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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00401-022-02424-5

Association between copy number alterations estimated using low-pass whole genome sequencing of formalin-fixed paraffin-embedded prostate tumor tissue and cancer-specific clinical parameters

Abstract Copy number alterations (CNAs) are frequently observed in early-stage prostate cancer and are associated with disease recurrence and tumor aggressiveness. Cost-effective assessment of CNAs could enhance clinical utility of CNAs. Here, we combined the cost-effectiveness of low-pass (low coverage) whole genome sequencing (LPWGS) and the routine availability of formalin-fixed paraffin-embedded (FFPE) tumor tissue for assessing CNAs in a cohort of 187 men with early-stage localised prostate cancer. We detected well known CNAs in 8p, 8q, 13q and 16q and recurrent gains of the oncogene MYC and losses of the tumor suppressor genes NKX3-1, PTEN and RB1, indicating assay reliability. The estimated burden of CNAs was significantly associated with Gleason score, pathological T stage, surgical margin status and biochemical recurrence. Further, genomic losses or gains in specific chromosomal arms were significantly associated with worse BCR-free survival. Copy number signatures extracted from the LPWGS data showed potential for risk stratifying patients, where signatures S1 and S2 showed significant association to worse BCR-free survival compared to S3. Our study provides clinical validation of the associations between CNAs and tumor aggressiveness in an independent and representative RP cohort, while demonstrating the feasibility of performing LPWGS of FFPE tumor tissue for cost-effective assessment of CNAs

Impact of new molecular criteria on diagnosis and survival of adult glioma patients

The fifth edition WHO classification of Tumors of the Central nervous system (WHO-CNS5) integrated new molecular parameters to refine CNS tumor classification. This study aimed to reclassify a retrospective cohort of adult glioma patients according to WHO-CNS5, and assess if overall survival (OS) correlated with the revised diagnosis. Further, the diagnostic impact of methylation profiling (MP) was evaluated. Adult gliomas diagnosed according to 2016 WHO-CNS (n = 226) were evaluated according to WHO-CNS5 criteria. All patients had diagnostic NGS performed. 29 patients had 850k MP performed due to challenging tumor cases. OS was analyzed using Kaplan-Meier plots and log-rank test. 19 patients were reclassified. Specifically, diffuse astrocytic glioma, IDH-wildtype, with molecular features of glioblastoma (DAG-G) were reclassified as glioblastoma (n = 15). Shifts to glioblastoma were because of TERT promoter (TERTp) mutation (n = 9), EGFR amplification (n = 2), EGFR amplification and TERTp mutation (n = 1), and TERTp mutation with gain of chromosome 7, but uncertain chromosome 10 status due to lack of NGS coverage (n = 3). Lower grade IDH-mutant astrocytomas were reclassified as astrocytoma IDH-mutant, WHO grade 4 due to CDKN2A/B homozygous deletion (n = 4). No significant difference in OS was found for reclassified DAG-G in whole group (p = 0.59) and for TERTp mutation only (p = 0.44), compared to glioblastoma. MP resulted in revised diagnosis (n = 2), confirmed diagnosis (n = 15) and no match (n = 12). Our study showed similar overall survival for glioblastoma and DAG patients, supporting that isolated TERTp mutation may have a prognostic role in IDH-wildtype gliomas. Further, our study suggests MP is useful for confirming the diagnoses in challenging tumors

Double-immunofluorescence labeling of human glioma tissue.

<p>Robust anti-CD15 (red) and anti-AQP9 co-labeling was observed in myelomonocytic cells (asterisk). In 4 patient-biopsies weaker CD15 immunolabeling was also observed in a number of larger cells, likely identifying these cells as BTSCs (examples marked by arrowheads). Non-myelomonocytic AQP9 positive cells (examples marked by arrows) were located near putative BTSCs, but no clear examples of AQP9 and CD15 co-expression were observed in these cells. Two areas are shown as overlay images (left), green channel (center) and red channel (right). Nuclei (blue). Scale bar  = 20 µm.</p

AQP9 mRNA expression levels in four types of glioblastoma.

<p>Expression levels of AQP9 in 173 patients were retrieved from the TCGA database and compared to patient descriptors that have previously been associated with four types of glioblastoma: classical, mesenchymal, neural and proneural <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075764#pone.0075764-Verhaak1" target="_blank">[25]</a>. Chi-square analysis suggests uneven AQP9 expression levels among these four tumor types (P<0.0001). High AQP9 expression (>200%) appears to be associated primarily with mesenchymal tumors. X-axis labels refer to <i>AQP9</i> expression in specific patients compared to average <i>AQP9</i> expression in TCGA glioma samples.</p