The diagnosis of ependymoma has moved from a purely histopathological review with limited prognostic value to an integrated diagnosis, relying heavily on molecular information. However, as the integrated approach is still novel and some molecular ependymoma subtypes are quite rare, few studies have correlated integrated pathology and clinical outcome, often focusing on small series of single molecular types. We collected data from 2023 ependymomas as classified by DNA methylation profiling, consisting of 1736 previously published and 287 unpublished methylation profiles. Methylation data and clinical information were correlated, and an integrated model was developed to predict progression-free survival. Patients with EPN-PFA, EPN-ZFTA, and EPN-MYCN tumors showed the worst outcome with 10-year overall survival rates of 56%, 62%, and 32%, respectively. EPN-PFA harbored chromosome 1q gains and/or 6q losses as markers for worse survival. In supratentorial EPN-ZFTA, a combined loss of CDKN2A and B indicated worse survival, whereas a single loss did not. Twelve out of 200 EPN-ZFTA (6%) were located in the posterior fossa, and these tumors relapsed or progressed even earlier than supratentorial tumors with a combined loss of CDKN2A/B. Patients with MPE and PF-SE, generally regarded as non-aggressive tumors, only had a 10-year progression-free survival of 59% and 65%, respectively. For the prediction of the 5-year progression-free survival, Kaplan-Meier estimators based on the molecular subtype, a Support Vector Machine based on methylation, and an integrated model based on clinical factors, CNV data, and predicted methylation scores achieved balanced accuracies of 66%, 68%, and 73%, respectively. Excluding samples with low prediction scores resulted in balanced accuracies of over 80%. In sum, our large-scale analysis of ependymomas provides robust information about molecular features and their clinical meaning. Our data are particularly relevant for rare and hardly explored tumor subtypes and seemingly benign variants that display higher recurrence rates than previously believed.

Medulloblastoma patients with a sub-total surgical resection (STR; >1.5 cm2 primary tumour residuum post-surgery) typically receive intensified treatment. However, the association of STR with poor outcomes has not been observed consistently, questioning the validity of STR as a high-risk disease feature.

Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Molecular markers are necessary for accurate risk stratification in the non-WNT subgroups due to the underlying heterogeneity in genetic alterations and overall survival. MiR-204 expression was evaluated in molecularly classified 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC cohort, SickKids, Canada. Low expression of miR-204 in the Group 3 / Group 4 tumors identify a highly aggressive subset of tumors having poor overall survival, in the two independent cohorts of medulloblastomas. Downregulation of miR-204 expression correlates with poor survival within the Group 4 as well indicating it as a valuable risk-stratification marker in the subgroup. Restoration of miR-204 expression in multiple medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. IGF2R was identified as a novel target of miR-204. MiR-204 expression resulted in downregulation of both M6PR and IGF2R that transport lysosomal proteases from the Golgi apparatus to the lysosomes. Consistent with this finding, miR-204 expression resulted in reduction in the levels of the lysosomal proteases in medulloblastoma cells. MiR-204 expression also resulted in inhibition of autophagy that is known to be dependent on the lysosomal degradation pathway and LC3B, a known miR-204 target. Treatment with HDAC inhibitors resulted in upregulation of miR-204 expression in medulloblastoma cells, suggesting therapeutic role for these inhibitors in the treatment of medulloblastomas. In summary, miR-204 is not only a valuable risk stratification marker in the combined cohort of Group 3 / Group 4 medulloblastomas as well as in the Group 4 itself, that has paucity of good prognostication markers, but also has therapeutic potential as indicated by its tumor suppressive effect on medulloblastoma cells.

TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6%±8.7%, respectively (p<0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89%±2% vs. 57.4%±1.8% (p<0.01)). In contrast, β-catenin mutation sensitized TP53 mutant cells to radiation (p<0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5%±1.5% in lithium treated cells vs. 56.6±3% (p<0.01)) accompanied by increased number of γH2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33%±8% for lithium treated cells vs. 27%±3% for untreated controls (p=0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.

Primitive neuroectodermal tumors of the central nervous system (CNS-PNETs) are highly aggressive, poorly differentiated embryonal tumors occurring predominantly in young children but also affecting adolescents and adults. Herein, we demonstrate that a significant proportion of institutionally diagnosed CNS-PNETs display molecular profiles indistinguishable from those of various other well-defined CNS tumor entities, facilitating diagnosis and appropriate therapy for patients with these tumors. From the remaining fraction of CNS-PNETs, we identify four new CNS tumor entities, each associated with a recurrent genetic alteration and distinct histopathological and clinical features. These new molecular entities, designated "CNS neuroblastoma with FOXR2 activation (CNS NB-FOXR2)," "CNS Ewing sarcoma family tumor with CIC alteration (CNS EFT-CIC)," "CNS high-grade neuroepithelial tumor with MN1 alteration (CNS HGNET-MN1)," and "CNS high-grade neuroepithelial tumor with BCOR alteration (CNS HGNET-BCOR)," will enable meaningful clinical trials and the development of therapeutic strategies for patients affected by poorly differentiated CNS tumors.

A detailed analysis of the expression of 440 cancer-related genes was performed after the combined treatment of medulloblastoma cells with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). The combinations of retinoids and celecoxib as a COX-2 inhibitor were reported to be effective in some regimens of metronomic therapy of relapsed solid tumors with poor prognosis. Our previous findings on neuroblastoma cells using expression profiling showed that LOX/COX inhibitors have the capability of enhancing the differentiating action of ATRA. Presented study focused on the continuation of our previous work to confirm the possibility of enhancing ATRA-induced cell differentiation in these cell lines via the application of LOX/COX inhibitors. This study provides more detailed information concerning the mechanisms of the enhancement of the ATRA-induced differentiation of medulloblastoma cells.

OTX2 is a potent oncogene that promotes tumor growth in Group 3 medulloblastoma. However, the mechanisms by which OTX2 represses neural differentiation are not well characterized. Here, we perform extensive multiomic analyses to identify an OTX2 regulatory network that controls Group 3 medulloblastoma cell fate. OTX2 silencing modulates the repressive chromatin landscape, decreases levels of PRC2 complex genes and increases the expression of neurodevelopmental transcription factors including PAX3 and PAX6. Expression of PAX3 and PAX6 is significantly lower in Group 3 medulloblastoma patients and is correlated with reduced survival, yet only PAX3 inhibits self-renewal in vitro and increases survival in vivo. Single cell RNA sequencing of Group 3 medulloblastoma tumorspheres demonstrates expression of an undifferentiated progenitor program observed in primary tumors and characterized by translation/elongation factor genes. Identification of mTORC1 signaling as a downstream effector of OTX2-PAX3 reveals roles for protein synthesis pathways in regulating Group 3 medulloblastoma pathogenesis.

Young children with brain tumours are at high risk of developing treatment-related sequelae. We aimed to assess neuropsychological outcomes 5 years after treatment. This cross-sectional study included children under 4 years of age with medulloblastoma (MB) or ependymoma (EP) enrolled in the German brain tumour trials HIT2000 and HIT-REZ2005. Testing was performed using the validated Wuerzburg Intelligence Diagnostics (WUEP-D), which includes Kaufman-Assessment-Battery, Coloured Progressive Matrices, Visual-Motor Integration, finger tapping "Speed", and the Continuous Performance Test. Of 104 patients in 47 centres, 72 were eligible for analyses. We assessed whether IQ was impacted by disease extent, disease location, patient age, gender, age at surgery, and treatment (chemotherapy with our without craniospinal irradiation [CSI] or local radiotherapy [LRT]). Median age at surgery was 2.3 years. Testing was performed at a median of 4.9 years after surgery. Patients with infratentorial EPs (treated with LRT) scored highest in fluid intelligence (CPM 100.9±16.9, mean±SD); second best scores were achieved by patients with MB without metastasis treated with chemotherapy alone (CPM 93.9±13.2), followed by patients with supratentorial EPs treated with LRT. In contrast, lowest scores were achieved by patients that received chemotherapy and CSI, which included children with metastasised MB and those with relapsed MB M0 (CPM 71.7±8.0 and 73.2±21.8, respectively). Fine motor skills were reduced in all groups. Multivariable analysis revealed that type of treatment had an impact on IQ, but essentially not age at surgery, time since surgery or gender. Our results confirm previous reports on the detrimental effects of CSI in a larger cohort of children. Comparable IQ scores in children with MB treated only with chemotherapy and in children with EP suggest that this treatment strategy represents an attractive option for children who have a high chance to avoid application of CSI. Longitudinal follow-up examinations are warranted to assess long-term neuropsychological outcomes.

The identification of a heritable tumor predisposition often leads to changes in management and increased surveillance of individuals who are at risk; however, for many rare entities, our knowledge of heritable predisposition is incomplete.

Sonic Hedgehog (SHH) medulloblastomas are brain tumours that arise in the posterior fossa. Cancer-propagating cells (CPCs) provide a reservoir of cells capable of tumour regeneration and relapse post-treatment. Understanding and targeting the mechanisms by which CPCs are maintained and expanded in SHH medulloblastoma could present novel therapeutic opportunities. We identified the aryl hydrocarbon receptor (AHR) pathway as a potent tumour suppressor in a SHH medulloblastoma mouse model. Ahr-deficient tumours and CPCs grown in vitro, showed elevated activation of the TGFβ mediator, SMAD3. Pharmacological inhibition of the TGFβ/SMAD3 signalling axis was sufficient to inhibit the proliferation and promote the differentiation of Ahr-deficient CPCs. Human SHH medulloblastomas with high expression of the AHR repressor (AHRR) exhibited a significantly worse prognosis compared to AHRRlow tumours in two independent patient cohorts. Together, these findings suggest that reduced AHR pathway activity promotes SHH medulloblastoma progression, consistent with a tumour suppressive role for AHR. We propose that TGFβ/SMAD3 inhibition may represent an actionable therapeutic approach for a subset of aggressive SHH medulloblastomas characterised by reduced AHR pathway activity.

Recent research demonstrated that fear of progression (FoP) is a major burden for adult cancer survivors. However, knowledge on FoP in parents of childhood cancer survivors is scarce. This study aimed to determine the proportion of parents who show dysfunctional levels of FoP, to investigate gender differences, and to examine factors associated with FoP in mothers and fathers.

Cancer metabolism influences multiple aspects of tumorigenesis and causes diversity across malignancies. Although comprehensive research has extended our knowledge of molecular subgroups in medulloblastoma (MB), discrete analysis of metabolic heterogeneity is currently lacking. This study seeks to improve our understanding of metabolic phenotypes in MB and their impact on patients' outcomes.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

Medulloblastoma (MB) is the most common malignant brain tumor in children and requires intensive multimodal therapy. Long-term survival is still dissatisfying and, most importantly, survivors frequently suffer from severe treatment-associated morbidities. The sonic hedgehog pathway (SHH) in SHH MB provides a promising target for specific therapeutic agents. The small molecule Vismodegib allosterically inhibits SMO, the main upstream activator of SHH. Vismodegib has proven effective in the treatment of MB in mice and in clinical studies. However, due to irreversible premature epiphyseal growth plate fusions after systemic application to infant mice and children, its implementation to pediatric patients has been limited. Intraventricular Vismodegib application might provide a promising novel treatment strategy for pediatric medulloblastoma patients.

Pediatric high-grade gliomas of the subclass MYCN (HGG-MYCN) are highly aggressive tumors frequently carrying MYCN amplifications, TP53 mutations, or both alterations. Due to their rarity, such tumors have only recently been identified as a distinct entity, and biological as well as clinical characteristics have not been addressed specifically. To gain insights into tumorigenesis and molecular profiles of these tumors, and to ultimately suggest alternative treatment options, we generated a genetically engineered mouse model by breeding hGFAP-cre::Trp53Fl/Fl::lsl-MYCN mice. All mice developed aggressive forebrain tumors early in their lifetime that mimic human HGG-MYCN regarding histology, DNA methylation, and gene expression. Single-cell RNA sequencing revealed a high intratumoral heterogeneity with neuronal and oligodendroglial lineage signatures. High-throughput drug screening using both mouse and human tumor cells finally indicated high efficacy of Doxorubicin, Irinotecan, and Etoposide as possible therapy options that children with HGG-MYCN might benefit from.

Sonic Hedgehog (SHH) medulloblastomas (MBs) exhibit an intermediate prognosis and extensive intertumoral heterogeneity. While SHH pathway antagonists are effective in post-pubertal patients, younger patients exhibit significant side effects, and tumors that harbor mutations in downstream SHH pathway genes will be drug resistant. Thus, novel targeted therapies are needed. Here, we performed preclinical testing of the potent MEK inhibitor (MEKi) trametinib on tumor properties across 2 human and 3 mouse SHH MB models in vitro and in 3 orthotopic MB xenograft models in vivo. Trametinib significantly reduces tumorsphere size, stem/progenitor cell proliferation, viability, and migration. RNA-sequencing on human and mouse trametinib treated cells corroborated these findings with decreased expression of cell cycle, stem cell pathways and SHH-pathway related genes concomitant with increases in genes associated with cell death and ciliopathies. Importantly, trametinib also decreases tumor growth and increases survival in vivo. Cell cycle related E2F target gene sets are significantly enriched for genes that are commonly downregulated in both trametinib treated tumorspheres and primary xenografts. However, IL6/JAK STAT3 and TNFα/NFκB signaling gene sets are specifically upregulated following trametinib treatment in vivo indicative of compensatory molecular changes following long-term MEK inhibition. Our study reveals a novel role for trametinib in effectively attenuating SHH MB tumor progression and warrants further investigation of this potent MEK1/2 inhibitor either alone or in combination with other targeted therapies for the treatment of SHH MB exhibiting elevated MAPK pathway activity.