Clear cell meningioma represents an uncommon variant of meningioma that typically affects children and young adults. Although an enrichment of loss-of-function mutations in the SMARCE1 gene has been reported for this subtype, comprehensive molecular investigations are lacking. Here we describe a molecularly distinct subset of tumors (n = 31), initially identified through genome-wide DNA methylation screening among a cohort of 3093 meningiomas, of which most were diagnosed histologically as clear cell meningioma. This cohort was further supplemented by an additional 11 histologically diagnosed clear cell meningiomas for analysis (n = 42). Targeted DNA sequencing revealed SMARCE1 mutations in 33/34 analyzed samples, accompanied by a nuclear loss of expression determined via immunohistochemistry and a decreased SMARCE1 transcript expression in the tumor cells. Analysis of time to progression or recurrence of patients within the clear cell meningioma group (n = 14) in comparison to those with meningioma WHO grade 2 (n = 220) revealed a similar outcome and support the assignment of WHO grade 2 to these tumors. Our findings indicate the existence of a highly distinct epigenetic signature of clear cell meningiomas, separate from all other variants of meningiomas, with recurrent mutations in the SMARCE1 gene. This suggests that these tumors may arise from a different precursor cell population than the broad spectrum of the other meningioma subtypes.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

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.

The optimal treatment for patients with low-grade glioma (LGG) WHO grade II remains controversial. Overall survival ranges from 2 to over 15 years depending on molecular and clinical factors. Hence, risk-adjusted treatments are required for optimizing outcome and quality of life. We aim at identifying mechanisms and associated molecular markers predictive for benefit from radiotherapy (RT) or temozolomide (TMZ) in LGG patients treated in the randomized phase III trial EORTC 22033. As candidate biomarkers for these genotoxic treatments, we considered the DNA methylome of 410 DNA damage response (DDR) genes. We first identified 62 functionally relevant CpG sites located in the promoters of 24 DDR genes, using the LGG data from The Cancer Genome Atlas. Then we tested their association with outcome [progression-free survival (PFS)] depending on treatment in 120 LGG patients of EORTC 22033, whose tumors were mutant for isocitrate dehydrogenase 1 or 2 (IDHmt), the molecular hallmark of LGG. The results suggested that seven CpGs of four DDR genes may be predictive for longer PFS in one of the treatment arms that comprised MGMT, MLH3, RAD21, and SMC4. Most interestingly, the two CpGs identified for MGMT are the same, previously selected for the MGMT-STP27 score that is used to determine the methylation status of the MGMT gene. This score was higher in the LGG with 1p/19q codeletion, in this and other independent LGG datasets. It was predictive for PFS in the TMZ, but not in the RT arm of EORTC 22033. The results support the hypothesis that a high score predicts benefit from TMZ treatment for patients with IDHmt LGG, regardless of the 1p/19q status. This MGMT methylation score may identify patients who benefit from first-line treatment with TMZ, to defer RT for long-term preservation of cognitive function and quality of life.

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. Patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H IDH1/2 mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of the site of origin. For 1p/19q non-codeleted glioma (astrocytoma) patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring tumours with IDH mutations other than IDH1R132H have a better outcome (hazard ratio 0.41, 95% CI [0.24, 0.71], p = 0.0013). Such non-R132H IDH1/2-mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p < 0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H IDH1/2-mutated astrocytomas have a more favourable prognosis than their IDH1R132H mutated counterpart indicates that not all IDH-mutations are identical. This difference is clinically relevant and should be taken into account for patient prognostication.

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

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

Immunodeficiency-associated primary CNS lymphoma (PCNSL) represents a distinct clinicopathological entity, which is typically Epstein-Barr virus-positive (EBV+) and carries an inferior prognosis. Genetic alterations that characterize EBV-related CNS lymphomagenesis remain unclear precluding molecular classification and targeted therapies. In this study, a comprehensive genetic analysis of 22 EBV+ PCNSL, therefore, integrated clinical and pathological information with exome and RNA sequencing (RNASeq) data. EBV+ PCNSL with germline controls carried a median of 55 protein-coding single nucleotide variants (SNVs; range 24-217) and 2 insertions/deletions (range 0-22). Genetic landscape was largely shaped by aberrant somatic hypermutation with a median of 41.01% (range 31.79-53.49%) of SNVs mapping to its target motifs. Tumors lacked established SNVs (MYD88, CD79B, PIM1) and copy number variants (CDKN2A, HLA loss) driving EBV- PCNSL. Instead, EBV+ PCNSL were characterized by SOCS1 mutations (26%), predicted to disinhibit JAK/STAT signaling, and mutually exclusive gain-of-function NOTCH pathway SNVs (26%). Copy number gains were enriched on 11q23.3, a locus directly targeted for chromosomal aberrations by EBV, that includes SIK3 known to protect from cytotoxic T-cell responses. Losses covered 5q31.2 (STING), critical for sensing viral DNA, and 17q11 (NF1). Unsupervised clustering of RNASeq data revealed two distinct transcriptional groups, that shared strong expression of CD70 and IL1R2, previously linked to tolerogenic tumor microenvironments. Correspondingly, deconvolution of bulk RNASeq data revealed elevated M2-macrophage, T-regulatory cell, mast cell and monocyte fractions in EBV+ PCNSL. In addition to novel insights into the pathobiology of EBV+ PCNSL, the data provide the rationale for the exploration of targeted therapies including JAK-, NOTCH- and CD70-directed approaches.

Oligodendrogliomas are defined at the molecular level by the presence of an IDH mutation and codeletion of chromosomal arms 1p and 19q. In the past, case reports and small studies described gliomas with sarcomatous features arising from oligodendrogliomas, so called oligosarcomas. Here, we report a series of 24 IDH-mutant oligosarcomas from 23 patients forming a distinct methylation class. The tumors were recurrences from prior oligodendrogliomas or developed de novo. Precursor tumors of 12 oligosarcomas were histologically and molecularly indistinguishable from conventional oligodendrogliomas. Oligosarcoma tumor cells were embedded in a dense network of reticulin fibers, frequently showing p53 accumulation, positivity for SMA and CALD1, loss of OLIG2 and gain of H3K27 trimethylation (H3K27me3) as compared to primary lesions. In 5 oligosarcomas no 1p/19q codeletion was detectable, although it was present in the primary lesions. Copy number neutral LOH was determined as underlying mechanism. Oligosarcomas harbored an increased chromosomal copy number variation load with frequent CDKN2A/B deletions. Proteomic profiling demonstrated oligosarcomas to be highly distinct from conventional CNS WHO grade 3 oligodendrogliomas with consistent evidence for a smooth muscle differentiation. Expression of several tumor suppressors was reduced with NF1 being lost frequently. In contrast, oncogenic YAP1 was aberrantly overexpressed in oligosarcomas. Panel sequencing revealed mutations in NF1 and TP53 along with IDH1/2 and TERT promoter mutations. Survival of patients was significantly poorer for oligosarcomas as first recurrence than for grade 3 oligodendrogliomas as first recurrence. These results establish oligosarcomas as a distinct group of IDH-mutant gliomas differing from conventional oligodendrogliomas on the histologic, epigenetic, proteomic, molecular and clinical level. The diagnosis can be based on the combined presence of (a) sarcomatous histology, (b) IDH-mutation and (c) TERT promoter mutation and/or 1p/19q codeletion, or, in unresolved cases, on its characteristic DNA methylation profile.

Glioblastoma (GBM) derived from the "stem cell" rich subventricular zone (SVZ) may constitute a therapy-refractory subgroup of tumors associated with poor prognosis. Risk stratification for these cases is necessary but is curtailed by error prone imaging-based evaluation. Therefore, we aimed to establish a robust DNA methylome-based classification of SVZ GBM and subsequently decipher underlying molecular characteristics. MRI assessment of SVZ association was performed in a retrospective training set of IDH-wildtype GBM patients (n = 54) uniformly treated with postoperative chemoradiotherapy. DNA isolated from FFPE samples was subject to methylome and copy number variation (CNV) analysis using Illumina Platform and cnAnalysis450k package. Deep next-generation sequencing (NGS) of a panel of 130 GBM-related genes was conducted (Agilent SureSelect/Illumina). Methylome, transcriptome, CNV, MRI, and mutational profiles of SVZ GBM were further evaluated in a confirmatory cohort of 132 patients (TCGA/TCIA). A 15 CpG SVZ methylation signature (SVZM) was discovered based on clustering and random forest analysis. One third of CpG in the SVZM were associated with MAB21L2/LRBA. There was a 14.8% (n = 8) discordance between SVZM vs. MRI classification. Re-analysis of these patients favored SVZM classification with a hazard ratio (HR) for OS of 2.48 [95% CI 1.35-4.58], p = 0.004 vs. 1.83 [1.0-3.35], p = 0.049 for MRI classification. In the validation cohort, consensus MRI based assignment was achieved in 62% of patients with an intraclass correlation (ICC) of 0.51 and non-significant HR for OS (2.03 [0.81-5.09], p = 0.133). In contrast, SVZM identified two prognostically distinct subgroups (HR 3.08 [1.24-7.66], p = 0.016). CNV alterations revealed loss of chromosome 10 in SVZM- and gains on chromosome 19 in SVZM- tumors. SVZM- tumors were also enriched for differentially mutated genes (p < 0.001). In summary, SVZM classification provides a novel means for stratifying GBM patients with poor prognosis and deciphering molecular mechanisms governing aggressive tumor phenotypes.