Ependymoma with RELA fusion has been defined as a novel entity of the revised World Health Organization 2016 classification of tumors of the central nervous system (CNS), characterized by fusion transcripts of the RELA gene and consequent pathological activation of the NFkB pathway. These tumors represent the majority of supratentorial ependymomas in children. The validation of diagnostic tools to identify this clinically relevant ependymoma entity is essential. Here, we have used interphase fluorescent in situ hybridization (FISH) for C11orf95 and RELA, immunohistochemistry (IHC) for p65-RelA and the recently developed DNA methylation-based classification besides conventional histopathology, and compared the precision of the methods in 40 supratentorial pediatric brain tumors diagnosed as ependymomas in the past years. Reverse transcription PCR (RT-PCR) and RNA sequencing were performed to explore discordant cases. Furthermore, we integrated imaging and clinical features as additional layers of information. The concordance between nuclear RelA expression by IHC and RELA FISH was 100%. Concordance between IHC and DNA methylation profiling, and between FISH and DNA methylation profiling was also high (96.4% and 95.2%, respectively). Thirty-four out of 40 (85%) cases were confirmed by integrated diagnoses as ependymal tumors, including 22 RELA-fused ependymomas (71% of ependymal tumors), two YAP1-fused ependymomas (6%), six non-RELA/non-YAP1 ependymomas (18%) and four ependymal/subependymal mixed tumors (12%). Ependymal/subependymal mixed tumors had an excellent clinical outcome despite the presence of histopathological signs of malignancy, suggesting that these tumors should not be diagnosed as classic ependymomas. DNA methylation profiling helped in the differential diagnosis of RELA-fused ependymomas. IHC and FISH, which are available in the majority of pathology laboratories, are valuable tools to identify RELA-fused ependymomas.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant tumor that may not only contain rhabdoid tumor cells but also poorly differentiated small-round-blue cells as well as areas with mesenchymal or epithelial differentiation. Little is known on factors associated with histopathological diversity. Recent studies demonstrated three molecular subgroups of AT/RT, namely ATRT-TYR, ATRT-SHH, and ATRT-MYC. We thus aimed to investigate if morphological patterns might be related to molecular subgroup status. Hematoxylin-eosin stained sections of 114 AT/RT with known molecular subgroup status were digitalized and independently categorized by nine blinded observers into four morphological categories, that is, "rhabdoid," "small-round-blue," "epithelial," and "mesenchymal." The series comprised 48 ATRT-SHH, 40 ATRT-TYR, and 26 ATRT-MYC tumors. Inter-observer agreement was moderate but significant (Fleiss' kappa = 0.47; 95% C.I. 0.41-0.53; p < 0.001) and there was a highly significant overall association between morphological categories and molecular subgroups for each of the nine observers (p < 0.0001). Specifically, the category "epithelial" was found to be over-represented in ATRT-TYR (p < 0.000001) and the category "small-round-blue" to be over-represented in ATRT-SHH (p < 0.01). The majority of ATRT-MYC was categorized as "mesenchymal" or "rhabdoid," but this association was less compelling. The specificity of the category "epithelial" for ATRT-TYR was highest and accounted for 97% (range: 88-99%) whereas sensitivity was low [49% (range: 35%-63%)]. In line with these findings, cytokeratin-positivity was highly overrepresented in ATRT-TYR. In conclusion, morphological features of AT/RT might reflect molecular alterations and may also provide a first hint on molecular subgroup status, which will need to be confirmed by DNA methylation profiling.

Meningiomas are the most frequent primary intracranial tumors. The considerable variety of histological subtypes has been expanded by the definition of molecular alterations, which can improve both diagnostic accuracy and determination of individual patient's outcome. According to the upcoming WHO classification of brain tumors, the in-time analysis of frequent molecular events in meningiomas may become mandatory to define meningioma subtypes. We have compiled a custom-made amplicon-based next generation sequencing (NGS) meningioma panel covering the most frequent known recurrent mutations in 15 different genes. In an unselected consecutive meningioma cohort (109 patients) analyzed over a period of 12 months, we detected mutations in 11 different genes, with most frequent alterations in NF2 (43%), AKT1E17K (15%), and TRAF7 (13%). In 39 tumors (36%), two different mutations were detected, with NF2 and SUFU (n = 5) and KLF4 and TRAF7 (n = 5) being the most frequent combinations. No alterations were found in POLR2A, CDKN2A, CDKN2B, and BAP1, and no homozygous CDKN2A/B deletion was detected. NF2 mutations were found in tumors of all WHO grades, whereas mutations in KLF4, TRAF7, and SMO were restricted to WHO grade I meningiomas. In contrast, SMARCE1 and TERT mutations were associated with WHO grade II meningiomas (according to the WHO classification 2016). The distribution of mutations across histological subtypes or tumor localization was in line with the existing literature, with typical combinations like KLF4K409Q /TRAF7 for secretory meningiomas and preferential skull base localization of meningiomas harboring SMO and AKT1E17K mutations. Thus, we present a custom-made NGS meningioma panel providing a time and cost-efficient reliable detection of relevant somatic molecular alterations in meningiomas suitable for daily routine.

The molecular biology of ependymomas is not well understood and this is particularly true for ependymoma relapses. We aimed at finding out if and to which extent, relapses differ from their corresponding primary tumors on the morphological, chromosomal and epigenetic level. We investigated 24 matched ependymoma primary and relapsed tumor samples and, as a first step, compared cell density, necrosis, vessel proliferation, Ki67 proliferative index, trimethylation at H3K27 and expression of CXorf67. For the investigation of global methylation profiles, we used public data in order to analyze copy number variation profiles, differential methylation, methylation status and fractions of hypo- and hypermethylated CpGs in different epigenomic substructures. Morphologically, we found a significant increase with relapse in cell density and proliferation. H3K27 trimethylation and CXorf67 expression remained stable between primary and relapse tumor samples, and the analysis of DNA methylation profiles neither revealed significant differences in copy number variations nor differentially methylated regions. Significant differences in the methylation status were found for CpG islands, but also in N Shelves or S Shelves, depending on the molecular subgroup. The fraction of probes changing their methylation in the epigenomic substructures appeared subgroup-specific. Most changes occur in CpG islands, for which relapsed tumors demonstrate higher methylation values than primary tumors. The morphological differences reflect increased aggressiveness upon ependymoma relapse, but, despite slight changes, this observation does not appear to be sufficiently explained by epigenetic changes.

FET:CREB fusions have been described in a variety of tumors from various phenotypes. Recently, these fusion transcripts were reported in intracranial tumors, variably named intracranial mesenchymal myxoid tumors or angiomatoid fibrous histiocytomas. Controversy remains concerning the terminology for these tumors. Here, we report 11 cases of central nervous system mesenchymal tumors with proven FET:CREB fusion. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor or Sarcoma Classifier (v11b4/v12.2). However, by using unsupervised t-SNE and hierarchical clustering analyses, six of the cases constituted a distinct cluster. The remaining four tumors showed no obvious relation to any of the other referenced classes but were close to the clusters of extra-CNS angiomatoid fibrous histiocytomas (n = 1), clear cell sarcomas (n = 1), or solitary fibrous tumors (n = 2). Our findings confirm that intracranial FET:CREB-fused tumors do not represent a single molecular tumor entity, although most samples clustered close to each other, indicating the existence of a distinct epigenetic group that could potentially be partially masked by the low number of cases included. Further analyses are needed to characterize intracranial FET:CREB fused-defined tumors in more detail.