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.

非典型畸胎样/横纹肌样肿瘤 (ATRT) 是一种侵袭性中枢神经系统肿瘤，其特征是 SMARCB1/INI1 蛋白表达缺失，包含三个不同的分子组，ATRT-TYR、ATRT-MYC 和 ATRT-SHH。ATRT-SHH 代表最大的分子群，并且在年龄、肿瘤位置和表观遗传特征方面具有异质性。因此，我们旨在调查 ATRT-SHH 内的异质性是否也具有生物学和临床重要性。DNA 甲基化谱的共识聚类和 65 个 ATRT-SHH 的验证性 t-SNE 分析产生了三个稳健的分子亚群，即 SHH-1A、SHH-1B 和 SHH-2。这些亚组的中位发病年龄（SHH-1A：18 个月，SHH-1B：107 个月，SHH-2：13 个月）和肿瘤位置（SHH-1A：88% 幕上；SHH-1B：85% 幕上； SHH-2：93% 幕下，常延伸至松果体区）。亚组表现出可比性SMARCB1突变谱，但与 SHH-1A (20%) 和 SHH-1B (0%) 相比，SHH-2 (63%) 中的致病性/可能致病性SMARCB1种系变异过多。原神经标志物 ASCL1（富含 SHH-1B）和胶质标志物 OLIG2 和 GFAP（在 SHH-2 中不存在）的蛋白质表达以及整体 mRNA 表达模式不同，但所有亚组的特征是 SHH 以及 Notch 通路成员的过表达. 在果蝇模型中，敲除 Snr1 （ SMARCB1的苍蝇同源物）) 在刺猬激活细胞中，不仅改变了刺猬信号，而且还导致异常的 Notch 信号和肿瘤样结构的形成。最后，在生存分析中，分子亚组和发病年龄（但不是 ASCL1 染色状态）与总生存率独立相关，携带 SHH-1B 的老年患者（> 3 岁）的结果相对有利。总之，ATRT-SHH 包含三个以 SHH 和 Notch 通路激活为特征的亚组，但分子和临床特征不同。我们的数据表明，ATRT-SHH 的分子亚组具有预后相关性，可能有助于在未来的临床试验中对患者进行分层。