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Supplementary Data from Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma

Supplementary Figure from Combination of Ribociclib and Gemcitabine for the Treatment of Medulloblastoma

Sign-tracking is a conditioned response where animals interact with reward-predictive cues and can be used as a means to approximate a cue’s motivational value. The nucleus accumbens core (NAc) has been highly implicated in mediating the sign-tracking response. Additionally, acetylcholine (ACh) transmission throughout the striatum broadly has been attributed to both incentive motivation and behavioral flexibility. Here, we show that sign-tracking responses are indeed flexible in the face of a contingency change in the form of an omission schedule, and that this flexibility is mediated by NAc ACh. Using behavioral and pharmacological methods, we show that blockade of NAc nicotinic receptors (nAChRs) augmented sign-tracking persistence, while blockade of muscarinic receptors (mAChRs) enhanced response flexibility following introduction of the omission schedule. Further, we detail how mAChR or nAChR antagonism impacted the microstructure of sign-tracking responses. These results indicate that NAc ACh receptors have opposing roles in the regulation of sign-tracking response flexibility without altering the motivational value of the cue.

Medulloblastoma, a malignant childhood cerebellar tumor, molecularly segregates into biologically distinct subgroups warranting personalized therapy(1). Murine modeling and cross-species genomics have provided mounting evidence of discrete, subgroup-specific developmental origins(2). However, human-specific anatomic and cellular complexity(3), particularly within the rhombic lip germinal zone that produces all glutamatergic neuronal lineages prior to internalization into the cerebellar nodulus, complicates prior murine-derived inferences. Here, we utilized multi-omics to resolve medulloblastoma subgroup origins 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 maintained in Group 3 and Group 4 medulloblastoma, implicating convergent basis. Systematic diagnostic imaging review of a prospective institutional cohort localized the putative anatomic origins of Group 3 and Group 4 tumors to the nodulus. Our results connect molecular and phenotypic features of clinically challenging medulloblastoma subgroups to their unified beginnings in the early human rhombic lip.

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.

Current treatments for obesity do not reliably reduce body weight over time. New interventional strategies, including chemogenetics, carry promise based on preclinical animal studies. Here, we focused on the ventral pallidum (VP) due to its clearly established role in eating behavior. Chronic inhibitory or excitatory chemogenetic activation was used to modulate the activity of VP-targeted neurons in rats on an obesogenic diet. Based on studies using acute VP manipulations, we hypothesized that VP inhibition would decrease weight gain, while VP stimulation would increase weight. Instead, both manipulations caused weight gain over time, and in a manner not clearly linked to consumption levels. We theorize that the complex reciprocal feedback between ventral striatal structures and metabolic centers likely underpin our unexpected findings. Regardless, this study suggests that the result of strategies to prevent obesity with chronic neuromodulation could be difficult to predict from prior preclinical studies that have used acute interventions.

A comprehensive data quality assessment is necessary to expand a nursing database that is designed for evaluating the impact of implementing Best Practice Guidelines (BPG) developed by the Registered Nurses’ Association of Ontario (RNAO). This case report presents a method to standardize data quality assessments of the Nursing Quality Indicators for Reporting and Evaluation (NQuIRE) database by developing a data quality framework (DQF) and assessing key dimensions of the framework using a data quality index (DQI). The data quality index is a single key performance metric for assessing the quality of the database. The aims of sharing this case report are 2-fold: (1) to promote best practices for assessing data quality by developing and implementing a data quality framework and (2) to demonstrate an unprecedented method of assessing the data quality of a nursing database.

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.

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., Although the hedgehog (HH) pathway is known to be deregulated in medulloblastoma, inhibitors of the pathway have shown disappointing clinical benefit. Using single-cell sequencing in a mouse model of the disease, the authors show that the response to the HH pathway inhibitor vismodegib is cell-type specific.

Medulloblastoma is a high-risk embryonal brain tumor arising in the cerebellum. Genomic profiling has revealed a striking molecular heterogeneity between medulloblastoma patients, yet treatment regimens are mostly uniform. Many children with medulloblastoma die from their disease and surviving patients often face severe long-term side effects, highlighting an urgent need for more effective treatment options. We and others have recently identified pronounced intra-tumoral heterogeneity and defined cellular hierarchies within medulloblastoma tumors. The functional role of these cellular hierarchies remains unknown. We now hypothesize the existence of an inter-cellular communication network that is maintained by receptor/ligand interactions. To test our hypothesis, we use our medulloblastoma single-cell RNA sequencing dataset of 25 patients, as well as bulk RNA sequencing, DNA methylation array, and genome sequencing data across molecular subtypes. Single-cell RNA sequencing data are analyzed to dissect cell compartments characterized by high expression of potentially oncogenic receptors and their respective ligands. Consequently, cell type-specific roles in auto- or paracrine signal transduction within the cellular community are explored. We further investigate downstream oncogenic signaling pathways by approximating transcription factor activity and explore genetic and epigenetic activation mechanisms by matched genome sequencing and DNA methylation profiling, respectively. Our findings will be applied to deconvolute bulk RNA sequencing data, thus identifying therapeutically relevant signaling networks in larger cohorts of medulloblastoma patients. Eventually, candidate targets will be validated on patient-derived cell models and xenografts by overexpression and inhibition studies. Together, here we aim at identifying tumor-driving receptor/ligand interactions in medulloblastoma, with the goal to define targets susceptible to precision oncology approaches.

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-cm2residual 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

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.

Summary: Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.

BACKGROUND: Given the vast molecular heterogeneity present within medulloblastoma (MB) and considerable differences in therapy, we performed a meta-analysis of three large, recently published, prospective clinical trials (ACNS0331, SJMB03, ACNS0332) comprising 898 children with newly-diagnosed MB to shape future therapy. METHODS: Molecular subgroups, subtypes, and copy number variations were uniformly procured from DNA methylation profiles and mutations from next-generation sequencing. Patients were stratified into six clinically homogeneous groups for cross-trial comparisons: (1) ACNS0331_LDCSI - patients with non-metastatic (M0), non-residual (R0), non-anaplastic MB treated with low-dose (LD) craniospinal irradiation(CSI); (2) ACNS0331_SDCSI - patients with M0R0 non-anaplastic MB treated with standard-dose(SD) CSI; (3) SJMB03_SDCSI - patients with M0R0 non-anaplastic MB treated with SDCSI; (4) SJMB03_HDCSI - patients with metastatic (M+) MB treated with high-dose (HD) CSI; (5) ACNS0332_HDCSI - patients with M+ MB treated with HDCSI; (6) ACNS0332_HDCSI_Carbo - patients with M+ MB treated with HDCSI and carboplatin. RESULTS: 803 (WNT=125, SHH=122, G3=189, G4=367) of 898 patients formed the cohort. No significant difference was observed between the event-free survival (EFS) from ACNS0331_SDCSI and SJMB03_SDCSI or from SJMB03_HDCSI and ACNS0332_HDCSI when analyzed as a whole or by subgroup. ACNS0331_LDCSI outcome was inferior to the combined ACNS0331_SDCSI + SJMB03_SDCSI cohorts (p90% EFS) and M0R0 G3/G4_subtype III as high risk ( CONCLUSION: The comparable results observed across trials presents a welcome opportunity to reduce toxicity by eliminating excessive doses of chemotherapy (i.e. vincristine, cisplatin, and cyclophosphamide) from therapy. Furthermore, these results support molecularly driven risk classification as the means for a better, more-refined, treatment stratification.

Medulloblastoma is the most common malignant brain tumor in children. Despite the use of contemporary multi-modal therapy, up to one-third of patients will succumb to disease progression. Conventional response monitoring is based on magnetic resonance imaging and cerebrospinal fluid (CSF) cytology and a marker for measurable residual disease (MRD) is lacking. In this study, we show the clinical utility of profiling CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD based on a sizable cohort of children with medulloblastoma enrolled on a prospective, multi-center, risk-adapted trial (SJMB03; NCT00085202). A total of 476 CSF samples were serially collected from 123 patients by lumbar puncture at post-operative baseline, during therapy, and at regular surveillance after completion of therapy. Using low-coverage whole-genome sequencing (lcWGS), tumor-associated copy-number alterations in CSF-derived cfDNA were investigated as an MRD surrogate for correlation with clinical features and outcomes. MRD was detected in post-operative baseline CSF samples for 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declined with therapy, yet those with persistent MRD detected at the end of therapy (20 of 68 CSFs at this timepoint) had significantly higher risk of disease progression (hazard ratio 8.94, 95%CI 4.10-19.49; log-rank p< 0.0001). Importantly, MRD-positivity from routine surveillance samples often preceded radiographic progression by more than 3 months. Comparative analyses of copy-number variations obtained from serial CSF samples enabled identification of relapse-specific alterations and early detection of relapse-dominant clones. Overall, our findings advocate for the routine incorporation of CSF-derived liquid biopsies in future medulloblastoma trials.

© 2021 by American Society of Clinical Oncology. Creative Commons Attribution Non-Commercial No Derivatives 4.0 License: https://creativecommons.org/licenses/by-nc-nd/4.0/, 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.

When pursuing desirable outcomes, one must make the decision between exploring possible actions to obtain those outcomes and exploiting known strategies to maximize efficiency. The dorsolateral striatum (DLS) has been extensively studied with respect to how actions can develop into habits and has also been implicated as an area involved in governing exploitative behavior. Surprisingly, prior work has shown that DLS cholinergic interneurons (ChIs) are not involved in the canonical habit formation function ascribed to the DLS but are instead modulators of behavioral flexibility after initial learning. To further probe this, we evaluated the role of DLS ChIs in behavioral exploration during a brief instrumental training experiment. Through designer receptors exclusively activated by designer drugs (DREADDs) in ChAT-Cre rats, ChIs in the DLS were inhibited during specific phases of the experiment: instrumental training, free-reward delivery, at both times, or never. Without ChI activity during instrumental training, animals biased their responding toward an "optimal" strategy while continuing to work efficiently. This effect was observed again when contingencies were removed as animals with ChIs offline during that phase, regardless of ChI inhibition previously, decreased responding more than animals with ChIs intact. These findings build upon a growing body of literature implicating ChIs in the striatum as gate-keepers of behavioral flexibility and exploration.

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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1007/s00401-020-02171-5) contains supplementary material, which is available to authorized users.

Cancer genomics has illuminated a wide spectrum of genes and core molecular processes contributing to human malignancy. Still, the genetic and molecular basis of many cancers remains only partially explained. Genetic predisposition accounts for 5-10% of cancer diagnoses(1,2) and genetic events cooperating with known somatic driver events are poorly understood. Analyzing established cancer predisposition genes in medulloblastoma (MB), a malignant childhood brain tumor, we recently identified pathogenic germline variants that account for 5% of all MB patients(3). Here, by extending our previous analysis to include all protein-coding genes, we discovered and replicated rare germline loss-of-function (LoF) variants across Elongator Complex Protein 1 (ELP1) on 9q31.3 in 15% of pediatric MB(SHH) cases, thus implicating ELP1 as the most common MB predisposition gene and increasing genetic predisposition to 40% for pediatric MB(SHH). Inheritance was verified based on parent-offspring and pedigree analysis, which identified two families with a history of pediatric MB. ELP1-associated MBs were restricted to the molecular SHHα subtype(4) and were characterized by universal biallelic inactivation of ELP1 due to somatic loss of chromosome 9q. The majority of ELP1-associated MBs exhibited co-occurring somatic PTCH1 (9q22.32) alterations, suggesting that ELP1-deficiency predisposes to tumor development in combination with constitutive activation of SHH signaling. ELP1 is an essential subunit of the evolutionary conserved Elongator complex, whose primary function is to enable efficient translational elongation through tRNA modifications at the wobble (U(34)) position(5,6). Biochemical, transcriptional, and proteomic analyses revealed that ELP1-associated MB(SHH) are characterized by a destabilized core Elongator complex, loss of Elongator-dependent tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response (UPR), consistent with deregulation of protein homeostasis due to Elongator-deficiency in model systems(7–9). Our findings suggest that genetic predisposition to proteome instability is a previously underappreciated determinant in the pathogenesis of pediatric brain cancer. These results provide a strong rationale for further investigating the role of protein homeostasis in other cancer types and potential opportunities for novel therapeutic interference.

Motivationally attractive cues can draw in behavior in a phenomenon termed incentive salience. Incentive cue attraction is an important model for animal models of drug seeking and relapse. One question of interest is the extent to which the pursuit of motivationally attractive cues is related to the value of the paired outcome or can become unrelated and habitual. We studied this question using a sign-tracking (ST) paradigm in rats, in which a lever stimulus preceding food reward comes to elicit conditioned lever-interaction behavior. We asked whether reinforcer devaluation by means of conditioned taste aversion, a classic test of habitual behavior, can modify ST to incentive cues, and whether this depends upon the manner in which reinforcer devaluation takes place. In contrast to several recent reports, we conclude that ST is indeed sensitive to reinforcer devaluation. However, this effect depends critically upon the congruence between the context in which taste aversion is learned and the context in which it is tested. When the taste aversion successfully transfers to the testing context, outcome value strongly influences ST behavior, both when the outcome is withheld (in extinction) and when animals can learn from outcome feedback (reacquisition). When taste aversion does not transfer to the testing context, ST remains high. In total, the extent to which ST persists after outcome devaluation is closely related to the extent to which that outcome is truly devalued in the task context. We believe this effect of context on devaluation can reconcile contradictory findings about the flexibility/inflexibility of ST. We discuss this literature and relate our findings to the study of habits generally.

Current treatments for obesity are unable to reliably reduce weight over time and alternative treatments need to address this challenge. New interventions that target the nervous system could manipulate brain networks underlying reward, metabolic rate and behavior. Here, the ventral pallidum was evaluated as a target of manipulation due to its established role in the networks underlying motivation, pleasure and behavioral output. Chronic inhibitory or excitatory chemogenetic activation was used to modulate the activity of ventral pallidum (VP) targeted neurons in rats on an obesogenic diet. We hypothesized that inhibition of VP activity would decrease the salience of the rats’ high-sugar, high-fat diet and lead to reduced food consumption and weight gain over time. Paradoxically, measurements of weight, water and food consumption revealed significantly increased weight gain in both groups receiving VP targeted manipulation that was not readily explained by food or water consumption. We theorize that the complex reciprocal feedback between ventral striatal structures (e.g., VP) and metabolic centers of the hypothalamus and brainstem, demonstrated by prior research, likely underpin our findings. This study suggests that the treatment of appetitive disorders (e.g., obesity) with chronic neuromodulation-based interventions could be burdened by the delayed onset of outcomes that are difficult to predict from related prior studies that used acute interventions.

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.

Summary Background Young children with medulloblastoma have a poor overall survival compared with older children, due to use of radiation-sparing therapy in young children. Radiotherapy is omitted or reduced in these young patients to spare them from debilitating long-term side-effects. We aimed to estimate event-free survival and define the molecular characteristics associated with progression-free survival in young patients with medulloblastoma using a risk-stratified treatment strategy designed to defer, reduce, or delay radiation exposure. Methods In this multicentre, phase 2 trial, we enrolled children younger than 3 years with newly diagnosed medulloblastoma at six centres in the USA and Australia. Children aged 3–5 years with newly diagnosed, non-metastatic medulloblastoma without any high-risk features were also eligible. Eligible patients were required to start therapy within 31 days from definitive surgery, had a Lansky performance score of at least 30, and did not receive previous radiotherapy or chemotherapy. Patients were stratified postoperatively by clinical and histological criteria into low-risk, intermediate-risk, and high-risk treatment groups. All patients received identical induction chemotherapy (methotrexate, vincristine, cisplatin, and cyclophosphamide), with high-risk patients also receiving an additional five doses of vinblastine. Induction was followed by risk-adapted consolidation therapy: low-risk patients received cyclophosphamide (1500 mg/m 2 on day 1), etoposide (100 mg/m 2 on days 1 and 2), and carboplatin (area under the curve 5 mg/mL per min on day 2) for two 4-week cycles; intermediate-risk patients received focal radiation therapy (54 Gy with a clinical target volume of 5 mm over 6 weeks) to the tumour bed; and high-risk patients received chemotherapy with targeted intravenous topotecan (area under the curve 120–160 ng-h/mL intravenously on days 1–5) and cyclophosphamide (600 mg/m 2 intravenously on days 1–5). After consolidation, all patients received maintenance chemotherapy with cyclophosphamide, topotecan, and erlotinib. The coprimary endpoints were event-free survival and patterns of methylation profiling associated with progression-free survival. Outcome and safety analyses were per protocol (all patients who received at least one dose of induction chemotherapy); biological analyses included all patients with tissue available for methylation profiling. This trial is registered with ClinicalTrials.gov, number NCT00602667, and was closed to accrual on April 19, 2017. Findings Between Nov 27, 2007, and April 19, 2017, we enrolled 81 patients with histologically confirmed medulloblastoma. Accrual to the low-risk group was suspended after an interim analysis on Dec 2, 2015, when the 1-year event-free survival was estimated to be below the stopping rule boundary. After a median follow-up of 5·5 years (IQR 2·7–7·3), 5-year event-free survival was 31·3% (95% CI 19·3–43·3) for the whole cohort, 55·3% (95% CI 33·3–77·3) in the low-risk cohort (n=23) versus 24·6% (3·6–45·6) in the intermediate-risk cohort (n=32; hazard ratio 2·50, 95% CI 1·19–5·27; p=0·016) and 16·7% (3·4–30·0) in the high-risk cohort (n=26; 3·55, 1·66–7·59; p=0·0011; overall p=0·0021). 5-year progression-free survival by methylation subgroup was 51·1% (95% CI 34·6–67·6) in the sonic hedgehog (SHH) subgroup (n=42), 8·3% (95% CI 0·0–24·0%) in the group 3 subgroup (n=24), and 13·3% (95% CI 0·0–37·6%) in the group 4 subgroup (n=10). Within the SHH subgroup, two distinct methylation subtypes were identified and named iSHH-I and iSHH-II. 5-year progression-free survival was 27·8% (95% CI 9·0–46·6; n=21) for iSHH-I and 75·4% (55·0–95·8; n=21) for iSHH-II. The most common adverse events were grade 3–4 febrile neutropenia (48 patients [59%]), neutropenia (21 [26%]), infection with neutropenia (20 [25%]), leucopenia (15 [19%]), vomiting (15 [19%]), and anorexia (13 [16%]). No treatment-related deaths occurred. Interpretation The risk-adapted approach did not improve event-free survival in young children with medulloblastoma. However, the methylation subgroup analyses showed that the SHH subgroup had improved progression-free survival compared with the group 3 subgroup. Moreover, within the SHH subgroup, the iSHH-II subtype had improved progression-free survival in the absence of radiation, intraventricular chemotherapy, or high-dose chemotherapy compared with the iSHH-I subtype. These findings support the development of a molecularly driven, risk-adapted, treatment approach in future trials in young children with medulloblastoma. Funding American Lebanese Syrian Associated Charities, St Jude Children's Research Hospital, NCI Cancer Center, Alexander and Margaret Stewart Trust, Sontag Foundation, and American Association for Cancer Research.

Our knowledge of the brain changes that enable habits to be formed continues to grow rapidly. As a key hub for habits, many studies have focused on neurobiological processes related to habits in the striatum. Attention has been paid to the contributions of the direct and indirect pathways, interneurons, dopaminergic inputs, and potential cortical and amygdala influences. We highlight this research here and conclude with a discussion of several additional topics that are also being addressed to propel our understanding of habits further forward.

Protected areas are increasingly being viewed and acknowledged within broader social-ecological landscapes as providing a range of ecosystem services, which offer an important connection between nature and society. We explore non-mechanised adventure racing as a form of nature-based tourism, how this activity enables access to a suite of cultural ecosystem services, and its facilitation by a network of relatively open-access protected landscapes. An international adventure race, set within the Garden Route, South Africa, was used as a case study. The physical setting (appreciating nature’s beauty and experiencing the environment in a different way) played the most important role as a motivating factor for participation within adventure racing. Mountainous scenery, rugged coastlines and encounters with iconic species along with the challenge, physical exertion and social bonding also contributed strongly towards the overall experience. Social media and live tracking provided an opportunity for broad exposure and a virtual experience of cultural ecosystem services at a range of spatial scales. In this manuscript we contribute to profiling adventure tourism within a cultural ecosystem service framework, and highlight some implications for protected area management.

Understanding the interplay between normal development and tumorigenesis, including the identification and characterization of lineage-specific origins of MB, is a fundamental challenge in the field. Recent studies have highlighted novel associations between biologically distinct MB subgroups and diverse murine cerebellar lineages via cross-species single-cell transcriptomics. Specifically, Group 4-MB correlated with the unipolar brush cell lineage and Group 3-MB resembled Nestin+ stem cells of the early cerebellum. However, these analyses were hampered by low resolution due to the sparsity of pertinent cerebellar cell types and the cross-species nature of the approach. Herein, we profoundly expand the depth of these rare developmental populations in the murine cerebellum using a combination of lineage tracing and integrative multi-omics. Isolation and enrichment of spatially and temporally unique developmental trajectories of key rhombic lip-derived glutamatergic lineages provided an enhanced reference for mapping MB subgroups based on molecular overlap, especially for poorly defined Group 3- and Group 4-MB. Further comparisons to a novel single-cell atlas of the human fetal cerebellum, companioned with laser-capture microdissected transcriptional and epigenetic datasets, reinforced developmental insights extracted from the mouse. Characterization of compartment-specific transcriptional programs and co-expression networks identified in the human upper rhombic lip implicated convergent cellular correlates of Group 3- and Group 4-MB, suggestive of a common developmental link. Together, our results strongly implicate developmental lineages of the upper rhombic lip as the probable origins of poorly defined Group 3- and Group 4-MB. These important findings will shape future efforts to accurately model the biological heterogeneity underlying these subgroups and provide unprecedented opportunities to explore their cellular and mechanistic basis.

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 (CNVs) 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 decline 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.

Medulloblastoma (MB), a common malignant pediatric brain tumor, comprises at least four distinct molecular entities: WNT, SHH, Group 3, and Group 4. SHH-MB is driven by aberrant activation of the Sonic hedgehog (SHH) pathway in granule neuron progenitors (GNPs) and is associated with hereditary cancer predisposition syndromes including Li Fraumeni and Gorlin. We recently identified germline loss of function (LoF) mutations affecting IKBKAP/ELP1, the primary scaffolding subunit of the Elongator complex in a subset of SHH-MB patients. Germline ELP1 mutations account for ~15% of all pediatric SHH-MBs and position ELP1 as the most prevalent hereditary predisposition gene in MB. We genetically engineered Elp1 LoF in mouse GNPs to determine Elp1 function in cerebellar development and SHH-MB. Results of both mechanistic and phenotypic experiments demonstrate that GNPs harboring Elp1 loss exhibit ribosome pausing and protein aggregation, reinforcing the critical role of Elp1 in translational elongation and protein homeostasis. Further, we generated new transgenic mouse models mimicking germline ELP1 LoF mutations observed in SHH-MB patients. Elp1+/- transgenic mice exhibit purkinje cell degeneration and an increased DNA damage response. These mice are currently being evaluated for their capacity to recapitulate ELP1-associated SHH-MB. Additional analyses carried out on SHH-MB patient-derived xenografts showed that ELP1-mutant tumor cells specifically exhibit defects in tRNA biogenesis. Therefore, the function of ELP1 as a translational regulator is severely impaired in ELP1-mutant SHH-MBs. Our ongoing molecular and functional studies will provide important insights into the most common MB predisposition gene and will lay the foundation for future preclinical studies.

BACKGROUND Our previous analysis of established cancer predisposition genes in medulloblastoma (MB) identified pathogenic germline variants in ~5% of all patients. Here, we extended our analysis to include all protein-coding genes. METHODS Case-control analysis performed on 795 MB patients against >118,000 cancer-free children and adults was performed to identify an association between rare germline variants and MB. RESULTS Germline loss-of-function variants of Elongator Complex Protein 1 (ELP1; 9q31.3) were strongly associated with SHH subgroup (MBSHH). ELP1-associated-MBs accounted for ~15% (29/202) of pediatric MBSHH cases and were restricted to the SHHα subtype. ELP1-associated-MBs demonstrated biallelic inactivation of ELP1 due to somatic chromosome 9q loss and most tumors exhibited co-occurring somatic PTCH1 (9q22.32) alterations. Inheritance was verified by parent-offspring sequencing (n=3) and pedigree analysis identified two families with a history of pediatric MB. ELP1-associated-MBSHH were characterized by desmoplastic/nodular histology (76%; 13/17) and demonstrated a favorable clinical outcome when compared to TP53-associated-MBSHH (5-yr OS 92% vs 20%; p-value=1.3e-6) despite both belonging to the SHHα subtype. ELP1 is a subunit of the Elongator complex, that promotes efficient translational elongation through tRNA modifications at the wobble (U34) position. Biochemical, transcriptional, and proteomic analyses revealed ELP1-associated-MBs exhibit destabilization of the core Elongator complex, loss of tRNA wobble modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response. CONCLUSIONS We identified ELP1 as the most common MB predisposition gene, increasing the total genetic predisposition for pediatric MBSHH to 40%. These results mark MBSHH as an overwhelmingly genetically-predisposed disease and implicate disruption of protein homeostasis in MBSHH development.

Nuclear organization of genomic DNA affects DNA damage and repair processes, and yet its impact on mutational landscapes in cancer genomes remains unclear. Here we analyzed genome-wide somatic mutations from 366 samples of 6 cancer types. We found that lamina-associated regions, which are typically localized at the nuclear periphery, displayed higher somatic mutation frequencies compared to the inter-lamina regions at the nuclear core. This effect remained even after adjusting for features such as GC%, chromatin, and replication timing. Furthermore, mutational signatures differed between the nuclear core and periphery, indicating differences in the patterns of DNA damage and/or DNA repair processes. For instance, smoking and UV-related signatures were more enriched in the nuclear periphery. Substitutions at certain motifs were also more common in the nuclear periphery. Taken together, we found that the nuclear architecture influences mutational landscapes in cancer genomes beyond the effects already captured by chromatin and replication timing.

Salt appetite, in which animals can immediately seek out salt when under a novel state of sodium deprivation, is a classic example of how homeostatic systems interface with learned associations to produce an on-the-fly updating of motivated behavior. Neural activity in the ventral pallidum (VP) has been shown to encode changes in the value of salt under such conditions, both the value of salt itself (Tindell et al., 2006) and the motivational value of its predictive cues (Tindell et al., 2009; Robinson and Berridge, 2013). However, it is not known whether the VP is necessary for salt appetite in terms of seeking out salt or consuming salt following sodium depletion. Here, we used a conditioned place-preference procedure to investigate the effects of optogenetically inhibiting the VP on context-driven salt seeking and the consumption of salt following deprivation. Male rats learned to associate one context with sucrose and another context with less-desirable salt. Following sodium depletion, and in the absence of either sucrose or salt, we found that inhibiting the VP selectively reduced the elevation in time spent in the salt-paired context. VP inhibition had minimal effects on the consumption of salt once it was made available. To our knowledge, this is the first evidence that the VP or any brain region is necessary for the ability to use contextual cues to guide salt seeking. These results highlight a dissociation between deficit-driven reward seeking and reward consumption to replenish those deficits, with the former process being particularly sensitive to on-line VP activity. SIGNIFICANCE STATEMENT Salt appetite, in which rats will immediately seek out a once-undesirable concentrated salt solution after being depleted of bodily sodium despite never having tasted salt as a positive reward, is a phenomenon showing how animals can update their motivational goals without any new learning or conditioning. This salt-seeking behavior is also observed when the animal is presented with salt-paired cues. The neural circuitry necessary for context-driven salt-seeking behavior is unknown. We used a novel conditioned place preference procedure to show that optogenetic inhibition of the ventral pallidum (VP), a region known for processing reward, impairs context-driven salt seeking and has minimal effects on the consumption of salt itself following sodium depletion. These results highlight the importance of the VP in context-driven reward-seeking behavior.

INTRODUCTION Recurrent medulloblastoma (MB) confers an abysmal prognosis with ~10% 5-year overall survival. Optimal treatment paradigms for relapsed disease are largely unknown. Conservation of molecular subgroup at relapse has been described and divergent clonal evolution implicated, yet multi-dimensional molecular characterization of larger cohorts are warranted to substantiate these findings and to disclose potential mechanisms underlying treatment failure and disease recurrence. METHODS A multi-institutional series of 85 patient-matched, primary MBs and their relapses was profiled by DNA methylation array (Illumina 450K/850K). Entity and molecular subgroup classifications were assigned using random forest tumor classifiers (Molecular Neuropathology v.11b4). Genome-wide copy-number aberrations were also inferred from these data while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (1%), SHH (41%), Group 3 (22%), Group 4 (35%), primary tumors largely retained subgroup affiliation at relapse with the notable exception of 14% of cases. The majority (8/12) of discrepant classifications were determined to be secondary glioblastomas, while three Group 4 primary tumors relapsed as Group 3. Amongst conserved pairs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with an average of 0.8 (range 0–5) primary-specific and 2.0 (range 0–11) relapse-specific cytogenetic alterations. Despite high global methylation correlations between primary/relapse pairs (median 0.95), epigenetic evolution at relapse is suggested by the lower degree of conservation amongst promoter-associated probes (median 0.83, p < 2.2e-16). CONCLUSION Secondary malignancy, particularly glioblastoma, may masquerade as relapsed MB, highlighting the utility of molecular characterization for relapsed disease and necessity for vigilant clinical surveillance. By deciphering the evolution of MB from diagnosis to relapse, a fundamental understanding of disease pathogenesis may be garnered, motivating rational and targeted clinical interventions in early therapy and at the time of relapse.

Medulloblastoma is a malignant childhood cerebellar tumor comprised of 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. We used single-cell transcriptomics to investigate intra- and inter-tumoral heterogeneity in twenty-five medulloblastomas spanning all molecular subgroups. WNT, SHH, and Group 3 tumors comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumors were exclusively comprised of differentiated neuronal-like neoplastic cells. SHH tumors closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumors exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, whose relative proportions 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 novel insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

Despite clear evidence linking the basal ganglia to the control of outcome insensitivity (i.e., habit) and behavioral vigor (i.e., its behavioral speed/fluidity), it remains unclear whether or how these functions relate to one another. Here, using male Long–Evans rats in response-based and cue-based maze-running tasks, we demonstrate that phasic dorsolateral striatum (DLS) activity occurring at the onset of a learned behavior regulates how vigorous and habitual it is. In a response-based task, brief optogenetic excitation at the onset of runs decreased run duration and the occurrence of deliberative behaviors, whereas midrun stimulation carried little effect. Outcome devaluation showed these runs to be habitual. DLS inhibition at run start did not produce robust effects on behavior until after outcome devaluation. At that time, when the DLS was plausibly most critically required for performance (i.e., habitual), inhibition reduced performance vigor measures and caused a dramatic loss of habitual responding (i.e., animals quit the task). In a second cue-based “beacon” task requiring behavior initiation at the start of the run and again in the middle of the run, DLS excitation at both time points could improve the vigor of runs. Postdevaluation testing showed behavior on the beacon task to be habitual as well. This pattern of results suggests that one role for phasic DLS activity at behavior initiation is to promote the execution of the behavior in a vigorous and habitual fashion by a diverse set of measures.SIGNIFICANCE STATEMENTOur research expands the literature twofold. First, we find that features of a habitual behavior that are typically studied separately (i.e., maze response performance, deliberation movements, running vigor, and outcome insensitivity) are quite closely linked together. Second, efforts have been made to understand “what” the dorsolateral striatum (DLS) does for habitual behavior, and our research provides a key set of results showing “when” it is important (i.e., at behavior initiation). By showing such dramatic control over habits by DLS activity in a phasic time window, plausible real-world applications could involve more informed DLS perturbations to curb intractably problematic habits.

BACKGROUND Metastasis, residual disease, and diffuse anaplasia are high-risk features in medulloblastoma. METHODS This was a randomized phase 3 study. Patients age 3–21 years with high-risk medulloblastoma received (+/-) daily carboplatin with 36Gy craniospinal radiation and weekly Vincristine followed by six cycles of maintenance chemotherapy with Cisplatin, Cyclophosphamide and Vincristine (+/) 12 cycles of isotretinoin during and following maintenance. The primary endpoint was event-free survival, with exact log-rank test to compare arms. Retrospective molecular analysis included DNA methylation and exome sequencing. RESULTS Of 294 medulloblastoma patients enrolled, 261 were eligible by central review of radiology and pathology, median age 8.6 years (range 3.3–21.2), 70% male, 189 (72%) with metastatic disease, 58 (22%) with diffuse anaplasia, 14 (5%) with >1.5cm2 residual disease. The 5-year EFS and OS for all subjects was 63%+4 and 73%+3, respectively. Isotretinoin randomization was closed due to futility. 5-year EFS was 66 + 5 with carboplatin versus 59 + 5 without (p=0.11), with effect exclusively observed in Group 3 subtype: 73%+8 with carboplatin versus 54%+9 without (p CONCLUSIONS Therapy intensification with carboplatin improved survival for high-risk group 3 medulloblastoma. These findings further support an integrated clinical and molecular risk stratification for medulloblastoma.

INTRODUCTION The next generation of clinical trials for relapsed medulloblastoma demands a thorough understanding of the clinical behavior of relapsed tumors as well as the molecular relationship to their diagnostic counterparts. METHODS A multi-institutional molecular cohort of patient-matched (n=126 patients) diagnostic MBs and relapses/subsequent malignancies was profiled by DNA methylation array. Entity, subgroup classification, and genome-wide copy-number aberrations were assigned while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (2%), SHH (41%), Group 3 (18%), Group 4 (39%), primary tumors retained subgroup affiliation at relapse with the notable exception of 10% of cases. The majority (8/13) of discrepant classifications were determined to be secondary glioblastomas. Additionally, rare (n=3) subgroup-switching events of Group 4 primary tumors to Group 3 relapses were identified coincident with MYC/MYCN pathway alterations. Amongst truly relapsing MBs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with Group 3 (55% of alterations shared) and Group 4 tumors (63% alterations shared) sharing a larger proportion of cytogenetic alterations compared to SHH tumors (42% alterations shared; Chi-square p-value < 0.001). Subgroup- and gene-specific patterns of conservation and divergence amongst putative driver genes were also observed. CONCLUSION Integrated molecular analysis of relapsed MB discloses potential mechanisms underlying treatment failure and disease recurrence while motivating rational implementation of relapse-specific therapies. The degree of genetic divergence between primary and relapsed MBs varied by subgroup but suggested considerably higher conservation than prior estimates.

OBJECTIVE To evaluate differences in time to recurrence among molecular subgroups of medulloblastoma treated on a single protocol and to identify factors associated with survival after first recurrence. METHODS Time to recurrence following SJMB03 treatment was compared across methylation subgroups among relapsed patients. Therapies received subsequent to relapse were noted. Kaplan-Meier methods and log-rank tests were used for statistical analyses. RESULTS 74 of 330 medulloblastoma patients developed recurrence after initial therapy. (38 Standard-Risk; 36 High-Risk). The 2- and 5-year survival after first recurrence was 30.4% and 14.6% respectively. DNA methylation-based subgroups from initial diagnosis were SHH (n=14), Group 3 (n=24), Group 4 (n=26), and unclassified (n=8). None of the pts with WNT MB had recurrent disease. Median time to first recurrence was 1.23, 0.91, and 3.09 years in SHH, Group3, and Group 4 respectively. Group 4 patients had longer post-recurrence survival than others (p-value=0.0169). Clinical risk at diagnosis (p-value=0.337), anaplasia (p-value=0.4032), TP53 (p-value=0.1969), MYC (p-value=0.8967), and MYCN (p value = 0.9404) abnormalities were not associated with post progression survival. Patients who received any therapeutic modality (chemotherapy, re-radiation and second surgery) had longer survival and those who had all three (n=10) had the best outcome (p-value CONCLUSION Outcome after recurrence in medulloblastoma is dismal, however, association with subgroups is still present. Group 4 patients had a longer time to recurrence and post progression survival. No other prognostic factor at initial diagnosis was associated with outcome after recurrence. Patients who received all 3 types of conventional therapy had better survival.

The COG conducted a randomized trial for average-risk medulloblastoma (AR-MB). Patients age 3–21 years were randomized to a radiation boost to the whole posterior fossa (PFRT) or an involved field volume (IFRT) after receiving CSI. Patients age 3–7 years were also randomized to standard dose CSI (23.4Gy, SDCSI) or low dose CSI (18Gy, LDCSI). 464 evaluable patients were available to compare PFRT vs. IFRT and 226 for SDCSI vs. LDCSI. 380 cases had sufficient tissue for DNA methylation-based molecular classification: 362 confirmed medulloblastoma; 6 non-medulloblastoma; 12 inconclusive. Molecular subgrouping confirmed the following representation amongst the evaluable cohort: 156 Group 4 (43.1%), 76 Group 3 (21.0%), 66 SHH (18.2%), 64 WNT (17.7%). Five-year event-free survival (EFS) estimates were 82.5±2.7% and 80.5±2.7% for IFRT and PFRT, respectively (p=0.44). Five-year EFS estimates were 71.4±4.4% and 82.9±3.7% for LDCSI and SDCSI, respectively (p=0.028). EFS distributions differed significantly by subgroup (p

Evaluating outcomes of behavior is a central function of the striatum. In circuits engaging the dorsomedial striatum, sensitivity to goal value is accentuated during learning, whereas outcome sensitivity is thought to be minimal in the dorsolateral striatum and its habit-related corticostriatal circuits. However, a distinct population of projection neurons in the dorsolateral striatum exhibits selective sensitivity to rewards. Here, we evaluated the outcome-related signaling in such neurons as rats performed an instructional T-maze task for two rewards. As the rats formed maze-running habits and then changed behavior after reward devaluation, we detected outcome-related spike activity in 116 units out of 1,479 recorded units. During initial training, nearly equal numbers of these units fired preferentially either after rewarded runs or after unrewarded runs, and the majority were responsive at only one of two reward locations. With overtraining, as habits formed, firing in nonrewarded trials almost disappeared, and reward-specific firing declined. Thus error-related signaling was lost, and reward signaling became generalized. Following reward devaluation, in an extinction test, postgoal activity was nearly undetectable, despite accurate running. Strikingly, when rewards were then returned, postgoal activity reappeared and recapitulated the original early response pattern, with nearly equal numbers responding to rewarded and unrewarded runs and to single rewards. These findings demonstrate that outcome evaluation in the dorsolateral striatum is highly plastic and tracks stages of behavioral exploration and exploitation. These signals could be a new target for understanding compulsive behaviors that involve changes to dorsal striatum function., National Institute of Mental Health (U.S.) (Grant R01-MH-060379), United States. Office of Naval Research (Grant N00014-04-1-0208)