Unified rhombic lip origins of group 3 and group 4 medulloblastoma.

Medulloblastoma, a malignant childhood cerebellar tumour, segregates molecularly into biologically distinct subgroups, suggesting that a personalized approach to therapy would be beneficial1. Mouse modelling and cross-species genomics have provided increasing evidence of discrete, subgroup-specific developmental origins2. However, the anatomical and cellular complexity of developing human tissues3-particularly within the rhombic lip germinal zone, which produces all glutamatergic neuronal lineages before internalization into the cerebellar nodulus-makes it difficult to validate previous inferences that were derived from studies in mice. Here we use multi-omics to resolve the origins of medulloblastoma subgroups in the developing human cerebellum. Molecular signatures encoded within a human rhombic-lip-derived lineage trajectory aligned with photoreceptor and unipolar brush cell expression profiles that are maintained in group 3 and group 4 medulloblastoma, suggesting a convergent basis. A systematic diagnostic-imaging review of a prospective institutional cohort localized the putative anatomical origins of group 3 and group 4 tumours to the nodulus. Our results connect the molecular and phenotypic features of clinically challenging medulloblastoma subgroups to their unified beginnings in the rhombic lip in the early stages of human development.

Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma.

Group3 (G3) medulloblastoma (MB) is one of the deadliest forms of the disease for which novel treatment is desperately needed. Here we evaluate ribociclib, a highly selective CDK4/6 inhibitor, with gemcitabine in mouse and human G3MBs. Ribociclib central nervous system (CNS) penetration was assessed by in vivo microdialysis and by IHC and gene expression studies and found to be CNS-penetrant. Tumors from mice treated with short term oral ribociclib displayed inhibited RB phosphorylation, downregulated E2F target genes, and decreased proliferation. Survival studies to determine the efficacy of ribociclib and gemcitabine combination were performed on mice intracranially implanted with luciferase-labeled mouse and human G3MBs. Treatment of mice with the combination of ribociclib and gemcitabine was well tolerated, slowed tumor progression and metastatic spread, and increased survival. Expression-based gene activity and cell state analysis investigated the effects of the combination after short- and long-term treatments. Molecular analysis of treated versus untreated tumors showed a significant decrease in the activity and expression of genes involved in cell-cycle progression and DNA damage response, and an increase in the activity and expression of genes implicated in neuronal identity and neuronal differentiation. Our findings in both mouse and human patient-derived orthotopic xenograft models suggest that ribociclib and gemcitabine combination therapy warrants further investigation as a treatment strategy for children with G3MB.

Low-coverage whole-genome sequencing of cerebrospinal-fluid-derived cell-free DNA in brain tumor patients.

This protocol summarizes the pipeline for analysis of tumor-derived cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) using low-coverage whole-genome sequencing (lcWGS). This approach enables resolution of chromosomal and focal copy-number variations (CNVs) as oncologic signatures, particularly for patients with central nervous system tumors. Our strategy tolerates sub-nanogram cfDNA input and is thus optimized for CSF samples where cfDNA yields are typically low. Overall, the detection of tumor-specific signatures in CSF-derived cfDNA is a promising biomarker for personalization of brain-tumor therapy. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).

Vorinostat and isotretinoin with chemotherapy in young children with embryonal brain tumors: A report from the Pediatric Brain Tumor Consortium (PBTC-026).

BACKGROUND: Embryonal tumors of the CNS are the most common malignant tumors occurring in the first years of life. This study evaluated the feasibility and safety of incorporating novel non-cytotoxic therapy with vorinostat and isotretinoin to an intensive cytotoxic chemotherapy backbone. METHODS: PBTC-026 was a prospective multi-institutional clinical trial for children <48 months of age with newly diagnosed embryonal tumors of the CNS. Treatment included three 21-day cycles of induction therapy with vorinostat and isotretinoin, cisplatin, vincristine, cyclophosphamide, and etoposide; three 28-day cycles of consolidation therapy with carboplatin and thiotepa followed by stem cell rescue; and twelve 28-day cycles of maintenance therapy with vorinostat and isotretinoin. Patients with M0 medulloblastoma (MB) received focal radiation following consolidation therapy. Molecular classification was by DNA methylation array. RESULTS: Thirty-one patients with median age of 26 months (range 6-46) received treatment on study; 19 (61%) were male. Diagnosis was MB in 20 and supratentorial CNS embryonal tumor in 11. 24/31 patients completed induction therapy within a pre-specified feasibility window of 98 days. Five-year progression-free survival (PFS) and overall survival (OS) for all 31 patients were 55 ± 15 and 61 ± 13, respectively. Five-year PFS was 42 ± 13 for group 3 MB (n = 12); 80 ± 25 for SHH MB (n = 5); 33 ± 19 for embryonal tumor with multilayered rosettes (ETMR, n = 6). CONCLUSION: It was safe and feasible to incorporate vorinostat and isotretinoin into an intensive chemotherapy regimen. Further study to define efficacy in this high-risk group of patients is warranted.

Serial assessment of measurable residual disease in medulloblastoma liquid biopsies.

Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.

Subgroup and subtype-specific outcomes in adult medulloblastoma.

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed.

Efficacy of Carboplatin and Isotretinoin in Children With High-risk Medulloblastoma: A Randomized Clinical Trial From the Children's Oncology Group.

IMPORTANCE: Brain tumors are the leading cause of disease-related death in children. Medulloblastoma is the most common malignant embryonal brain tumor, and strategies to increase survival are needed. OBJECTIVE: To evaluate therapy intensification with carboplatin as a radiosensitizer and isotretinoin as a proapoptotic agent in children with high-risk medulloblastoma in a randomized clinical trial and, with a correlative biology study, facilitate planned subgroup analysis according to World Health Organization consensus molecular subgroups of medulloblastoma. DESIGN, SETTING, AND PARTICIPANTS: A randomized clinical phase 3 trial was conducted from March 2007 to September 2018. Analysis was completed in September 2020. Patients aged 3 to 21 years with newly diagnosed high-risk medulloblastoma from Children's Oncology Group institutions within the US, Canada, Australia, and New Zealand were included. High-risk features included metastasis, residual disease, or diffuse anaplasia. INTERVENTIONS: Patients were randomized to receive 36-Gy craniospinal radiation therapy and weekly vincristine with or without daily carboplatin followed by 6 cycles of maintenance chemotherapy with cisplatin, cyclophosphamide, and vincristine with or without 12 cycles of isotretinoin during and following maintenance. MAIN OUTCOMES AND MEASURES: The primary clinical trial end point was event-free survival, using the log-rank test to compare arms. The primary biology study end point was molecular subgroup classification by DNA methylation array. RESULTS: Of 294 patients with medulloblastoma, 261 were evaluable after central radiologic and pathologic review; median age, 8.6 years (range, 3.3-21.2); 183 (70%) male; 189 (72%) with metastatic disease; 58 (22%) with diffuse anaplasia; and 14 (5%) with greater than 1.5-cm2 residual disease. For all participants, the 5-year event-free survival was 62.9% (95% CI, 55.6%-70.2%) and overall survival was 73.4% (95% CI, 66.7%-80.1%). Isotretinoin randomization was closed early owing to futility. Five-year event-free survival was 66.4% (95% CI, 56.4%-76.4%) with carboplatin vs 59.2% (95% CI, 48.8%-69.6%) without carboplatin (P = .11), with the effect exclusively observed in group 3 subgroup patients: 73.2% (95% CI, 56.9%-89.5%) with carboplatin vs 53.7% (95% CI, 35.3%-72.1%) without (P = .047). Five-year overall survival differed by molecular subgroup (P = .006): WNT pathway activated, 100% (95% CI, 100%-100%); SHH pathway activated, 53.6% (95% CI, 33.0%-74.2%); group 3, 73.7% (95% CI, 61.9%-85.5%); and group 4, 76.9% (95% CI, 67.3%-86.5%). CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, therapy intensification with carboplatin improved event-free survival by 19% at 5 years for children with high-risk group 3 medulloblastoma. These findings further support the value of an integrated clinical and molecular risk stratification for medulloblastoma. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00392327.

Children's Oncology Group Phase III Trial of Reduced-Dose and Reduced-Volume Radiotherapy With Chemotherapy for Newly Diagnosed Average-Risk Medulloblastoma.

PURPOSE: Children with average-risk medulloblastoma (MB) experience survival rates of ≥ 80% at the expense of adverse consequences of treatment. Efforts to mitigate these effects include deintensification of craniospinal irradiation (CSI) dose and volume. METHODS: ACNS0331 (ClinicalTrials.gov identifier: NCT00085735) randomly assigned patients age 3-21 years with average-risk MB to receive posterior fossa radiation therapy (PFRT) or involved field radiation therapy (IFRT) following CSI. Young children (3-7 years) were also randomly assigned to receive standard-dose CSI (SDCSI; 23.4 Gy) or low-dose CSI (LDCSI; 18 Gy). Post hoc molecular classification and mutational analysis contextualized outcomes according to known biologic subgroups (Wingless, Sonic Hedgehog, group 3, and group 4) and genetic biomarkers. Neurocognitive changes and ototoxicity were monitored over time. RESULTS: Five hundred forty-nine patients were enrolled on study, of which 464 were eligible and evaluable to compare PFRT versus IFRT and 226 for SDCSI versus LDCSI. The five-year event-free survival (EFS) was 82.5% (95% CI, 77.2 to 87.8) and 80.5% (95% CI, 75.2 to 85.8) for the IFRT and PFRT regimens, respectively, and 71.4% (95% CI, 62.8 to 80) and 82.9% (95% CI, 75.6 to 90.2) for the LDCSI and SDCSI regimens, respectively. IFRT was not inferior to PFRT (hazard ratio, 0.97; 94% upper CI, 1.32). LDCSI was inferior to SDCSI (hazard ratio, 1.67%; 80% upper CI, 2.10). Improved EFS was observed in patients with Sonic Hedgehog MB who were randomly assigned to the IFRT arm (P = .018). Patients with group 4 MB receiving LDCSI exhibited inferior EFS (P = .047). Children receiving SDCSI exhibited greater late declines in IQ (estimate = 5.87; P = .021). CONCLUSION: Reducing the radiation boost volume in average-risk MB is safe and does not compromise survival. Reducing CSI dose in young children with average-risk MB results in inferior outcomes, possibly in a subgroup-dependent manner, but is associated with better neurocognitive outcome. Molecularly informed patient selection warrants further exploration for children with MB to be considered for late-effect sparing approaches.

Clinical Outcomes and Patient-Matched Molecular Composition of Relapsed Medulloblastoma.

PURPOSE: We sought to investigate clinical outcomes of relapsed medulloblastoma and to compare molecular features between patient-matched diagnostic and relapsed tumors. METHODS: Children and infants enrolled on either SJMB03 (NCT00085202) or SJYC07 (NCT00602667) trials who experienced medulloblastoma relapse were analyzed for clinical outcomes, including anatomic and temporal patterns of relapse and postrelapse survival. A largely independent, paired molecular cohort was analyzed by DNA methylation array and next-generation sequencing. RESULTS: A total of 72 of 329 (22%) SJMB03 and 52 of 79 (66%) SJYC07 patients experienced relapse with significant representation of Group 3 and wingless tumors. Although most patients exhibited some distal disease (79%), 38% of patients with sonic hedgehog tumors experienced isolated local relapse. Time to relapse and postrelapse survival varied by molecular subgroup with longer latencies for patients with Group 4 tumors. Postrelapse radiation therapy among previously nonirradiated SJYC07 patients was associated with long-term survival. Reirradiation was only temporizing for SJMB03 patients. Among 127 patients with patient-matched tumor pairs, 9 (7%) experienced subsequent nonmedulloblastoma CNS malignancies. Subgroup (96%) and subtype (80%) stabilities were largely maintained among the remainder. Rare subgroup divergence was observed from Group 4 to Group 3 tumors, which is coincident with genetic alterations involving MYC, MYCN, and FBXW7. Subgroup-specific patterns of alteration were identified for driver genes and chromosome arms. CONCLUSION: Clinical behavior of relapsed medulloblastoma must be contextualized in terms of up-front therapies and molecular classifications. Group 4 tumors exhibit slower biological progression. Utility of radiation at relapse is dependent on patient age and prior treatments. Degree and patterns of molecular conservation at relapse vary by subgroup. Relapse tissue enables verification of molecular targets and identification of occult secondary malignancies.

Outcomes by Clinical and Molecular Features in Children With Medulloblastoma Treated With Risk-Adapted Therapy: Results of an International Phase III Trial (SJMB03).

PURPOSE: SJMB03 (ClinicalTrials.gov identifier: NCT00085202) was a phase III risk-adapted trial that aimed to determine the frequency and clinical significance of biological variants and genetic alterations in medulloblastoma. PATIENTS AND METHODS: Patients 3-21 years old were stratified into average-risk and high-risk treatment groups based on metastatic status and extent of resection. Medulloblastomas were molecularly classified into subgroups (Wingless [WNT], Sonic Hedgehog [SHH], group 3, and group 4) and subtypes based on DNA methylation profiles and overlaid with gene mutations from next-generation sequencing. Coprimary study end points were (1) to assess the relationship between ERBB2 protein expression in tumors and progression-free survival (PFS), and (2) to estimate the frequency of mutations associated with WNT and SHH tumors. Clinical and molecular risk factors were evaluated, and the most robust were used to model new risk-classification categories. RESULTS: Three hundred thirty eligible patients with medulloblastoma were enrolled. Five-year PFS was 83.2% (95% CI, 78.4 to 88.2) for average-risk patients (n = 227) and 58.7% (95% CI, 49.8 to 69.1) for high-risk patients (n = 103). No association was found between ERBB2 status and PFS in the overall cohort (P = .74) or when patients were stratified by clinical risk (P = .71). Mutations in CTNNB1 (96%), DDX3X (37%), and SMARCA4 (24%) were most common in WNT tumors and PTCH1 (38%), TP53 (21%), and DDX3X (19%) in SHH tumors. Methylome profiling classified 53 WNT (17.4%), 48 SHH (15.7%), 65 group 3 (21.3%), and 139 group 4 (45.6%) tumors. A comprehensive clinicomolecular risk factor analysis identified three low-risk groups (WNT, low-risk SHH, and low-risk combined groups 3 and 4) with excellent (5-year PFS > 90%) and two very high-risk groups (high-risk SHH and high-risk combined groups 3 and 4) with poor survival (5-year PFS < 60%). CONCLUSION: These results establish a new risk stratification for future medulloblastoma trials.

Deconstructing Sonic Hedgehog Medulloblastoma: Molecular Subtypes, Drivers, and Beyond.

Medulloblastoma (MB) is a highly malignant cerebellar tumor predominantly diagnosed during childhood. Driven by pathogenic activation of sonic hedgehog (SHH) signaling, SHH subgroup MB (SHH-MB) accounts for nearly one-third of diagnoses. Extensive molecular analyses have identified biologically and clinically relevant intertumoral heterogeneity among SHH-MB tumors, prompting the recognition of novel subtypes. Beyond germline and somatic mutations promoting constitutive SHH signaling, driver alterations affect a multitude of pathways and molecular processes, including TP53 signaling, chromatin modulation, and post-transcriptional gene regulation. Here, we review recent advances in the underpinnings of SHH-MB in the context of molecular subtypes, clarify novel somatic and germline drivers, highlight cellular origins and developmental hierarchies, and describe the composition of the tumor microenvironment and its putative role in tumorigenesis.

Functional loss of a noncanonical BCOR-PRC1.1 complex accelerates SHH-driven medulloblastoma formation.

Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10 ) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/- ;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/- ;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.

Patient-derived orthotopic xenografts of pediatric brain tumors: a St. Jude resource.

Pediatric brain tumors are the leading cause of cancer-related death in children. Patient-derived orthotopic xenografts (PDOX) of childhood brain tumors have recently emerged as a biologically faithful vehicle for testing novel and more effective therapies. Herein, we provide the histopathological and molecular analysis of 37 novel PDOX models generated from pediatric brain tumor patients treated at St. Jude Children's Research Hospital. Using a combination of histopathology, whole-genome and whole-exome sequencing, RNA-sequencing, and DNA methylation arrays, we demonstrate the overall fidelity and inter-tumoral molecular heterogeneity of pediatric brain tumor PDOX models. These models represent frequent as well as rare childhood brain tumor entities, including medulloblastoma, ependymoma, atypical teratoid rhabdoid tumor, and embryonal tumor with multi-layer rosettes. PDOX models will be valuable platforms for evaluating novel therapies and conducting pre-clinical trials to accelerate progress in the treatment of brain tumors in children. All described PDOX models and associated datasets can be explored using an interactive web-based portal and will be made freely available to the research community upon request.

DDX3X Suppresses the Susceptibility of Hindbrain Lineages to Medulloblastoma.

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma-the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes.

Bridging the treatment gap in infant medulloblastoma: molecularly informed outcomes of a globally feasible regimen.

BACKGROUND: Infant medulloblastoma represents an enormous challenge in neuro-oncology, due to their simultaneous high-risk of recurrence and high risk of severe neurodevelopmental sequelae with craniospinal irradiation. Currently infant medulloblastoma are treated with intensified protocols, either comprising intraventricular methotrexate or autologous transplant, both of which carry significant morbidity and are not feasible in the majority of the world. We sought to evaluate the molecular predictors of outcome in a cohort of infants homogeneously treated with induction chemotherapy, focal radiation and maintenance chemotherapy. METHODS: In a retrospective analysis, 29 young children treated with a craniospinal irradiation sparing strategy from Hospital Garrahan in Buenos Aires were profiled using Illumina HumanMethylationEPIC arrays, and correlated with survival. RESULTS: Twenty-nine children (range, 0.3-4.6 y) were identified, comprising 17 sonic hedgehog (SHH), 10 Group 3/4, and 2 non-medulloblastomas. Progression-free survival (PFS) across the entire cohort was 0.704 (95% CI: 0.551-0.899). Analysis by t-distributed stochastic neighbor embedding revealed 3 predominant groups, SHHß, SHHγ, and Group 3. Survival by subtype was highly prognostic with SHHγ having an excellent 5-year PFS of 100% (95% CI: 0.633-1) and SHHß having a PFS of 0.56 (95% CI: 0.42-1). Group 3 had a PFS of 0.50 (95% CI: 0.25-1). Assessment of neurocognitive outcome was performed in 11 patients; the majority of survivors fell within the low average to mild intellectual disability, with a median IQ of 73.5. CONCLUSIONS: We report a globally feasible and effective strategy avoiding craniospinal radiation in the treatment of infant medulloblastoma, including a robust molecular correlation along with neurocognitive outcomes.

Germline Elongator mutations in Sonic Hedgehog medulloblastoma.

Cancer genomics has revealed many genes and core molecular processes that contribute to human malignancies, but the genetic and molecular bases of many rare cancers remains unclear. Genetic predisposition accounts for 5 to 10% of cancer diagnoses in children1,2, and genetic events that cooperate with known somatic driver events are poorly understood. Pathogenic germline variants in established cancer predisposition genes have been recently identified in 5% of patients with the malignant brain tumour medulloblastoma3. Here, by analysing all protein-coding genes, we identify and replicate rare germline loss-of-function variants across ELP1 in 14% of paediatric patients with the medulloblastoma subgroup Sonic Hedgehog (MBSHH). ELP1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among paediatric patients with MBSHH. Parent-offspring and pedigree analyses identified two families with a history of paediatric medulloblastoma. ELP1-associated medulloblastomas were restricted to the molecular SHHα subtype4 and characterized by universal biallelic inactivation of ELP1 owing to somatic loss of chromosome arm 9q. Most ELP1-associated medulloblastomas also exhibited somatic alterations in PTCH1, which suggests that germline ELP1 loss-of-function variants predispose individuals to tumour development in combination with constitutive activation of SHH signalling. ELP1 is the largest subunit of the evolutionarily conserved Elongator complex, which catalyses translational elongation through tRNA modifications at the wobble (U34) position5,6. Tumours from patients with ELP1-associated MBSHH were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis due to Elongator deficiency in model systems7-9. Thus, genetic predisposition to proteome instability may be a determinant in the pathogenesis of paediatric brain cancers. These results support investigation of the role of protein homeostasis in other cancer types and potential for therapeutic interference.

SWI/SNF complex heterogeneity is related to polyphenotypic differentiation, prognosis, and immune response in rhabdoid tumors.

BACKGROUND: Rhabdoid tumors (RTs) arise within (atypical teratoid/rhabdoid tumor [AT/RT]) or outside the brain (extra [e]CNS-RT) and are driven mainly by inactivation of the SWItch/sucrose nonfermentable (SWI/SNF) complex subunit SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1). A pathognomonic hallmark of RTs is heterogeneous multilineage differentiation, including anomalous neuronal differentiation in some eCNS-RTs. Because remodeling of the SWI/SNF complex regulates differentiation, we hypothesized that SWI/SNF Brahma-associated factors (BAF) and polybromo-associated BAF (PBAF) complex heterogeneity are related to both multilineage differentiation and clinical outcome. METHODS: We performed an integrated analysis of SWI/SNF complex alterations in the developing kidney and cerebellum (most common regions of RT origin) in comparison to eCNS-RT (n = 14) and AT/RT (n = 25) tumors. RT samples were interrogated using immunohistochemistry, DNA methylation, and gene expression analyses. RESULTS: The SWI/SNF BAF paralogs actin-like protein (ACTL)6A and ACTL6B were expressed in a mutually exclusive manner in the developing cerebellum and kidney. In contrast, a subset of eCNS-RTs lost mutual exclusivity and coexpressed both subunits. These tumors showed aberrant DNA methylation of genes that regulate neuronal and renal development and demonstrated immunohistochemical evidence of neuronal differentiation. In addition, low expression of the PBAF subunit polybromo-1 (PBRM1) identified a group of AT/RTs in younger children with better overall prognosis. PBRM1-low AT/RT and eCNS-RTs showed altered DNA methylation and gene expression in immune-related genes. PBRM1 knockdown resulted in lowering immunosuppressive cytokines, and PBRM1 levels in tumor samples showed an inverse relationship with cluster of differentiation (CD)8 cytotoxic T-cell infiltration. CONCLUSIONS: Heterogeneity in SWI/SNF BAF (ACTL6A/ACTL6B) and PBAF (PBRM1) subunits is related to histogenesis, contributes to the immune microenvironment and prognosis in RTs, and may inform opportunities to develop immunotherapies.

scRNA-seq in medulloblastoma shows cellular heterogeneity and lineage expansion support resistance to SHH inhibitor therapy.

Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.

Resolving medulloblastoma cellular architecture by single-cell genomics.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.