymomas discovered so far, at least two more supratentorial ependymoma types (RELA-like and tanycytic) exist.

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.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Retinoblastoma and other eye tumors in childhood are rare diseases. Many eye tumors are the first signs of a genetic tumor predisposition syndrome and the affected children carry a higher risk of developing other cancers later in life. Clinical and genetic data of all children with eye tumors diagnosed between 2013-2018 in Germany and Austria were collected in a multicenter prospective observational study. In five years, 300 children were recruited into the study: 287 with retinoblastoma, 7 uveal melanoma, 3 ciliary body medulloepithelioma, 2 retinal astrocytoma, 1 meningioma of the optic nerve extending into the eye. Heritable retinoblastoma was diagnosed in 44% of children with retinoblastoma. One child with meningioma of the optic nerve extending into the eye was diagnosed with neurofibromatosis 2. No pathogenic constitutional variant in DICER1 was detected in a child with medulloepithelioma while two children did not receive genetic analysis. Because of the known association with tumor predisposition syndromes, genetic counseling should be offered to all children with eye tumors. Children with a genetic predisposition to cancer should receive a tailored surveillance including detailed history, physical examinations and, if indicated, imaging to screen for other cancer. Early detection of cancers may reduce mortality.

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.

Posterior fossa type A (PF-EPN-A, PFA) ependymoma are aggressive tumors that mainly affect children and have a poor prognosis. Histopathology shows significant intratumoral heterogeneity, ranging from loose tissue to often sharply demarcated, extremely cell-dense tumor areas. To determine molecular differences in morphologically different areas and to understand their clinical significance, we analyzed 113 PF-EPN-A samples, including 40 corresponding relapse samples. Cell-dense areas ranged from 0 to 100% of the tumor area and displayed a higher proportion of proliferating tumor cells (p < 0.01). Clinically, cell density was associated with poor progression-free and overall survival (pPFS = 0.0026, pOS < 0.01). Molecularly, tumor areas with low and high cell density showed diverging DNA methylation profiles regarding their similarity to distinct previously discovered PF-EPN-A subtypes in 9/21 cases. Prognostically relevant chromosomal changes at 1q and 6q showed spatial heterogeneity within single tumors and were significantly enriched in cell-dense tumor areas as shown by single-cell RNA (scRNA)-sequencing as well as copy number profiling and fluorescence in situ hybridization (FISH) analyses of different tumor areas. Finally, spatial transcriptomics revealed cell-dense areas of different tumors to be more similar than various different areas of the same tumor. High-density areas distinctly overexpressed genes encoding histone proteins, WNT5A, TGFB1, or IGF2. Relapsing tumors displayed a higher proportion of cell-dense areas (p = 0.036), a change in PF-EPN-A methylation subtypes (13/32 patients), and novel chromosome 1q gains and 6q losses (12/32 cases) compared to corresponding primary tumors. Our data suggest that PF-EPN-A ependymomas habor a previously unrecognized intratumoral heterogeneity with clinical implications, which has to be accounted for when selecting diagnostic material, inter alia, by histological evaluation of the proportion of cell-dense areas.

Combination concepts of radiotherapy and immune checkpoint inhibition are currently of high interest. We examined imaging findings, acute toxicity, and local control in patients with melanoma brain metastases receiving programmed death 1 (PD-1) inhibitors and/or robotic stereotactic radiosurgery (SRS). Twenty-six patients treated with SRS alone (n = 13; 20 lesions) or in combination with anti-PD-1 therapy (n = 13; 28 lesions) were analyzed. Lesion size was evaluated three and six months after SRS using a volumetric assessment based on cranial magnetic resonance imaging (cMRI) and acute toxicity after 12 weeks according to the Common Terminology Criteria for Adverse Events (CTCAE). Local control after six months was comparable (86%, SRS + anti-PD-1, and 80%, SRS). All toxicities reported were less than or equal to grade 2. One metastasis (5%) in the SRS group and six (21%) in the SRS + anti-PD-1 group increased after three months, whereas four (14%) of the six regressed during further follow-ups. This was rated as pseudoprogression (PsP). Three patients (23%) in the SRS + anti-PD-1 group showed characteristics of PsP. Treatment with SRS and anti-PD-1 antibodies can be combined safely in melanoma patients with cerebral metastases. Early volumetric progression of lesions under simultaneous treatment may be related to PsP; thus, the evaluation of combined radioimmunotherapy remains challenging and requires experienced teams.

Standards for chemotherapy against choroid plexus tumors (CPT) have not yet been established.

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system with a poor prognosis. In the treatment of GBM tumors, radiotherapy plays a major role. Typically, GBM tumors cannot be cured by irradiation because of intrinsic resistance machanisms. An escalation of the irradiation dose in the GBM tumor is difficult due to the high risk of severe side effects in the brain. In the last decade, the development of new irradiation techniques, including proton-based irradiation, promised new chances in the treatment of brain tumors. In contrast to conventional radiotherapy, irradiation with protons allows a dosimetrically more confined dose deposition in the tumor while better sparing the normal tissue surrounding the tumor. A systematic comparison of both irradiation techniques on glioblastoma cells has not been performed so far. Despite the improvements in radiotherapy, it remains challenging to predict the therapeutical response of GBM tumors. Recent publications suggest extracellular vesicles (EVs) as promising markers predicting tumor response. Being part of an ancient intercellular communication system, virtually all cells release specifically composed EVs. The assembly of EVs varies between cell types and depends on environmental parameters. Here, we compared the impact of photon-based with proton-based radiotherapy on cell viability and phenotype of four different glioblastoma cell lines. Furthermore, we characterized EVs released by different glioblastoma cells and correlated released EVs with the cellular response to radiotherapy. Our results demonstrated that glioblastoma cells reacted more sensitive to irradiation with protons than photons, while radiation-induced cell death 72 h after single dose irradiation was independent of the irradiation modality. Moreover, we detected CD9 and CD81-positive EVs in the supernatant of all glioblastoma cells, although at different concentrations. The amount of released CD9 and CD81-positive EVs increased after irradiation when cells became apoptotic. Although secreted EVs of non-irradiated cells were not predictive for radiosensitivity, their increased EV release after irradiation correlated with the cytotoxic response to radiotherapy 72 h after irradiation. Thus, our data suggest a novel application of EVs in the surveillance of anti-cancer therapies.