DistSNE: Distributed computing and online visualization of DNA methylation-based central nervous system tumor classification.

The current state-of-the-art analysis of central nervous system (CNS) tumors through DNA methylation profiling relies on the tumor classifier developed by Capper and colleagues, which centrally harnesses DNA methylation data provided by users. Here, we present a distributed-computing-based approach for CNS tumor classification that achieves a comparable performance to centralized systems while safeguarding privacy. We utilize the t-distributed neighborhood embedding (t-SNE) model for dimensionality reduction and visualization of tumor classification results in two-dimensional graphs in a distributed approach across multiple sites (DistSNE). DistSNE provides an intuitive web interface (https://gin-tsne.med.uni-giessen.de) for user-friendly local data management and federated methylome-based tumor classification calculations for multiple collaborators in a DataSHIELD environment. The freely accessible web interface supports convenient data upload, result review, and summary report generation. Importantly, increasing sample size as achieved through distributed access to additional datasets allows DistSNE to improve cluster analysis and enhance predictive power. Collectively, DistSNE enables a simple and fast classification of CNS tumors using large-scale methylation data from distributed sources, while maintaining the privacy and allowing easy and flexible network expansion to other institutes. This approach holds great potential for advancing human brain tumor classification and fostering collaborative precision medicine in neuro-oncology.

Leveraging Attention-Based Convolutional Neural Networks for Meningioma Classification in Computational Histopathology.

Convolutional neural networks (CNNs) are becoming increasingly valuable tools for advanced computational histopathology, promoting precision medicine through exceptional visual decoding abilities. Meningiomas, the most prevalent primary intracranial tumors, necessitate accurate grading and classification for informed clinical decision-making. Recently, DNA methylation-based molecular classification of meningiomas has proven to be more effective in predicting tumor recurrence than traditional histopathological methods. However, DNA methylation profiling is expensive, labor-intensive, and not widely accessible. Consequently, a digital histology-based prediction of DNA methylation classes would be advantageous, complementing molecular classification. In this study, we developed and rigorously assessed an attention-based multiple-instance deep neural network for predicting meningioma methylation classes using tumor methylome data from 142 (+51) patients and corresponding hematoxylin-eosin-stained histological sections. Pairwise analysis of sample cohorts from three meningioma methylation classes demonstrated high accuracy in two combinations. The performance of our approach was validated using an independent set of 51 meningioma patient samples. Importantly, attention map visualization revealed that the algorithm primarily focuses on tumor regions deemed significant by neuropathologists, offering insights into the decision-making process of the CNN. Our findings highlight the capacity of CNNs to effectively harness phenotypic information from histological sections through computerized images for precision medicine. Notably, this study is the first demonstration of predicting clinically relevant DNA methylome information using computer vision applied to standard histopathology. The introduced AI framework holds great potential in supporting, augmenting, and expediting meningioma classification in the future.

Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

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 5th 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.

DNA methylation-based classification of sinonasal tumors.

The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs.

ATRT-SHH comprises three molecular subgroups with characteristic clinical and histopathological features and prognostic significance.

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.

Tumor treating fields therapy is feasible and safe in a 3-year-old patient with diffuse midline glioma H3K27M - a case report.

Since high grade gliomas are aggressive brain tumors, intensive search for new treatment options is ongoing. For adult patients with newly diagnosed (ndGBM) and recurrent glioblastoma (rGBM), low intensity intermediate frequency alternating electric fields, known as tumor treating fields (TTFields) have been established as a new treatment modality. Tumor treating fields significantly increase survival rates in combination with adjuvant temozolomide (TMZ) in adult and GBM patients. Here, we report about feasibility and safety of treatment on a pediatric patient with diffuse midline glioma who is receiving TTFields therapy in combination with temozolomide.

Oligosarcomas, IDH-mutant are distinct and aggressive.

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.

SangeR: the high-throughput Sanger sequencing analysis pipeline.

Summary: In the era of next generation sequencing and beyond, the Sanger technique is still widely used for variant verification of inconclusive or ambiguous high-throughput sequencing results or as a low-cost molecular genetical analysis tool for single targets in many fields of study. Many analysis steps need time-consuming manual intervention. Therefore, we present here a pipeline-capable high-throughput solution with an optional Shiny web interface, that provides a binary mutation decision of hotspots together with plotted chromatograms including annotations via flat files. Availability and implementation: SangeR is freely available at https://github.com/Neuropathology-Giessen/SangeR and https://hub.docker.com/repository/docker/kaischmid/sange_r. Contact: Kai.Schmid@patho.med.uni-giessen.de or Daniel.Amsel@patho.med.uni-giessen.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Integrated Molecular-Morphologic Meningioma Classification: A Multicenter Retrospective Analysis, Retrospectively and Prospectively Validated.

PURPOSE: Meningiomas are the most frequent primary intracranial tumors. Patient outcome varies widely from benign to highly aggressive, ultimately fatal courses. Reliable identification of risk of progression for individual patients is of pivotal importance. However, only biomarkers for highly aggressive tumors are established (CDKN2A/B and TERT), whereas no molecularly based stratification exists for the broad spectrum of patients with low- and intermediate-risk meningioma. METHODS: DNA methylation data and copy-number information were generated for 3,031 meningiomas (2,868 patients), and mutation data for 858 samples. DNA methylation subgroups, copy-number variations (CNVs), mutations, and WHO grading were analyzed. Prediction power for outcome was assessed in a retrospective cohort of 514 patients, validated on a retrospective cohort of 184, and on a prospective cohort of 287 multicenter cases. RESULTS: Both CNV- and methylation family-based subgrouping independently resulted in increased prediction accuracy of risk of recurrence compared with the WHO classification (c-indexes WHO 2016, CNV, and methylation family 0.699, 0.706, and 0.721, respectively). Merging all risk stratification approaches into an integrated molecular-morphologic score resulted in further substantial increase in accuracy (c-index 0.744). This integrated score consistently provided superior accuracy in all three cohorts, significantly outperforming WHO grading (c-index difference P = .005). Besides the overall stratification advantage, the integrated score separates more precisely for risk of progression at the diagnostically challenging interface of WHO grade 1 and grade 2 tumors (hazard ratio 4.34 [2.48-7.57] and 3.34 [1.28-8.72] retrospective and prospective validation cohorts, respectively). CONCLUSION: Merging these layers of histologic and molecular data into an integrated, three-tiered score significantly improves the precision in meningioma stratification. Implementation into diagnostic routine informs clinical decision making for patients with meningioma on the basis of robust outcome prediction.

Recurrent fusions in PLAGL1 define a distinct subset of pediatric-type supratentorial neuroepithelial tumors.

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.

PATZ1 fusions define a novel molecularly distinct neuroepithelial tumor entity with a broad histological spectrum.

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.

IDH2 R172 Mutations Across Poorly Differentiated Sinonasal Tract Malignancies: Forty Molecularly Homogenous and Histologically Variable Cases With Favorable Outcome.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

Glioblastomas with primitive neuronal component harbor a distinct methylation and copy-number profile with inactivation of TP53, PTEN, and RB1.

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.

Case Report of Complete Radiological Response of a Thalamic Glioblastoma After Treatment With Proton Therapy Followed by Temozolomide and Tumor-Treating Fields.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. We present a case of a 42-year-old male patient presenting with headache and vomiting. Imaging demonstrated obstructive hydrocephalus and a ring-enhancing lesion in the right posterior thalamus. After endoscopic third ventriculostomy and stereotactic biopsy, the histopathologic diagnosis of a malignant glioma was confirmed by DNA methylation array as GBM isocitrate dehydrogenase wild type. The patient was treated with combined treatment of chemoradiation with temozolomide (TMZ) including proton boost, TMZ maintenance, and tumor-treating fields. In this case report, complete radiological response was observed 1 year after the end of radiation therapy.

Nonmetastatic Medulloblastoma of Early Childhood: Results From the Prospective Clinical Trial HIT-2000 and An Extended Validation Cohort.

PURPOSE: The HIT-2000-BIS4 trial aimed to avoid highly detrimental craniospinal irradiation (CSI) in children < 4 years of age with nonmetastatic medulloblastoma by systemic chemotherapy, intraventricular methotrexate, and risk-adapted local radiotherapy. PATIENTS AND METHODS: From 2001-2011, 87 patients received systemic chemotherapy and intraventricular methotrexate. Until 2006, CSI was reserved for nonresponse or progression. After 2006, local radiotherapy was introduced for nonresponders or patients with classic medulloblastoma (CMB) or large-cell/anaplastic medulloblastoma (LCA). DNA methylation profiles of infantile sonic hedgehog-activated medulloblastoma (SHH-INF) were subdivided into iSHH-I and iSHH-II subtypes in the HIT-2000-BIS4 cohort and a validation cohort (n = 71) from the HIT group and Russia. RESULTS: Five years after diagnosis, patients with desmoplastic medulloblastoma (DMB) or medulloblastoma with extensive nodularity (MBEN; n = 42) had 93% progression-free survival (5y-PFS), 100% overall survival (5y-OS), and 93% CSI-free (5y-CSI-free) survival. Patients with CMB/LCA (n = 45) had 37% 5y-PFS, 62% 5y-OS, and 39% 5y-CSI-free survival. Local radiotherapy did not improve survival in patients with CMB/LCA. All DMB/MBEN assessed by DNA methylation profiling belonged to the SHH-INF subgroup. Group 3 patients (5y-PFS, 36%; n = 14) relapsed more frequently than the SHH-INF group (5y-PFS, 93%; n = 28) or group 4 patients (5y-PFS, 83%; n = 6; P < .001). SHH-INF split into iSHH-I and iSHH-II subtypes in HIT-2000-BIS4 and the validation cohort, without prognostic impact (5y-PFS: iSHH-I, 73%, v iSHH-II, 83%; P = .25; n = 99). Intelligence quotient (IQ) was significantly lower in patients after CSI (mean IQ, 90 [no radiotherapy], v 74 [CSI]; P = .012). CONCLUSION: Systemic chemotherapy and intraventricular methotrexate led to favorable survival in both iSHH subtypes of SHH-activated DMB/MBEN with acceptable neurotoxicity. Survival in patients with non-wingless (WNT)/non-SHH disease with CMB/LCA was not improved by local radiotherapy. Patients with group 4 disease had more favorable survival rates than those with group 3 medulloblastoma.

KLF4K409Q-mutated meningiomas show enhanced hypoxia signaling and respond to mTORC1 inhibitor treatment.

Meningioma represents the most common primary brain tumor in adults. Recently several non-NF2 mutations in meningioma have been identified and correlated with certain pathological subtypes, locations and clinical observations. Alterations of cellular pathways due to these mutations, however, have largely remained elusive. Here we report that the Krueppel like factor 4 (KLF4)-K409Q mutation in skull base meningiomas triggers a distinct tumor phenotype. Transcriptomic analysis of 17 meningioma samples revealed that KLF4K409Q mutated tumors harbor an upregulation of hypoxia dependent pathways. Detailed in vitro investigation further showed that the KLF4K409Q mutation induces HIF-1α through the reduction of prolyl hydroxylase activity and causes an upregulation of downstream HIF-1α targets. Finally, we demonstrate that KLF4K409Q mutated tumors are susceptible to mTOR inhibition by Temsirolimus. Taken together, our data link the KLF4K409Q mediated upregulation of HIF pathways to the clinical and biological characteristics of these skull base meningiomas possibly opening new therapeutic avenues for this distinct meningioma subtype.

[CLIPPERS syndrome - a disorder with many faces- a case report]. / CLIPPERS-Syndrom: Eine Erkrankung mit vielen Gesichtern ­ ein Fallbericht.

The CLIPPERS syndrome is a chronic, inflammatory disorder of the central nervous system of unknown etiology, which was first described in 2010 by Pittock and colleagues. It is characterized by typical magnetic resonance imaging (MRI) changes with lesions mainly in the brainstem, a perivascular, lymphohistiocytic inflammatory process and significant improvement under glucocorticoid therapy. Here we describe the case of a 40-year-old male who presented initially with typical clinical and radiological signs of CLIPPERS syndrome and who achieved complete remission under immunosuppressive therapy. Two years later, he presented with severe headaches. The MRI showed a reappearance of the lesion in the cerebellum, but now with a confluent character. The first brain biopsy showed lymphoproliferation. A second brain biopsy could finally confirm the suspected diagnosis of a primary CNS lymphoma.

Tumors diagnosed as cerebellar glioblastoma comprise distinct molecular entities.

In this multi-institutional study we compiled a retrospective cohort of 86 posterior fossa tumors having received the diagnosis of cerebellar glioblastoma (cGBM). All tumors were reviewed histologically and subjected to array-based methylation analysis followed by algorithm-based classification into distinct methylation classes (MCs). The single MC containing the largest proportion of 25 tumors diagnosed as cGBM was MC anaplastic astrocytoma with piloid features representing a recently-described molecular tumor entity not yet included in the WHO Classification of Tumours of the Central Nervous System (WHO classification). Twenty-nine tumors molecularly corresponded to either of 6 methylation subclasses subsumed in the MC family GBM IDH wildtype. Further we identified 6 tumors belonging to the MC diffuse midline glioma H3 K27 M mutant and 6 tumors allotted to the MC IDH mutant glioma subclass astrocytoma. Two tumors were classified as MC pilocytic astrocytoma of the posterior fossa, one as MC CNS high grade neuroepithelial tumor with BCOR alteration and one as MC control tissue, inflammatory tumor microenvironment. The methylation profiles of 16 tumors could not clearly be assigned to one distinct MC. In comparison to supratentorial localization, the MC GBM IDH wildtype subclass midline was overrepresented, whereas the MCs GBM IDH wildtype subclass mesenchymal and subclass RTK II were underrepresented in the cerebellum. Based on the integration of molecular and histological findings all tumors received an integrated diagnosis in line with the WHO classification 2016. In conclusion, cGBM does not represent a molecularly uniform tumor entity, but rather comprises different brain tumor entities with diverse prognosis and therapeutic options. Distinction of these molecular tumor classes requires molecular analysis. More than 30% of tumors diagnosed as cGBM belong to the recently described molecular entity of anaplastic astrocytoma with piloid features.

Anaplastic astrocytoma with piloid features, a novel molecular class of IDH wildtype glioma with recurrent MAPK pathway, CDKN2A/B and ATRX alterations.

Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding (t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5 years. The most frequent localization was the posterior fossa (74%). Deletions of CDKN2A/B (66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting NF1, followed by BRAF and FGFR1) and mutations of ATRX or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were IDH1/2 wildtype. The MGMT promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than IDH wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of CDKN2A/B and ATRX, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.