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.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.

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.

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens.

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.

Atypical teratoid/rhabdoid tumor (ATRT) is an aggressive central nervous system tumor characterized by loss of SMARCB1/INI1 protein expression and comprises three distinct molecular groups, ATRT-TYR, ATRT-MYC and ATRT-SHH. ATRT-SHH represents the largest molecular group and is heterogeneous with regard to age, tumor location and epigenetic profile. We, therefore, aimed to investigate if heterogeneity within ATRT-SHH might also have biological and clinical importance. Consensus clustering of DNA methylation profiles and confirmatory t-SNE analysis of 65 ATRT-SHH yielded three robust molecular subgroups, i.e., SHH-1A, SHH-1B and SHH-2. These subgroups differed by median age of onset (SHH-1A: 18 months, SHH-1B: 107 months, SHH-2: 13 months) and tumor location (SHH-1A: 88% supratentorial; SHH-1B: 85% supratentorial; SHH-2: 93% infratentorial, often extending to the pineal region). Subgroups showed comparable SMARCB1 mutational profiles, but pathogenic/likely pathogenic SMARCB1 germline variants were over-represented in SHH-2 (63%) as compared to SHH-1A (20%) and SHH-1B (0%). Protein expression of proneural marker ASCL1 (enriched in SHH-1B) and glial markers OLIG2 and GFAP (absent in SHH-2) as well as global mRNA expression patterns differed, but all subgroups were characterized by overexpression of SHH as well as Notch pathway members. In a Drosophila model, knockdown of Snr1 (the fly homologue of SMARCB1) in hedgehog activated cells not only altered hedgehog signaling, but also caused aberrant Notch signaling and formation of tumor-like structures. Finally, on survival analysis, molecular subgroup and age of onset (but not ASCL1 staining status) were independently associated with overall survival, older patients (> 3 years) harboring SHH-1B experiencing relatively favorable outcome. In conclusion, ATRT-SHH comprises three subgroups characterized by SHH and Notch pathway activation, but divergent molecular and clinical features. Our data suggest that molecular subgrouping of ATRT-SHH has prognostic relevance and might aid to stratify patients within future clinical trials.

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.

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.

In 2012, an international consensus paper reported that medulloblastoma comprises four molecular subgroups (WNT, SHH, Group 3, and Group 4), each associated with distinct genomic features and clinical behavior. Independently, multiple recent reports have defined further intra-subgroup heterogeneity in the form of biologically and clinically relevant subtypes. However, owing to differences in patient cohorts and analytical methods, estimates of subtype number and definition have been inconsistent, especially within Group 3 and Group 4. Herein, we aimed to reconcile the definition of Group 3/Group 4 MB subtypes through the analysis of a series of 1501 medulloblastomas with DNA-methylation profiling data, including 852 with matched transcriptome data. Using multiple complementary bioinformatic approaches, we compared the concordance of subtype calls between published cohorts and analytical methods, including assessments of class-definition confidence and reproducibility. While the lowest complexity solutions continued to support the original consensus subgroups of Group 3 and Group 4, our analysis most strongly supported a definition comprising eight robust Group 3/Group 4 subtypes (types I-VIII). Subtype II was consistently identified across all component studies, while all others were supported by multiple class-definition methods. Regardless of analytical technique, increasing cohort size did not further increase the number of identified Group 3/Group 4 subtypes. Summarizing the molecular and clinico-pathological features of these eight subtypes indicated enrichment of specific driver gene alterations and cytogenetic events amongst subtypes, and identified highly disparate survival outcomes, further supporting their biological and clinical relevance. Collectively, this study provides continued support for consensus Groups 3 and 4 while enabling robust derivation of, and categorical accounting for, the extensive intertumoral heterogeneity within Groups 3 and 4, revealed by recent high-resolution subclassification approaches. Furthermore, these findings provide a basis for application of emerging methods (e.g., proteomics/single-cell approaches) which may additionally inform medulloblastoma subclassification. Outputs from this study will help shape definition of the next generation of medulloblastoma clinical protocols and facilitate the application of enhanced molecularly guided risk stratification to improve outcomes and quality of life for patients and their families.

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

This study aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in WNT-activated medulloblastoma (WNT-MB). In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by DNA sequencing. Chromosomal copy-number aberrations were assessed by molecular inversion probe technology (MIP), SNP6, or 850k methylation array hybridization. Prognostic impact was evaluated in 120 patients with follow-up data from the HIT2000 medulloblastoma trial or HIT registries. CTNNB1 mutations were present in 92.2%, and APC mutations in 6.8% of samples. One CTNNB1 wild-type tumor gained WNT activation due to homozygous FBXW7 deletion. Monosomy 6 was present in 78.6%, and more frequent in children than adults. 16.1% of tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. Loss of heterozygosity at the TP53 locus (chromosome 17p13.1) was found in 40.7% (11/27) of TP53 mutant tumor samples and in 12.6% of TP53 wild-type cases (13/103). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; 5-year-PFS 68% versus 93%, p = 0.001), and were enriched for chromosomes 17p (p = 0.001), 10, and 13 losses. Gains of OTX2 (14q22.3) occurred in 38.9% of samples and were associated with poor PFS and OS (5-year-PFS 72% versus 93%, p = 0.017 resp. 5-year-OS 83% versus 97%, p = 0.006). Multivariable Cox regression analysis for PFS/OS identified both genetic alterations as independent prognostic markers. Our data suggest that patients with WNT-MB carrying TP53 mutations or OTX2 gains (58.1%) are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

Gliomas demonstrate epigenetic dysregulation exemplified by the Glioma CpG Island Methylator Phenotype (G-CIMP) seen in IDH1 mutant tumors. 5-Hydroxymethylcytosine (5hmC) is implicated in glioma pathogenesis; however, its role in IDH1 mutant gliomas is incompletely understood. To characterize 5hmC in IDH1 mutant gliomas further, we examine 5hmC in a cohort of IDH1 mutant and wild-type high-grade gliomas (HGG) using a quantitative locus-specific approach. Regions demonstrating high 5hmC abundance and differentially hydroxymethylated regions (DHMR) enrich for enhancers implicated in glioma pathogenesis. Among these regions, IDH1 mutant tumors possess greater 5hmC compared to wild type. 5hmC contributes to overall methylation status of G-CIMP genes. 5hmC targeting gene body regions correlates significantly with increased gene expression. In particular, a strong correlation between increased 5hmC and increased gene expression is identified for genes highly expressed in the IDH1 mutant cohort. Overall, locus-specific gain of 5hmC targeting regulatory regions and associated with overexpressed genes suggests a significant role for 5hmC in IDH1 mutant HGG.

Medulloblastoma is curable in approximately 70% of patients. Over the past decade, progress in improving survival using conventional therapies has stalled, resulting in reduced quality of life due to treatment-related side effects, which are a major concern in survivors. The vast amount of genomic and molecular data generated over the last 5-10 years encourages optimism that improved risk stratification and new molecular targets will improve outcomes. It is now clear that medulloblastoma is not a single-disease entity, but instead consists of at least four distinct molecular subgroups: WNT/Wingless, Sonic Hedgehog, Group 3, and Group 4. The Medulloblastoma Down Under 2013 meeting, which convened at Bunker Bay, Australia, brought together 50 leading clinicians and scientists. The 2-day agenda included focused sessions on pathology and molecular stratification, genomics and mouse models, high-throughput drug screening, and clinical trial design. The meeting established a global action plan to translate novel biologic insights and drug targeting into treatment regimens to improve outcomes. A consensus was reached in several key areas, with the most important being that a novel classification scheme for medulloblastoma based on the four molecular subgroups, as well as histopathologic features, should be presented for consideration in the upcoming fifth edition of the World Health Organization's classification of tumours of the central nervous system. Three other notable areas of agreement were as follows: (1) to establish a central repository of annotated mouse models that are readily accessible and freely available to the international research community; (2) to institute common eligibility criteria between the Children's Oncology Group and the International Society of Paediatric Oncology Europe and initiate joint or parallel clinical trials; (3) to share preliminary high-throughput screening data across discovery labs to hasten the development of novel therapeutics. Medulloblastoma Down Under 2013 was an effective forum for meaningful discussion, which resulted in enhancing international collaborative clinical and translational research of this rare disease. This template could be applied to other fields to devise global action plans addressing all aspects of a disease, from improved disease classification, treatment stratification, and drug targeting to superior treatment regimens to be assessed in cooperative international clinical trials.

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.

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation.

In contrast to adults, meningiomas are uncommon tumors in childhood and adolescence. Whether adult and pediatric meningiomas differ on a molecular level is unclear. Here we report detailed genomic analyses of 37 pediatric meningiomas by sequencing and DNA methylation profiling. Histologically, the series was dominated by meningioma subtypes with aggressive behavior, with 70% of patients suffering from WHO grade II or III meningiomas. The most frequent cytogenetic aberrations were loss of chromosomes 22 (23/37 [62%]), 1 (9/37 [24%]), 18 (7/37 [19%]), and 14 (5/37 [14%]). Tumors with NF2 alterations exhibited overall increased chromosomal instability. Unsupervised clustering of DNA methylation profiles revealed separation into three groups: designated group 1 composed of clear cell and papillary meningiomas, whereas group 2A comprised predominantly atypical meningiomas and group 2B enriched for rare high-grade subtypes (rhabdoid, chordoid). Meningiomas from NF2 patients clustered exclusively within groups 1 and 2A. When compared with a dataset of 105 adult meningiomas, the pediatric meningiomas largely grouped separately. Targeted panel DNA sequencing of 34 tumors revealed frequent NF2 alterations, while other typical alterations found in adult non-NF2 tumors were absent. These data demonstrate that pediatric meningiomas are characterized by molecular features distinct from adult tumors.

Prognostic factors and standards of care for astrocytoma, isocitrate dehydrogenase (IDH)-mutant, CNS WHO grade 4, remain poorly defined. Here we sought to explore disease characteristics, prognostic markers, and outcome in patients with this newly defined tumor type. We determined molecular biomarkers and assembled clinical and outcome data in patients with IDH-mutant astrocytomas confirmed by central pathology review. Patients were identified in the German Glioma Network cohort study; additional cohorts of patients with CNS WHO grade 4 tumors were identified retrospectively at two sites. In total, 258 patients with IDH-mutant astrocytomas (114 CNS WHO grade 2, 73 CNS WHO grade 3, 71 CNS WHO grade 4) were studied. The median age at diagnosis was similar for all grades. Karnofsky performance status at diagnosis inversely correlated with CNS WHO grade (p < 0.001). Despite more intensive treatment upfront with higher grade, CNS WHO grade was strongly prognostic: median overall survival was not reached for grade 2 (median follow-up 10.4 years), 8.1 years (95% CI 5.4-10.8) for grade 3, and 4.7 years (95% CI 3.4-6.0) for grade 4. Among patients with CNS WHO grade 4 astrocytoma, median overall survival was 5.5 years (95% CI 4.3-6.7) without (n = 58) versus 1.8 years (95% CI 0-4.1) with (n = 12) homozygous CDKN2A deletion. Lower levels of global DNA methylation as detected by LINE-1 methylation analysis were strongly associated with CNS WHO grade 4 (p < 0.001) and poor outcome. MGMT promoter methylation status was not prognostic for overall survival. Histomolecular stratification based on CNS WHO grade, LINE-1 methylation level, and CDKN2A status revealed four subgroups of patients with significantly different outcomes. In conclusion, CNS WHO grade, global DNA methylation status, and CDKN2A homozygous deletion are prognostic in patients with IDH-mutant astrocytoma. Combination of these parameters allows for improved prediction of outcome. These data aid in designing upcoming trials using IDH inhibitors.