A highly abundant circular RNA from the RMST locus plays a role in posterior fossa ependymoma pathogenesis.

Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need low-cost techniques to promptly classify these molecular subtypes. Here, we applied low-cost methods to classify EPNs from a Brazilian cohort with 60 pediatric EPN patients. Fusion transcripts (C11orf95-RELA, YAP1-MAMLD1, and YAP1-FAM118B) were investigated in supratentorial EPN (ST-EPNs) samples through RT-PCR/Sanger sequencing and immunohistochemistry (IHC) for p65/L1CAM. qRT-PCR and IHC were used to evaluate expression profiling of CXorf67, LAMA2, NELL2, and H3K27me3 in posterior fossa EPN (PF-EPNs) samples. In silico analysis was performed using public microarray data to validate the molecular assignment PF-EPNs with LAMA2/NELL2 markers. RELA cases and YAP1-MAMLD1 fusions were identified in nine and four ST-EPNs, respectively. An additional RELA case was identified by IHC. Of note, LAMA2 and NELL2 gene expression and immunoprofiling were less accurate for classifying PF-EPNs, which were confirmed by in silico analysis. Yet, H3K27me3 staining was sufficient to classify PF-EPN subgroups. Our results emphasize the feasibility of a simplified strategy to molecularly classify EPNs in the vast majority of cases (49/60; 81.7%). A coordinated combination of simple methods can be effective to screen pediatric EPN with the available laboratory resources at most low-/mid-income countries, giving support for clinical practice in pediatric EPN. KEY MESSAGES: Low- and middle-income countries need effective low-cost approaches to promptly distinguish between EPN molecular subgroups. RT-PCR plus Sanger sequencing is able to recognize the most common types of RELA and YAP1 fusion transcripts in ST-EPNs. Genetic and protein expressions of LAMA2 and NELL2 are of limited value to accurately stratify PF-EPNs. Immunohistochemical staining for H3K27me3 may be used as a robust method to accurately diagnose PF-EPNs subgroups. A coordinated flow diagram based on these validated low-cost methods is proposed to help clinical-decision making and to reduce costs with NGS assessment outside research protocols.

INTRODUCTION: Ependymomas are rare glial tumors of the brain and spinal cord. Although common in children, posterior fossa (PF) ependymomas occur at any age and show marked clinical differences between children and adults. To better understand the biological basis for these observations, we performed a comparison of the expression profiles of PF ependymoma across age groups. METHOD: Our study included expression profiles of tumor samples from PF of 37 ependymoma patients with age ranging from 1 to 63 years. The change in gene expression among infants (0-3 years), children (4-17 years), and adults (≥18 years) was calculated using linear models based on Bayesian moderated statistics. RESULTS: We detected a total of 613 genes that show high expression levels in infants (adjusted P value, FDR < 0.05) and 444 genes in adults. In contrast, there was no significant gene expression alteration detected in Children. Similar trend of expression alterations was observed in an independent data set of 34 PF ependymomas (960 genes for infants; 434 for adults; and 1 gene for children). The infant PF ependymoma was enriched for genes associated with axon guidance, cell adhesion, ECM-receptor interaction, calcium, chemokine, MAPK, Neurotrophins, PPAR signaling pathways, whereas the adults were significantly enriched for genes associated with cilium organization and microtubule-based process. Markers of ECM signaling (LAMA2, log2 fold change (FC) = 2.82, FDR < 0.009; CD44, FC = 1.98, FDR < 0.007) were overexpressed in infants, while NELL2 (FC = 1.99, FDR < 0.002) was overexpressed in adult PF ependymomas. CONCLUSION: Our results showed substantial differences in gene expression landscapes underlying different age groups of PF ependymoma.

Ependymomas are the third most common childhood brain tumor. These tumors arise throughout the nervous system, but in children, are most common within posterior fossa (PF). Treatment consists of surgery and radiation, as chemotherapy has demonstrated little to no survival benefit. We have shown previously that PF ependymomas are divided into two clinically and molecularly distinct subtypes, termed Group A and Group B. While Group B tumors, which have a favorable outcome (5 year survival ∼95%) and are characterized by increased copy number alterations (CNAs), Group A tumours have a poor prognosis (5 year survival ∼20%) and have few somatic CNAs. To discover somatic SNVs of PF ependymomas, we performed whole genome-, and whole exome-sequencing of 47 PF ependymomas including matched germline DNA (27 PFA, and 20 PFB). In addition, we analysed DNA methylation patterns in a discovery cohort of 79 ependymomas using methyl-binding domain-2 (MBD2) protein recovery followed by hybridization to Nimblegen 385K CpG Island Promoter Plus microarrays (MBD2-chip). Unsupervised consensus clustering of CpG methylation profiles yielded in principle two distinct subtypes of posterior fossa ependymomas (Group A and B) respectively, in a pattern highly similar to that yielded by unsupervised clustering of gene expression profiles. We validated our findings in a non-overlapping cohort of 48 PF ependymomas using an orthogonal technology (Illumina Infinium450k methylation arrays). To uncover additional epigenetic alteration we performed whole genome bisulphite-sequencing in 6 tumors and H3K27me3 ChIP-seq in 11 primary PF ependymomas. Unlike some other childhood malignancies, the rate of somatic SNVs was extremely low in PF ependymomas, with an average of 5.0 somatic non-synonymous SNVs per exome across the entire cohort. While devoid of recurrent SNVs and focal copy number aberrations, poor prognosis Group A ependymomas exhibit a CpG island methylator phenotype (CIMP+). Further, transcriptional silencing driven by CpG methylation converges exclusively on targets of the polycomb repressor complex 2 (PRC2) that represses expression of differentiation genes through tri-methylation of H3K27. CIMP positive PF ependymomas (Group A) are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland. Since epigenetic modulators are already approved drugs, this therapeutic strategy could be rapidly repurposed to treat children with ependymoma. Citation Format: Hendrik Witt, Steve C. Mack, Stefan M. Pfister, Andrey Korshunov, Michael D. Taylor, Ependymoma Collaborative Consortium. Epigenomic alterations define lethal CIMP-positive ependymomas of infancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-198. doi:10.1158/1538-7445.AM2014-LB-198

Dose Escalation for Posterior Fossa Ependymomas: Analysis of Prospective Pediatric Proton/Photon Consortium Registry

Ependymomas originate in posterior fossa (PF), supratentorial (ST) or spinal cord (SC) compartments. At present, grading schemes are applied independent of anatomic site. We performed detailed histological examination on 238 World Health Organization grade II and III ependymomas. Among PF ependymomas, the presence of hypercellular areas, necrosis, microvascular proliferation and elevated mitotic rate (all P < 0.01) were significantly associated with worse progression-free survival (PFS), while extensive ependymal canal formation was not (P = 0.89). Similar to the PF tumors, microvascular proliferation (P = 0.01) and elevated mitotic rate (P = 0.03) were significantly associated with worse PFS in the ST tumors. However, in contrast to PF tumors, extensive ependymal canals (P = 0.03) were associated with worse clinical outcome in ST ependymomas, but hypercellularity (P = 0.57) and necrosis (P = 0.47) were not. On multivariate Cox regression, after adjusting for relevant clinical variables, individual histological factors and a composite histological score remained significant among ST and PF ependymoma. In contrast to both PF and ST ependymoma, histological features were not found to be associated with PFS in SC tumors. Taken together, the clinical relevance of specific histological features in ependymoma appears to be related to the anatomic site of origin and suggests that site-specific grading criteria be considered in future classification systems.

Background Posterior fossa brain ependymomas are one of the most devastating forms of human illnesses which are more common in children. Brainstem compression, herniation and death are the risks with tumours in this critical location. Patients and Methods: A retrospective study including 50 patients with posterior fossa ependymoma were performed at the Neurosurgery Department in Ait IDDIR University Hospital between the period of January 2005 and December 2015 . In each case, diagnosis was made clinically and confirmed radiologically and histo-pathologically.. All patients received the adjuvant treatment Results Out of 50 patients, 30 (60%) patients were males and 20 (40%) were females. The mean age was 24 years (ranged 5 months –47 years) ; we identified 29 (58%) children and 21 (42%) adults,. Ventriculo-peritoneal shunts were placed in all our patients, the total tumor excision was done to 35% and the partial tumor excision was done to 65% patients. The most common complications were as follows: Shunt malfunction : 4% , operative cavity hematoma 6%, CSF fistula 4%, deterioration of Cerebellar syndrome 6%, Cerebellar Mutism 2%, Mixed nerves palsy 4% and early post operative deaths 10%. Tumor architecture was classified as classic (Grade II) in 35 (70%) cases and anaplastic (Grade III) in 13(26%) cases. Adjuvant treatment regimens following resection included radiation therapy only (72%) for 36 patients including children above and adults and chemotherapy only (36%) for 9 children below 4 years and 9 recurrent tumors. During follow-up period, recurrence occurred in 27% 11patients out of 41patients .Five patients died (10%). Conclusion The surgical treatment of posterior fossa tumours still represents a challenge for neurosurgeons, Radical surgery with preservation of vital structures is the treatment of choice in patients with Posterior Cerebral Fossa ependymomas.. Our experience shows the accepted results, complications and surgical outcome in relation to previous clinical studies.

The ACNS0831 Children’s Oncology Group clinical trial enrolled patients from 2010 to 2019 with the primary aim of determining whether post-irradiation chemotherapy offers a survival benefit to children with intracranial ependymoma. Histopathological metrics to inform grade were collected from tumors of 449 patients, 283 (63%) with a posterior fossa (PF) ependymoma, and 166 (37%) with a supratentorial (ST) ependymoma. A retrospective analysis of molecular group (ZFTA, YAP1, PFA, PFB), H3K27-trimethylation status (PF tumors), and CNVs on chromosomes 1q and 6q (PF tumors) and at the CDKN2A locus (ST tumors) was undertaken on prospectively collected samples using methylation profiling, fluorescence in situ hybridization, and immunohistochemistry. Survival data were used to assess the prognostic value of histopathological and molecular variables. Of ST and PF ependymomas, 75% and 54% were WHO grade 3, respectively, the remainder being WHO grade 2. Grade was significantly (p&amp;lt;0.01) associated with event-free and overall survival in all patients with a gross total resection. Molecular group was established in 422/449 (94%) of cases. Most ependymomas were classified by methylation profiling as PFA (59%), followed by ZFTA (31%), PFB (8%), and YAP1 (2%). A relatively good survival was associated with PFB tumors, when compared to ZFTA and PFA tumors. Gain of 1q and loss of 6q were associated with a worse survival among PF and PFA tumors, but homozygous deletion of CDKN2A was not an adverse prognostic factor among ST tumors. Utilizing the histopathological and molecular data from PF ependymomas, it was possible to create ‘tumor risk-groups’ with significantly different survival outcomes. However, ST ependymoma molecular markers did not carry prognostic value. This is the first clinical trial of new therapeutic approaches for intracranial ependymoma that has prospectively collected tissue for molecular analysis. Trial data suggest that histopathological and molecular variables can be used in future trials for therapeutic stratification.

Brain tumors are the most common cause of cancer-related death in childhood. Ependymomas, are the third most common pediatric brain tumor. The disease remains incurable for about 45% of patients even after gross total resection and radiotherapy. Despite showing a very homogeneous histological picture, ependymomas display distinct molecular behavior, which supports the existence of several independent entities of the disease. We examined two non-overlapping cohorts of 102 and 75 ependymomas by mRNA expression profiling, on two different array platforms (Affymetrix, Agilent). When performing multiple statistical clustering methods (unsupervised consensus NMF and consensus HCL), we could consistently identify three major clusters, including two subgroups of posterior fossa (PF) ependymoma, a variant common in children and associated with heterogeneous clinical outcome. Subgroup-specific chromosome aberrations of PF tumors were detected by aCGH, and biological signaling pathways distinguishing PF subgroups were identified by gene set enrichment analysis and visualized in Cytoscape. We validated the most significantly classifying markers of each subgroup by immunohistochemistry on a tissue microarray containing an independent set of 265 PF ependymomas. Our findings delineate two subgroups of PF ependymoma (groups A and B) which are demographically, transcriptionally, genetically, and clinically distinct. Group A patients are younger, have laterally located tumors with a balanced genome, more frequently develop secondary metastases and are much more likely to have an extremely poor outcome as compared with group B patients. Based on a multi-variate Cox proportional-hazards model, our identified markers have the strongest independent prognostic value among demographic and molecular variables with Hazard ratios of 8.45 (PFS) and 10.55 (OS). Prognostic significance and predictive impact is being validated in the GPOH HIT2000 Ependymoma study. The identification of two distinct subgroups of PF ependymoma, and markers applicable for their clinical distinction, will allow for better prognostication of individual cases, independent of age, level of resection and WHO grade, and also for stratification in future ependymoma clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1432. doi:1538-7445.AM2012-1432

Background: Ependymoma (EPN) is the third most common malignant pediatric brain tumor. Current standard therapy include maximally safe surgical resection followed by radiation and lead to a 5-year overall survival of 50-71%. Recent molecular subgrouping of EPN has identified one group, posterior fossa A (PFA), which accounts for 45% of all EPN cases, to have one of the worst prognosis and it is driven by epigenetic changes, suggesting targeting epigenetic changes in PFA EPN can potentially be effective. In this study, we examined the therapeutic efficacy of SYC-836, a novel LSD-1 inhibitor compound developed at Baylor College of Medicine, both in vitro and in vivo in PDOX models of posterior fossa EPN. Methods: To examine in vitro anti-tumor activities, paired primary cultured cells (both as attached cells and neurospheres) from an established PDOX model of posterior fossa EPN (ICb-4423EPN) were subjected to SYC-836 at various concentrations (0-25uM). Cell viability and proliferation were measured using Cell Counting Kit-8 assay at 5 different time points over 14 days. To validate the drug’s in vivo efficacy, two established posterior fossa EPN PDOX models, ICb-4423EPN and ICb-2002EPN, were utilized. 40 eight weeks old SCID mice per model were implanted with tumor cells. They were divided into 4 treatment groups (10 mice/group) each: 1) control (DPBS, 10uL/kg IP daily x 28 days), 2) radiation/standard therapy (2 Gy focal XRT daily x 5 days), 3) SYC-836 only (15mg/kg IP daily x 28 days), and 4) combination (radiation + SYC-836 per regimen above). Animal survival times were analyzed using log rank analysis. Changes of histone lysine methylation were examined through western hybridization. Results: SYC-836 demonstrated effective cell killing in vitro against both attached and neurosphere cultured cells in both time- and dose-dependent manner. IC50 was ~7.5uM. In vivo experiment was completed in 1 of the 2 EPN PDOX models (ICb-2002EPN) with the second model ongoing. Median survival times for each group is as followed: control 136 days, radiation 148 days, SYC-836 only 136 days, combination 180 days. There were no survival benefit with either XRT only (P=0.205) or SYC-836 only (P=0.186) when compared to the control group; however, when used in combination, the treatment strategy lead to significant improvement in animal survival (P=0.004). SYC-836 was well tolerated in mice. Conclusion: Our data showed that combining SYC-836 with current standard therapy of radiation synergistically prolongs animal survival significantly, although as a single agent SYC-836 was not effective against posterior fossa ependymoma. Our data suggest that SYC-836 may have a role in the clinical setting by either reducing radiation dosages, or be a potential adjuvant agent to other chemotherapy drugs in our treatment approach for ependymoma. Citation Format: Sibo Zhao, Huiyuan Zhang, Lin Qi, Holly Lindsay, Yuchen Du, Mari Kogiso, Frank Braun, Sarah Injac, Laszlo Perlaky, Donald W. Parsons, Murali Chintagumpala, Adekunle Adesina, Yongcheng Song, Xiao-Nan Li. Lysine specific demethylase-1 (LSD-1) Inhibitor SYC-836 in combination with radiation prolongs animal survival in patient-derived posterior fossa ependymoma xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2017-5058

INTRODUCTION Ependymomas are a heterogenous group of central nervous system tumors, with significant variability in clinical outcomes influenced by molecular subtype, location, and extent of resection. Posterior fossa (PF) ependymomas represent a distinct clinical challenge due to variable prognosis and treatment response. The objective of this study was to determine whether recent advances in DNA methylation profiling which have enabled more precise molecular classification, will guide prognosis and therapy in posterior fossa ependymomas. METHODS We retrospectively analyzed 18 patients with ependymomas who underwent DNA methylation testing at our institution. The mean age was 37.9 years (17 months to 78 years). Tumor locations included the posterior fossa (n=11), spine (n=4), and supratentorial region (n=3). Tumor grading revealed WHO I (n=1), WHO II (n=7), WHO III (n=9), and one unassigned. Molecular subtypes identified were PF-EPN-A (n=3), PF-EPN-B (n=4), PF-SE (n=4), SP-MPE (n=2), SP-EPN (n=2), ST-EPN-RELA (n=3), and one unclassifiable case. RESULTS PF-EPN-A subtypes were associated with a higher recurrence rate (2 of 3), and poorer progression-free survival, even with adjuvant radiation, while PF-EPN-B showed no recurrence following gross total resection (GTR). This aligns with prior reports estimating 5-years progression-free survival rates ranging from 26.1% to 56.8% for PF-EPN-A and exceeding 70% for PF-EPN-B with complete resection. PF-EPN-B and PF-SE subtypes achieved better outcomes with GTR alone, whereas PF-EPN-A demonstrated limited responsiveness. CONCLUSION These findings align with previous studies supporting the integration of DNA methylation profiling into routine evaluation for ependymoma management. For posterior fossa tumors, both molecular subtype and extent of resection should guide postoperative management, emphasizing the need for subtype-specific therapeutic approaches, particularly for high-risk PF-EPN-A cases.

Better understanding of ependymoma (EPN) biology at relapse is needed to improve therapy at this critical event. Convincing data exist defining transcriptionally distinct posterior fossa (PF) sub-groups A and B at diagnosis. The clinical and biological consequence of these sub-groups at recurrence has not yet been defined. Genome and transcriptome microarray profiles and clinical variables of matched primary and first recurrent PF EPN pairs were used to identify biologically distinct patterns of progression between EPN sub-groups at recurrence. Key findings were validated by histology and immune function assays. Transcriptomic profiles were partially conserved at recurrence. However, 4 of 14 paired samples changed sub-groups at recurrence, and significant sub-group-specific transcriptomic changes between primary and recurrent tumors were identified, which were predominantly immune-related. Further examination revealed that Group A primary tumors harbor an immune gene signature and cellular functionality consistent with an immunosuppressive phenotype associated with tissue remodeling and wound healing. Conversely, Group B tumors develop an adaptive, antigen-specific immune response signature and increased T-cell infiltration at recurrence. Clinical distinctions between sub-groups become more apparent after first recurrence. Group A tumors were more often sub-totally resected and had a significantly shorter time to subsequent progression and worse overall survival. Minimal tumor-specific genomic changes were observed for either PF Groups A or B at recurrence. Molecular sub-groups of PF EPN convey distinct immunobiologic signatures at diagnosis and recurrence, providing potential biologic rationale to their disparate clinical outcomes. Immunotherapeutic approaches may be warranted, particularly in Group A PF EPN.

Data on frequency, clinical presentation, and outcome of primary metastatic intracranial ependymoma in children are scarce. Prospective data on patients younger than 21 years with metastatic intracranial ependymoma at first diagnosis, registered from 2001 to 2014 in the HIT-2000 trial and the HIT-2000 Interim Registry, were analyzed. Of 453 registered patients with intracranial ependymoma and central neuropathology review, initial staging included spinal magnetic resonance imaging in all patients and lumbar cerebrospinal fluid (CSF) analysis in 402 patients. Ten patients (2.2%) had metastatic disease, including three with microscopic CSF positivity only (M1 metastasis stage, 0.7% of patients with CSF staging). Location of the primary tumor was supratentorial in four patients (all supratentorial RELA-fused ependymoma [ST-EPN-RELA]) and within the posterior fossa in five patients (posterior fossa ependymoma type A [PF-EPN-A], n = 4; posterior fossa ependymoma not further classifiable, n = 1), and multifocal in one patient.All four patients with ST-EPN-RELA were alive in first or second complete remission (CR) 7.5-12.3 years after diagnosis. All four patients with macroscopic metastases of posterior fossa or multifocal ependymoma died. Three patients with initial M1 stage (ST-EPN-RELA, n = 1; PF-EPN-A, n = 2) received chemotherapy and local irradiation and were alive in second or third CR 3.0-9.7 years after diagnosis. Progression-free and overall survival of the entire cohort at 5 years was 13% (±6%), and 58% (±16%), respectively. Primary metastatic disease is rare in children with intracranial ependymoma. Prognosis may depend on molecular subgroup and extent of dissemination, and relevance of CSF analysis for initial staging remains to be clarified. Childhood ependymoma presenting with metastasis at first diagnosis is very rare with a frequency of 2.4% in this population-based, well-characterized cohort. Detection of microscopic metastases in the cerebrospinal fluid was extremely rare, and impact on prognosis and respective treatment decision on irradiation field remains unclear. Initial metastatic presentation occurs in both supratentorial RELA-fused ependymoma and posterior fossa ependymoma. Prognosis may differ according to extent of metastasis and biological subgroup, with poor prognosis in diffusely spread metastatic posterior fossa ependymoma even after combination therapy with both intensive chemotherapy and craniospinal irradiation, which may help to guide individual therapeutic decisions for future patients.

Adult intracranial ependymomas (EPNs) are extremely rare brain tumors. Currently, clinical and molecular factors that could inform individualized treatment strategies are still lacking for EPNs in this age group. The aim of this study was to investigate potential prognostic indicators and rational therapeutic management in a large cohort of adult intracranial EPNs. Adult patients who underwent resection of World Health Organization (WHO) grade II or III intracranial EPNs were included. The demographic features, clinicopathologic manifestations, molecular subgroups, and outcomes were retrospectively analyzed. Overall survival and progression-free survival were calculated using the Kaplan-Meier analysis. Potential prognostic indicators were identified using multivariable Cox proportional hazards model. This cohort included 236 adult patients with a mean age of 36.2 years (range: 18 to 72 y) at diagnosis. The tumor location was supratentorial (ST) in 102 (43.2%) and infratentorial in 134 (56.8%). Pathologic analysis revealed 43.1% of ST-EPNs with RELA fusion and 88.1% of posterior fossa ependymomas (PF-EPNs) with positive H3K27me3 staining. Gross total removal was achieved in 169 cases (71.6%). During follow-up, 97 (41.1%) patients had disease progression and 39 (16.5%) died. Kaplan-Meier analysis showed that patients with H3K27me3-positive PF-EPN had excellent survival, whereas patients with RELA fusion-positive ST-EPN or H3K27me3-negative PF-EPN had poor prognosis (progression-free survival: P=1.3E-16, overall survival: P=2.5E-12). Multivariate analysis showed that molecular subgroup, extent of resection, and Ki-67 index were strong independent prognostic indicators. In conclusion, our study provides essential information on the prognostic prediction of adult intracranial EPNs that will assist in establishing appropriate risk stratification and individualized treatment strategies in future clinical trials.

Epithelial-to-mesenchymal transition–related transcription factors are up-regulated in ependymomas and correlate with a poor prognosis

The Consortium to Inform Molecular and Practical Approaches to Central Nervous System Tumor Taxonomy (cIMPACT‐NOW) updates provide guidelines for the diagnosis of central nervous system (CNS) tumors and suggestions for future World Health Organization (WHO) classification. Following publication of the fifth edition WHO Classification of CNS Tumors (WHO CNS5) in 2021, the cIMPACT‐NOW working group “Clarification” reviewed WHO CNS5 and prioritized two topics for further elucidation: (a) distinction of Glioblastoma, IDH‐wildtype from Diffuse pediatric‐type high‐grade glioma, H3‐wildtype, and IDH‐wildtype and (b) clarification of subgroups of posterior fossa (PF) ependymal tumors. Recommendations regarding the IDH‐ and H3‐wildtype diffuse high‐grade gliomas include: (1) use caution assigning CNS WHO grade 4 (diagnosis of Glioblastoma, IDH‐wildtype) to a “TERT promoter only”, histologically low‐grade, IDH‐wildtype tumor; (2) EGFR gene amplification and +7/−10 chromosome copy number alterations should not be used as solitary defining features for diagnosing high‐grade gliomas as Glioblastoma, IDH‐wildtype in patients <40 years of age; (3) Diffuse pediatric‐type high‐grade glioma, H3‐wildtype, and IDH‐wildtype should be considered in the differential diagnosis in adults, especially those <40 years of age; (4) PDGFRA alteration, EGFR alteration, or MYCN amplification count as key molecular features of Diffuse pediatric‐type high‐grade glioma, H3‐wildtype, and IDH‐wildtype only in patients <25 years. Guidelines for improved diagnosis of posterior fossa ependymal tumors include: (1) immunohistochemical demonstration of nuclear EZHIP supports classification as PF group A ependymoma; (2) a PF ependymoma with retained nuclear H3 K27me3 expression and no nuclear EZHIP overexpression for which DNA methylation profiling is not performed should be considered as PF ependymoma, “not otherwise specified”; (3) for emerging tumors not included in WHO CNS5, “not elsewhere classified” (NEC) can be added to the diagnosis. Of note, these recommendations are not formal changes to the WHO definitions and diagnostic criteria but are intended to provide diagnostic guidance in advance of WHO CNS6.