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.

Aims Although inactivation of the von Hippel–Lindau gene (VHL) on chromosome 3p25 is considered to be the major cause of hereditary endolymphatic sac tumours (ELSTs), the genetic background of sporadic ELST is largely unknown. The aim of this study was to determine the prevalence of VHL mutations in sporadic ELSTs and compare their characteristics to VHL‐disease‐related tumours. Methods Genetic and epigenetic alterations were compared between 11 sporadic and 11 VHL‐disease‐related ELSTs by targeted sequencing and DNA methylation analysis. Results VHL mutations and small deletions detected by targeted deep sequencing were identified in 9/11 sporadic ELSTs (82%). No other cancer‐related genetic pathway was altered except for TERT promoter mutations in two sporadic ELST and one VHL‐disease‐related ELST (15%). Loss of heterozygosity of chromosome 3 was found in 6/10 (60%) VHL‐disease‐related and 10/11 (91%) sporadic ELSTs resulting in biallelic VHL inactivation in 8/10 (73%) sporadic ELSTs. DNA methylation profiling did not reveal differences between sporadic and VHL‐disease‐related ELSTs but reliably distinguished ELST from morphological mimics of the cerebellopontine angle. VHL patients were significantly younger at disease onset compared to sporadic ELSTs (29 vs. 52 years, p < 0.0001, Fisher's exact test). VHL‐disease status was not associated with an increased risk of recurrence, but the presence of clear cells was found to be associated with shorter progression‐free survival (p = 0.0002, log‐rank test). Conclusion Biallelic inactivation of VHL is the main mechanism underlying ELSTs, but unknown mechanisms beyond VHL may rarely be involved in the pathogenesis of sporadic ELSTs.

Hemangioblastomas (HBs) of the central nervous system are highly vascular neoplasms that occur sporadically or as a manifestation of von Hippel-Lindau (VHL) disease. Despite their benign nature, HBs are clinically heterogeneous and can be associated with significant morbidity due to mass effects of peritumoral cysts or tumor progression. Underlying molecular factors involved in HB tumor biology remain elusive. We investigated genome-wide DNA methylation profiles and clinical and histopathological features in a series of 47 HBs from 42 patients, including 28 individuals with VHL disease. Thirty tumors occurred in the cerebellum, 8 in the brainstem and 8 HBs were of spinal location, while 1 HB was located in the cerebrum. Histologically, 12 HBs (26%) belonged to the cellular subtype and exclusively occurred in the cerebellum, whereas 35 HBs were reticular (74%). Unsupervised clustering and dimensionality reduction of DNA methylation profiles revealed two distinct subgroups. Methylation cluster 1 comprised 30 HBs of mainly cerebellar location (29/30, 97%), whereas methylation cluster 2 contained 17 HBs predominantly located in non-cerebellar compartments (16/17, 94%). The sum of chromosomal regions being affected by copynumber alterations was significantly higher in methylation cluster 1 compared to cluster 2 (mean 262 vs. 109 Mb, p = 0.001). Of note, loss of chromosome 6 occurred in 9/30 tumors (30%) of methylation cluster 1 and was not observed in cluster 2 tumors (p = 0.01). No relevant methylation differences between sporadic and VHL-related HBs or cystic and non-cystic HBs could be detected. Deconvolution of the bulk DNA methylation profiles revealed four methylation components that were associated with the two methylation clusters suggesting cluster-specific celltype compositions. In conclusion, methylation profiling of HBs reveals 2 distinct subgroups that mainly associate with anatomical location, cytogenetic profiles and differences in cell type composition, potentially reflecting different cells of origin.