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We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5 th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

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., (© 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Background: Case reports have portrayed spinal cord atypical teratoid/rhabdoid tumor (spATRT) as an aggressive form of ATRT. We conducted a retrospective European survey to collect data on clinical characteristics, molecular biology, treatment, and outcome of children with intramedullary spATRT., Methods: Scrutinizing a French national series and the European Rhabdoid Registry database, we identified 13 patients (median age 32 months; metastatic disease at diagnosis, n = 6). Systemic postoperative chemotherapy was administered to all patients; three received intrathecal therapy and six were irradiated (craniospinal, n = 3; local, n = 3)., Results: Median observation time was 8 (range, 1-93) months. Progression-free and overall survival rates at 1 and (2 years) were 35.2% ± 13.9% (26.4% ± 12.9%) and 38.5% ± 13.5% (23.1% ± 11.7%). Four patients (ATRT-SHH, n = 2; ATRT-MYC, n = 1; DNA methylation subgroup not available, n = 1) achieved complete remission (CR); two of them are alive in CR 69 and 72 months from diagnosis. One patient relapsed after CR and is alive with progressive disease (PD) and one died of the disease. Three patients (ATRT-MYC, n = 2; subgroup not available, n = 1) died after 7 to 22 months due to PD after having achieved a partial remission (n = 1) or stabilization (n = 2). Five patients (ATRT-MYC, n = 2; subgroup not available, n = 3) developed early PD and died. One patient (ATRT-MYC) died of intracerebral hemorrhage prior to response evaluation., Conclusions: Long-term survival is achievable in selected patients with spATRT using aggressive multimodality treatment. Larger case series and detailed molecular analyses are needed to understand differences between spATRT and their inracranial counterparts and the group of extradural malignant rhabdoid tumors., (© 2019 Wiley Periodicals, Inc.)

Background: Recurrent specific mutations in evolutionarily conserved histone 3 (H3) variants drive pediatric high-grade gliomas (HGGs), but little is known about their downstream effects. The aim of this study was to identify genes involved in the detrimental effects of mutant H3.3-K27M, the main genetic driver in lethal midline HGG, in a transgenic Drosophila model., Methods: Mutant and wild-type histone H3.3-expressing flies were generated using a φC31-based integration system. Genetic modifier screens were performed by crossing H3.3-K27M expressing driver strains and 194 fly lines expressing short hairpin RNA targeting genes selected based on their potential role in the detrimental effects of mutant H3. Expression of the human orthologues of genes with functional relevance in the fly model was validated in H3-K27M mutant HGG., Results: Ubiquitous and midline glia-specific expression of H3.3-K27M but not wild-type H3.3 caused pupal lethality, morphological alterations, and decreased H3K27me3. Knockdown of 17 candidate genes shifted the lethal phenotype to later stages of development. These included histone modifying and chromatin remodeling genes as well as genes regulating cell differentiation and proliferation. Notably, several of these genes were overexpressed in mutant H3-K27M mutated HGG., Conclusions: Rapid screening, identification, and validation of relevant targets in "oncohistone" mediated pathogenesis have proven a challenge and a barrier to providing novel therapies. Our results provide further evidence on the role of chromatin modifiers in the genesis of H3.3-K27M. Notably, they validate Drosophila as a model system for rapid identification of relevant genes functionally involved in the detrimental effects of H3.3-K27M mutagenesis., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly encountered in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. A small group of ATRT stands out clinically, because these tumors are located in the sellar region of adults. To investigate if sellar region ATRT in adults represents a molecular distinct entity, we characterized molecular alterations in 7 sellar region ATRTs in adults as compared with 150 pediatric ATRTs and 47 pituitary adenomas using SMARCB1 sequencing, multiplex ligation-dependent probe amplification and fluorescence in situ hybridization as well as DNA methylation profiling. The median age of the 6 female and 1 male patients was 56 years. On histopathologic examination, all tumors were malignant rhabdoid tumors showing loss of SMARCB1/INI1 protein expression. Two cases displayed compound heterozygous SMARCB1 point mutations, 3 cases showed heterozygous SMARCB1 deletions with point mutations of the other allele and 1 case a homozygous SMARCB1 deletion; in 1 case, underlying SMARCB1 alterations could not be identified. On unsupervised hierarchical cluster analysis of DNA methylation profiles, sellar region ATRTs did not form a distinct group, but clustered with ATRT-MYC, 1 of 3 recently described molecular subgroups of ATRT. On analysis of DNA methylation array intensity data, only 1 sellar region ATRT showed characteristic features of pediatric ATRT-MYC, that is, major copy number losses affecting the SMARCB1 region. In conclusion, these results suggest that sellar region ATRTs in adults form a clinically distinct entity with a different mutational spectrum, but epigenetic similarities with pediatric ATRTs of the ATRT-MYC subgroup.

Aims: Molecular investigation of small-cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) has revealed that it is a monogenetic tumour characterized by alteration of SMARCA4 (BRG1), encoding a member of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodelling complex. A large majority of cases show loss of expression of the corresponding SMARCA4/BRG1 protein. Furthermore, three cases of SCCOHT with retained SMARCA4 protein expression showed loss of SMARCB1/INI1 expression. The aim of this study was to assess the sensitivity and specificity of loss of SMARCA4 expression as a diagnostic test for SCCOHT., Methods and Results: We performed SMARCA4 and SMARCB1 staining in 245 tumours, many of which were potentially in the differential diagnosis of SCCOHT. We also stained 56 cases of SCCOHT for SMARCA4 and 37 of these for SMARCB1. Fifty-four of the SCCOHT cases showed complete absence of SMARCA4 expression. The two cases with retained expression showed molecular alteration of SMARCA4. Of the 217 other neoplasms with interpretable staining, all retained SMARCA4 expression. Although the majority showed diffuse, strong nuclear expression, a heterogeneous, typically weak staining pattern was present in 13% of cases. All 37 cases of SCCOHT tested and all other neoplasms, apart from three malignant rhabdoid tumours, showed retained nuclear SMARCB1 expression. Loss of SMARCA4 expression had a sensitivity of 96.55% and specificity of 100%., Conclusions: Loss of SMARCA4 expression is sensitive and specific for SCCOHT. Although some mimics show heterogeneous expression, there is retention of nuclear staining in at least a part of the tumour; therefore, only complete loss of staining should be regarded as being supportive of SCCOHT., (© 2016 John Wiley & Sons Ltd.)

Pilocytic astrocytoma (PA) is the most common brain tumor in children but is rare in adults, and hence poorly studied in this age group. We investigated 222 PA and report increased aneuploidy in older patients. Aneuploid genomes were identified in 45% of adult compared with 17% of pediatric PA. Gains were non-random, favoring chromosomes 5, 7, 6 and 11 in order of frequency, and preferentially affecting non-cerebellar PA and tumors with BRAF V600E mutations and not with KIAA1549-BRAF fusions or FGFR1 mutations. Aneuploid PA differentially expressed genes involved in CNS development, the unfolded protein response, and regulators of genomic stability and the cell cycle (MDM2, PLK2),whose correlated programs were overexpressed specifically in aneuploid PA compared to other glial tumors. Thus, convergence of pathways affecting the cell cycle and genomic stability may favor aneuploidy in PA, possibly representing an additional molecular driver in older patients with this brain tumor.

Atypical teratoid rhabdoid tumor (AT/RT), a rare and highly malignant tumor entity of the central nervous system that presents in early childhood, has a poor prognosis. AT/RTs are characterized by biallelic inactivating mutations of the gene SMARCB1 in 98% of patients; these mutations may serve as molecular markers for residual tumor cell detection in liquid biopsies. We developed a marker-specific method to detect residual AT/RT cells. Seven of 150 patient samples were selected, each with a histological and genetically ascertained diagnosis of AT/RT. Tumor tissue was either formalin fixed or fresh frozen. DNA was extracted from the patients' peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF). Multiplex ligation-dependent probe amplification, DNA sequencing, and fluorescence in situ hybridization were used to characterize the tumors' mutations. Residual tumor cell detection used mutation-specific primers and real-time PCR. The detection limit for the residual tumor cell search was 1-18%, depending on the quality of the template provided. The residual tumor cell search in PBL and CSF was negative for all seven patients. The SMARCB1 region of chromosome 22 is prone to DNA double-strand breaks. The individual breakpoints and breakpoint-specific PCR offer the option to detect minimal residual tumor cells in CSF or blood. Even if we did not detect minimal residual tumor cells in the investigated material, proof of principle for this method was confirmed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Whole-exome sequencing (WES) is revolutionizing medical diagnostics and taxonomy. In less than 5 years since its first use, WES has revealed unexpected molecular drivers of numerous cancers. Here, we describe our use of WES to uncover the true nature of an enigmatic pathological entity, small-cell carcinoma of the ovary, hypercalcaemic type (SCCOHT), which has resisted definitive characterisation since it was first described in 1979. We conducted WES using three families with SCCOHT and identified deleterious mutations in the chromatin-remodelling gene SMARCA4 (encoding BRG1) in all cases. Follow-up of these findings, using both Sanger sequencing and WES of formalin-fixed paraffin-embedded tumours, showed that virtually all SCCOHTs we studied lacked functional SMARCA4/BRG1. Notably, this gene, and the related SMARCB1 gene, is mutated in most, if not all, atypical teratoid/rhabdoid tumours and malignant rhabdoid tumours. Other groups have similar findings. We review the relationship between these three neoplasms, discuss how they were distinguished from morphologically similar neoplasms, consider their similarities and show how WES has revealed that SCCOHTs are in fact rhabdoid tumours. We propose that SCCOHT be renamed 'malignant rhabdoid tumour of the ovary' (MRTO) to reflect these observations., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Embryonal tumor with multilayered rosettes (ETMR, previously known as ETANTR) is a highly aggressive embryonal CNS tumor, which almost exclusively affects infants and is associated with a dismal prognosis. Accurate diagnosis is of critical clinical importance because of its poor response to current treatment protocols and its distinct biology. Amplification of the miRNA cluster at 19q13.42 has been identified previously as a genetic hallmark for ETMR, but an immunohistochemistry-based assay for clinical routine diagnostics [such as INI-1 for atypical teratoid rhabdoid tumor (AT/RT)] is still lacking. In this study, we screened for an ETMR-specific marker using a gene-expression profiling dataset of more than 1,400 brain tumors and identified LIN28A as a highly specific marker for ETMR. The encoded protein binds small RNA and has been implicated in stem cell pluripotency, metabolism and tumorigenesis. Using an LIN28A specific antibody, we carried out immunohistochemical analysis of LIN28A in more than 800 childhood brain-tumor samples and confirmed its high specificity for ETMR. Strong LIN28A immunoexpression was found in all 37 ETMR samples tested, whereas focal reactivity was only present in a small (6/50) proportion of AT/RT samples. All other pediatric brain tumors were completely LIN28A-negative. In summary, we established LIN28A immunohistochemistry as a highly sensitive and specific, rapid, inexpensive diagnostic tool for routine pathological verification of ETMR.

Background: Studies in adults with malignant glioma suggest MGMT methylation as a stratification marker. Similar data for children are sparse. We investigated the impact of MGMT methylation and expression on survival of children with high-grade glioma (HGG) registered into the German HIT-GBM database receiving temozolomide (TMZ) as part of their treatment (n = 21 relapsed, n = 4 primary)., Procedure: Twenty-four patients were included retrospectively. Methylation specific PCR (MSP), calibrated combined bisulfite restriction analysis (COBRA), and immunohistochemistry (IHC) were applied. Survival analyses were performed by Kaplan-Meier and Cox proportional-hazards models., Results: MSP demonstrated DNA methylation in 77%. Patients with a methylated MGMT promoter had a sixfold longer median EFS (P = 0.015; 5.5 months vs. 0.9 months). Considering the results of calibrated COBRA, MGMT methylation was again associated with an elevated EFS (P = 0.05; 10.2 months vs. 2.6 months) and OS (P = 0.06; 18.7 months vs. 7.2 months) only if methylation was >14%. No difference in EFS and OS at all was noted between unmethylated and tumors methylated at low level (n = 9). Twenty-two tumors were positive by IHC, 10 showed low MGMT expression (IHC score 0-4). We did not detect any difference in EFS and OS between moderate/high-expressing tumors (IHC score 6-12) and those with low or no expression (IHC score 0-4)., Conclusion: DNA methylation, but not protein expression of MGMT was associated with an increased median EFS and OS of children with relapsed HGG. MGMT methylation status warrants prospective evaluation as a stratification marker for children with HGG., ((c) 2009 Wiley-Liss, Inc.)

Aims: The polycomb factor BMI-1 has recently been implicated in tumorigenesis of the central nervous system in several experimental animal models. However, the significance of BMI-1 in human glioma has not been investigated. Here we describe expression of the polycomb protein BMI-1 and its downstream targets p16(Ink4a) and MDM2 in both high- and low-grade human glioma., Methods: Tumour samples were collected from 305 adult patients treated for primary grades 2-4 gliomas between 1980 and 2006 in Finland and Germany. BMI-1, p16 and MDM2 expression was evaluated using immunohistochemistry in representative paraffin-embedded tumour tissue. The significance of observed immunoreactivity, age at onset, gender, histopathological findings and proliferative index was analysed in univariate and multivariate survival models., Results: BMI-1 was expressed in all histologic types of diffuse gliomas. We found a significant correlation (P = 0.007) between the frequency of BMI-1 immunoreactive tumour cells and poor survival in World Health Organization grades II-III oligodendrogliomas and oligoastrocytomas (n = 62). The median survival of patients grouped by low, intermediate or high frequency of BMI-1 immunoreactive tumour cells was 191 months, 151 months and 68 months, respectively. This association was also significant in the Cox multivariate regression model. Nuclear p16 immunopositivity predicted better survival in astrocytomas and an inverse correlation between p16 expression and the Ki-67 mitotic index was also observed., Conclusions: BMI-1 is found in all histological types of gliomas and the relative protein expression of BMI-1 is a novel independent prognostic marker in oligodendroglial tumours.

The histopathology of papillary tumours of the pineal region (PTPR) closely resembles that of ependymomas and choroid plexus tumours. Therefore, immunohistochemical staining profiles were investigated in a series of 15 PTPR. In addition to cytokeratin, synaptophysin and glial fibrillary acidic protein expression, PTPR were examined for the presence of dot- or ring-like epithelial membrane antigen (EMA) immunoreactivity typically encountered in ependymoma, staining for inwardly rectifying potassium channel Kir7.1 and stanniocalcin-1 (specifically expressed in choroid plexus tumours) as well as microtubule-associated protein-2 (MAP-2). Furthermore, comparative genomic hybridization was performed in five PTPR. Cytokeratin was expressed in all PTPR examined, whereas glial fibrillary acidic protein and synaptophysin staining were absent. Dot- or ring-like EMA immunoreactivity was only observed in 1 out of 15 PTPR. Membranous Kir7.1 and cytoplasmic stanniocalcin-1 staining were present in the minority of PTPR (3/15 and 4/15, respectively). In contrast, MAP-2 immunoreactivity was encountered in 13 out of 15 PTPR, but was significantly less frequently observed in a series of choroid plexus tumours (7/37). PTPR mainly presented with chromosomal losses affecting chromosomes 10 (4/5 cases) and 22q (3/5 cases) as well as gains on chromosomes 4 (4/5 cases), 8 (3/5 cases), 9 (3/5 cases) and 12 (3/5 cases). To conclude, the majority of PTPR can be distinguished from ependymomas and choroid plexus tumours by absent staining for epithelial membrane antigen, Kir7.1 and staniocalcin-1 as well as the presence of distinct MAP-2 immunoreactivity. Antibodies directed against these antigens are thus expected to be valuable markers in the diagnosis of papillary tumours located in the vicinity of the third ventricle.

To identify specific markers for the diagnosis of choroid plexus tumors, gene expression profiles of choroid plexus epithelial cells (n = 8) and ependymal cells (n = 6) microdissected from human autopsy brains as well as choroid plexus papilloma tissue were investigated using DNA microarrays. Protein expression of genes overexpressed in choroid plexus was evaluated in normal choroid plexus, choroid plexus papilloma, choroid plexus carcinoma, other primary brain tumors, and cerebral metastases. Forty-six genes found to be overexpressed in normal choroid plexus epithelial cells were also present in choroid plexus papilloma. Among those, 11 were further analyzed by immunohistochemistry. Expression of inward rectifier potassium channel Kir7.1 was confirmed in normal choroid plexus (34 of 35), choroid plexus papilloma (12 of 18), and choroid plexus carcinoma (5 of 5) but was not found in 100 other primary brain tumors and cerebral metastases. Similarly, stanniocalcin-1 stained normal choroid plexus (32 of 35), choroid plexus papilloma (16 of 18), and choroid plexus carcinoma (3 of 5), whereas staining was seen in only 2 of 100 other primary brain tumors and cerebral metastases. Transthyretin stained choroid plexus (33 of 35), choroid plexus papilloma (14 of 18), and plexus carcinoma (2 of 5), but its specificity was significantly lower. Antibodies directed against coagulation factor V, glutathione peroxidase 3, pigment epithelium derived factor, serotonin receptor 5-HTR2C, lumican, fibulin-1, plastin-1, and cytokeratin 18 revealed varying degrees of specificity and sensitivity. Our data suggest that antibodies directed against Kir7.1 and stanniocalcin-1 might serve as sensitive and specific diagnostic markers for choroid plexus tumors.

Pattern and extent of epithelial membrane antigen (EMA) immunoreactivity in ependymomas as compared to other glial tumors have only been investigated in small series. To determine sensitivity and specificity of EMA staining, 54 ependymomas were evaluated in comparison to 54 glioblastomas, 43 fibrillary astrocytomas and 21 oligodendrogliomas. Distinct punctate intracytoplasmic EMA immunoreactivity was observed in 48/54 ependymomas (89%), whereas ring-like EMA staining was observed in 17/54 ependymomas (31%). Apart from the absence in most myxopapillary ependymomas, neither staining pattern was related to tumor grade or localization. Dot-like EMA immunoreactivity was less frequently observed in glioblastomas [32/54 (59%), P<0.05 vs ependymomas], fibrillary astrocytomas [10/43 (23%), P<0.001 vs ependymomas] and oligodendrogliomas [2/21 (10%), P<0.001 vs ependymomas], whereas ring-like EMA staining was absent. Sensitivity and specificity of punctate EMA staining for the diagnosis of ependymoma as compared to other glial tumors were determined: A finding of 5 EMA dots/high-power field was associated with a sensitivity of 72% and a specificity of 81%. The presence of ring-like EMA positive structures was less sensitive (32%), but highly specific (100%). To conclude, distinct punctate and ring-like EMA staining might serve as sensitive and specific markers of ependymal differentiation in glial tumors and, thus, may aid the diagnosis of ependymoma.