Transcriptional immunogenomic analysis reveals distinct immunological clusters in paediatric nervous system tumours.

BACKGROUND: Cancer immunotherapies including immune checkpoint inhibitors and Chimeric Antigen Receptor (CAR) T-cell therapy have shown variable response rates in paediatric patients highlighting the need to establish robust biomarkers for patient selection. While the tumour microenvironment in adults has been widely studied to delineate determinants of immune response, the immune composition of paediatric solid tumours remains relatively uncharacterized calling for investigations to identify potential immune biomarkers. METHODS: To inform immunotherapy approaches in paediatric cancers with embryonal origin, we performed an immunogenomic analysis of RNA-seq data from 925 treatment-naïve paediatric nervous system tumours (pedNST) spanning 12 cancer types from three publicly available data sets. RESULTS: Within pedNST, we uncovered four broad immune clusters: Paediatric Inflamed (10%), Myeloid Predominant (30%), Immune Neutral (43%) and Immune Desert (17%). We validated these clusters using immunohistochemistry, methylation immune inference and segmentation analysis of tissue images. We report shared biology of these immune clusters within and across cancer types, and characterization of specific immune cell frequencies as well as T- and B-cell repertoires. We found no associations between immune infiltration levels and tumour mutational burden, although molecular cancer entities were enriched within specific immune clusters. CONCLUSIONS: Given the heterogeneity of immune infiltration within pedNST, our findings suggest personalized immunogenomic profiling is needed to guide selection of immunotherapeutic strategies.

Recurrent atypical teratoid/rhabdoid tumors (AT/RT) reveal discrete features of progression on histology, epigenetics, copy number profiling, and transcriptomics.

Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors manifesting in infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth quantitatively smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). Molecular characteristics of disease recurrence or metastatic spread, which go along with a particularly dismal outcome, are currently unclear. Here, we investigated tumor tissue from 26 patients affected by AT/RT to identify signatures of recurrences in comparison with matched primary tumor samples. Microscopically, AT/RT recurrences demonstrated a loss of architecture and significantly enhanced mitotic activity as compared to their related primary tumors. Based on DNA methylation profiling, primary tumor and related recurrence were grossly similar, but three out of 26 tumors belonged to a different molecular type or subtype after second surgery compared to related primary lesions. Copy number variations (CNVs) differed in six cases, showing novel gains on chromosome 1q or losses of chromosome 10 in recurrences as the most frequent alterations. To consolidate these observations, our cohort was combined with a data set of unmatched primary and recurrent AT/RT, which demonstrated chromosome 1q gain and 10 loss in 18% (n = 7) and 11% (n = 4) of the recurrences (n = 38) as compared to 7% (n = 3) and 0% (n = 0) in the primary tumors (n = 44), respectively. Similar to the observations made by DNA methylation profiling, RNA sequencing of our cohort revealed AT/RT primary tumors and matched recurrences clustering closely together. However, a number of genes showed significantly altered expression in AT/RT-SHH recurrences. Many of them are known tumor driving growth factors, involved in embryonal development and tumorigenesis, or are cell-cycle-associated. Overall, our work identifies subtle molecular changes that occur in the course of the disease and that may help define novel therapeutic targets for AT/RT recurrences.

Renal Medullary Carcinomas Harbor a Distinct Methylation Phenotype and Display Aberrant Methylation of Genes Related to Early Nephrogenesis.

Renal medullary carcinomas (RMC) are rare aggressive tumors of the kidneys, characterized by a loss of SMARCB1. Characteristically, these tumors arise in patients with sickle cell trait or other hemoglobinopathies. Recent characterization efforts have unraveled oncogenic pathways that drive tumorigenesis. Among these, gene sets that characterize replicative stress and the innate immune response are upregulated in RMCs. Despite comprehensive genetic and transcriptomic characterizations, commonalities or differences to other SMARCB1 deficient entities so far have not been investigated. We analyzed the methylome of seven primary RMC and compared it to other SMARCB1 deficient entities such as rhabdoid tumors (RT) and epithelioid sarcomas using 850 K methylation arrays. Moreover, we evaluated the differential gene expression of RMC using RNA-sequencing in comparison to other rhabdoid tumors. In accordance with previous gene expression data, we found that RMCs separate from other SMARCB1 deficient entities, pointing to a potentially different cell of origin and a role of additional genetic aberrations that may drive tumorigenesis and thus alter the methylome when compared to rhabdoid tumors. In a focused analysis of genes that are important for nephrogenesis, we particularly detected genes that govern early nephrogenesis such as FOXI1 to be hypomethylated and expressed at high levels in RMC. Overall, our analyses underscore the fact that RMCs represent a separate entity with limited similarities to rhabdoid tumors, warranting specific treatment tailored to the aggressiveness of the disease.

Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target.

Infants and Newborns with Atypical Teratoid Rhabdoid Tumors (ATRT) and Extracranial Malignant Rhabdoid Tumors (eMRT) in the EU-RHAB Registry: A Unique and Challenging Population.

Introduction: Malignant rhabdoid tumors (MRT) predominantly affect infants and young children. Patients below six months of age represent a particularly therapeutically challenging group. Toxicity to developing organ sites limits intensity of treatment. Information on prognostic factors, genetics, toxicity of treatment and long-term outcomes is sparse. Methods: Clinical, genetic, and treatment data of 100 patients (aged below 6 months at diagnosis) from 13 European countries were analyzed (2005-2020). Tumors and matching blood samples were examined for SMARCB1 mutations using FISH, MLPA and Sanger sequencing. DNA methylation subgroups (ATRT-TYR, ATRT-SHH, and ATRT-MYC) were determined using 450 k / 850 k-profiling. Results: A total of 45 patients presented with ATRT, 29 with extracranial, extrarenal (eMRT) and 9 with renal rhabdoid tumors (RTK). Seventeen patients demonstrated synchronous tumors (SYN). Metastases (M+) were present in 27% (26/97) at diagnosis. A germline mutation (GLM) was detected in 55% (47/86). DNA methylation subgrouping was available in 50% (31 / 62) with ATRT or SYN; for eMRT, methylation-based subgrouping was not performed. The 5-year overall (OS) and event free survival (EFS) rates were 23.5 ± 4.6% and 19 ± 4.1%, respectively. Male sex (11 ± 5% vs. 35.8 ± 7.4%), M+ stage (6.1 ± 5.4% vs. 36.2 ± 7.4%), presence of SYN (7.1 ± 6.9% vs. 26.6 ± 5.3%) and GLM (7.7 ± 4.2% vs. 45.7 ± 8.6%) were significant prognostic factors for 5-year OS. Molecular subgrouping and survival analyses confirm a previously described survival advantage for ATRT-TYR. In an adjusted multivariate model, clinical factors that favorably influence the prognosis were female sex, localized stage, absence of a GLM and maintenance therapy. Conclusions: In this cohort of homogenously treated infants with MRT, significant predictors of outcome were sex, M-stage, GLM and maintenance therapy. We confirm the need to stratify which patient groups benefit from multimodal treatment, and which need novel therapeutic strategies. Biomarker-driven tailored trials may be a key option.

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.

SMARCB1-deficient and SMARCA4-deficient Malignant Brain Tumors With Complex Copy Number Alterations and TP53 Mutations May Represent the First Clinical Manifestation of Li-Fraumeni Syndrome.

Atypical teratoid/rhabdoid tumor (AT/RT) is a malignant central nervous system tumor predominantly affecting infants. Mutations of SMARCB1 or (rarely) SMARCA4 causing loss of nuclear SMARCB1 or SMARCA4 protein expression are characteristic features, but further recurrent genetic alterations are lacking. Most AT/RTs occur de novo, but secondary AT/RTs arising from other central nervous system tumors have been reported. Malignant gliomas, IDH wild-type, arising in patients with Li-Fraumeni syndrome typically show somatic mutations of TP53 as well as complex copy number alterations, but little is known about the loss of SMARCB1 or SMARCA4 protein expression in this context. Here, we report 2 children in whom malignant supratentorial brain tumors with SMARCB1 deficiency, complex copy number alterations, and somatic TP53 mutations lead to the discovery of pathogenic/likely pathogenic TP53 variants in the germline. Screening of the molecularneuropathology.org dataset for cases with similar genetic and epigenetic alterations yielded another case with SMARCA4 deficiency in a young adult with Li-Fraumeni syndrome. In conclusion, SMARCB1-deficient or SMARCA4-deficient malignant brain tumors with complex copy number alterations and somatic TP53 mutations in children and young adults may represent the first clinical manifestation of Li-Fraumeni syndrome and should prompt genetic counseling and investigation for TP53 germline status.

Current and Emerging Therapeutic Approaches for Extracranial Malignant Rhabdoid Tumors.

Extracranial malignant rhabdoid tumors (extracranial MRT) are rare, highly aggressive malignancies affecting mainly infants and children younger than 3 years. Common anatomic sites comprise the kidneys (RTK - rhabdoid tumor of kidney) and other soft tissues (eMRT - extracranial, extrarenal malignant rhabdoid tumor). The genetic origin of these diseases is linked to biallelic pathogenic variants in the genes SMARCB1, or rarely SMARCA4, encoding subunits of the SWI/SNF chromatin-remodeling complex. Even if extracranial MRT seem to be quite homogeneous, recent epigenome analyses reveal a certain degree of epigenetic heterogeneity. Use of intensified therapies has modestly improved survival for extracranial MRT. Patients at standard risk profit from conventional therapies; most high-risk patients still experience a dismal course and often therapy resistance. Discoveries of clinical and molecular hallmarks and the exploration of experimental therapeutic approaches open exciting perspectives for clinical and molecularly stratified experimental treatment approaches. To ultimately improve the outcome of patients with extracranial MRTs, they need to be characterized and stratified clinically and molecularly. High-risk patients need novel therapeutic approaches including selective experimental agents in phase I/II clinical trials.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant: A clinical and histopathological distinct entity showing epigenetic similarity with ATRT-MYC.

Low-grade diffusely infiltrative tumour (LGDIT), SMARCB1-mutant, is a histopathological distinct low-grade lesion encountered in older children and young adults that shows epigenetic similarity with ATRT-MYC and has the potential for malignant progression.

Cui Bono? Identifying Patient Groups That May Benefit From Granulocyte Transfusions in Pediatric Hematology and Oncology.

INTRODUCTION: Granulocyte transfusions have long been used to bridge the time to neutrophil recovery in patients with neutropenia and severe infection. Recent randomized controlled trials did not prove a beneficial effect of granulocyte transfusions, but were likely underpowered and suffered from very heterogeneous study populations. METHODS: We retrospectively reviewed data of all patients treated with granulocyte transfusions at our pediatric center from 2004 to 2019. To identify parameters that predict the success of granulocyte transfusions, we stratified patients in 3 groups. Patients in group 1 cleared their infection, whereas patients in group 2 succumbed to an infection in neutropenia despite granulocyte transfusions. A third group included all patients who died of causes that were not related to infection. RESULTS: We demonstrate that patients without respiratory or cardiocirculatory insufficiency are enriched in group 1 and more likely to benefit from granulocyte transfusions than patients who already require these intensive care measures. The effect of granulocyte transfusions correlates with the cell dose per body weight applied per time. With our standard twice weekly dosing, patients with a body weight below 40 kg are more likely to achieve a sufficient leukocyte increment and clear their infection in comparison to patients with a higher body weight. DISCUSSION/CONCLUSIONS: We suggest that future studies on the benefits of granulocyte transfusions stratify patients according to clinical risk factors that include the need for respiratory or cardiocirculatory support and strive for a sufficient dose density of granulocyte transfusions.

Outcome for Pediatric Adreno-Cortical Tumors Is Best Predicted by the COG Stage and Five-Item Microscopic Score-Report from the German MET Studies.

Background: Adrenocortical tumors (ACTs) encompassing the adrenocortical adenoma (ACA), carcinoma (ACC), and tumors of undetermined malignant potential (ACx) are rare endocrine neoplasms with a poor prognosis. We report on pediatric ACT patients registered with the Malignant Endocrine Tumor studies and explore the EXPeRT recommendations for management. Patients: Data from the ACT patients (<18 years) were analyzed. For the risk prediction, the patients were retrospectively assigned to the COG stages and the five-item score. Results: By December 2021, 161 patients with ACT (ACA n = 51, ACx n = 19, and ACC n = 91) had been reported (the median age at the diagnosis was 4.3 years with a range of 0.1−17.8), with lymph node and distant metastases in 10.7% and 18.9% of the patients with ACC/ACx. The mean follow-up was 4.5 years (with a range of 0−16.7). The three-year overall (OS) and event-free survival (EFS) rates were 65.5% and 50.6%. In the univariate analyses, the OS was impaired for patients aged ≥ 4 years (p = 0.001) with the initial biopsy (p = 0.016), tumor spillage (p = 0.028), incomplete tumor resection (p < 0.001), unfavorable histology (p = 0.047), and COG stages III/IV (p = 0.002). Multivariate analysis revealed COG stages III/IV and an unfavorable five-item score as independent negative prognostic factors for the EFS and OS. Conclusions: Age defines the clinical presentation and prognosis in pediatric ACTs. The outcome is best predicted by the COG stage and five-item score.

Clinical response to nivolumab in an INI1-deficient pediatric chordoma correlates with immunogenic recognition of brachyury.

Poorly differentiated chordoma (PDC) is a recently recognized subtype of chordoma characterized by expression of the embryonic transcription factor, brachyury, and loss of INI1. PDC primarily affects children and is associated with a poor prognosis and limited treatment options. Here we describe the molecular and immune tumour microenvironment profiles of two paediatric PDCs produced using whole-genome, transcriptome and whole-genome bisulfite sequencing (WGBS) and multiplex immunohistochemistry. Our analyses revealed the presence of tumour-associated immune cells, including CD8+ T cells, and expression of the immune checkpoint protein, PD-L1, in both patient samples. Molecular profiling provided the rationale for immune checkpoint inhibitor (ICI) therapy, which resulted in a clinical and radiographic response. A dominant T cell receptor (TCR) clone specific for a brachyury peptide-MHC complex was identified from bulk RNA sequencing, suggesting that targeting of the brachyury tumour antigen by tumour-associated T cells may underlie this clinical response to ICI. Correlative analysis with rhabdoid tumours, another INI1-deficient paediatric malignancy, suggests that a subset of tumours may share common immune phenotypes, indicating the potential for a therapeutically targetable subgroup of challenging paediatric cancers.

The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets.

INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99-not applicable], compared with 117 days (95% CI, 106-143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases. SIGNIFICANCE: The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.

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.

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.

Atypical Teratoid/Rhabdoid Tumor (AT/RT) With Molecular Features of Pleomorphic Xanthoastrocytoma.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant central nervous system tumor predominantly occurring in infants that may also arise in older children and adults. Rare secondary AT/RT developing from other tumors such as pleomorphic xanthoastrocytoma (PXA) are on record, but AT/RT presenting with molecular features of PXA have not been described. Here, we report 3 malignant central nervous system tumors in children (10, 13, and 18 y old). All tumors were located in the temporal lobe. In 2 cases, there was no history of a low-grade precursor lesion; in 1 case anaplastic PXA had been diagnosed 3 months earlier. Histopathologically, all tumors were composed of RT cells and showed frank signs of malignancy as well as loss of nuclear SMARCB1/INI1 protein expression. Two cases displayed homozygous deletions of the SMARCB1 region while the third case showed an exon 7 mutation (c.849_850delGT; p.Met283Ilefs*77). Of note, DNA methylation profiles did not group with AT/RT or other tumor entities using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-distributed stochastic neighbor embedding analysis and hierarchical clustering analysis, however, all tumors clearly grouped with PXA. Genome-wide copy number analysis revealed homozygous CDNK2A/B deletions and gains of whole chromosome 7. BRAF V600E mutations could be demonstrated in all cases. In conclusion, the possibility of AT/RT with molecular features of PXA needs to be taken into account and warrants molecular characterization of AT/RT especially in older children. Since treatments targeting mutated BRAF are available, identification of such cases may also have therapeutic consequences.

Atypical teratoid/rhabdoid tumors (ATRTs) with SMARCA4 mutation are molecularly distinct from SMARCB1-deficient cases.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.