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.

Ependymoma with YAP1-MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1-MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1-MAMLD1 fusion was documented by RT-PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo-rosettes, small to medium-sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot-like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1-MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1-11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow-up, 4.84 years). In this to date, largest series of ependymomas with YAP1-MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings.

Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytoma predominantly affecting children and young adults. We performed comprehensive genomic characterization on a cohort of 67 patients with histologically defined PXA (n = 53, 79%) or anaplastic PXA (A-PXA, n = 14, 21%), including copy number analysis (ThermoFisher Oncoscan, n = 67), methylation profiling (Illumina EPIC array, n = 43) and targeted next generation sequencing (n = 32). The most frequent alterations were CDKN2A/B deletion (n = 63; 94%) and BRAF p.V600E (n = 51, 76.1%). In 7 BRAF p.V600 wild-type cases, alternative driver alterations were identified involving BRAF, RAF1 and NF1. Downstream phosphorylation of ERK kinase was uniformly present. Additional pathogenic alterations were rare, with TERT, ATRX and TP53 mutations identified in a small number of tumors, predominantly A-PXA. Methylation-based classification of 46 cases utilizing a comprehensive reference tumor allowed assignment to the PXA methylation class in 40 cases. A minority grouped with the methylation classes of ganglioglioma or pilocytic astrocytoma (n = 2), anaplastic pilocytic astrocytoma (n = 2) or control tissues (n = 2). In 9 cases, tissue was available from matched primary and recurrent tumors, including 8 with anaplastic transformation. At recurrence, two tumors acquired TERT promoter mutations and the majority demonstrated additional non-recurrent copy number alterations. Methylation class was preserved at recurrence. For 62 patients (92.5%), clinical follow-up data were available (median follow-up, 5.4 years). Overall survival was significantly different between PXA and A-PXA (5-year OS 80.8% vs. 47.6%; P = 0.0009) but not progression-free survival (5-year PFS 59.9% vs. 39.8%; P = 0.05). WHO grade remained a strong predictor of overall survival when limited to 38 cases defined as PXA by methylation-based classification. Our data confirm the importance of WHO grading in histologically and epigenetically defined PXA. Methylation-based classification may be helpful in cases with ambiguous morphology, but is largely confirmatory in PXA with well-defined morphology.