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.

Histopathological patterns in atypical teratoid/rhabdoid tumors are related to molecular subgroup.

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.

Inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1/INI1 protein in a molecular subset of atypical teratoid/rhabdoid tumors.

Loss of nuclear SMARCB1 (INI1/hSNF5/BAF47) protein expression due to biallelic mutations of the SMARCB1 tumor suppressor gene is a hallmark of atypical teratoid/rhabdoid tumors (ATRT), but the presence of cytoplasmic SMARCB1 protein in these tumors has not yet been described. In a series of 102 primary ATRT, distinct cytoplasmic SMARCB1 staining on immunohistochemistry was encountered in 19 cases (19%) and was highly over-represented in cases showing pathogenic sequence variants leading to truncation or mutation of the C-terminal part of SMARCB1 (15/19 vs. 4/83; Chi-square: 56.04, p = 1.0E-10) and, related to this, in tumors of the molecular subgroup ATRT-TYR (16/36 vs. 3/66; Chi-square: 24.47, p = 7.6E-7). Previous reports have indicated that while SMARCB1 lacks a bona fide nuclear localization signal, it harbors a masked nuclear export signal (NES) and that truncation of the C-terminal region results in unmasking of this NES leading to cytoplasmic localization. To determine if cytoplasmic localization found in ATRT is due to unmasking of NES, we generated GFP fusions of one of the SMARCB1 truncating mutations (p.Q318X) found in the tumors along with a p.L266A mutation, which was shown to disrupt the interaction of SMARCB1-NES with exportin-1. We found that while the GFP-SMARCB1(Q318X) mutant localized to the cytoplasm, the double mutant GFP-SMARCB1(Q318X;L266A) localized to the nucleus, confirming NES requirement for cytoplasmic localization. Furthermore, cytoplasmic SMARCB1(Q318X) was unable to cause senescence as determined by morphological observations and by senescence-associated ß-galactosidase assay, while nuclear SMARCB1(Q318X;L266A) mutant regained this function. Selinexor, a selective exportin-1 inhibitor, was effective in inhibiting the nuclear export of SMARCB1(Q318X) and caused rapid cell death in rhabdoid tumor cells. In conclusion, inhibition of nuclear export restores nuclear localization and residual tumor suppressor function of truncated SMARCB1. Therapies aimed at preventing nuclear export of mutant SMARCB1 protein may represent a promising targeted therapy in ATRT harboring truncating C-terminal SMARCB1 mutations.