Medulloblastoma Group Research Articles

IN VIVO APPLICATION OF LOCAL DRUG DELIVERY SYSTEMS AGAINST MEDULLOBLASTOMA GROUP 3 AND ATYPICAL/TERATOID/RHABOID TUMOURS

Abstract AIMS Local drug delivery systems (LDDSs) applied during neurosurgery offer a means of overcoming poor blood brain barrier (BBB) permeability and have been extensively investigated for the treatment of adult gliomas, but to the best of our knowledge, not for the treatment of paediatric cerebellar tumours. Here we report the applicability of an LDDS against medulloblastoma group 3 (MB3) and atypical teratoid/rhaboid tumours (AT/RT), both of which are associated with poor prognoses. METHOD We prepared and characterised an injectable and biodegradable poly(ethyleneglycol)-poly(caprolactone)- poly(ethyleneglycol) (PECE) hydrogel, which has been investigated with an array of therapeutic agents against numerous cancer types. Here, we loaded the PECE hydrogel with chemotherapeutics previously identified as being effective against primary MB3 and AT/RT in vitro, but which do not cross the BBB in vivo, namely CHIR99021, ribavirin and PG545. Biocompatibility and cytotoxicity of drug loaded hydrogels was assessed in vitro. LDDSs safety and efficacy was evaluated using patient derived MB3 and AT/RT intracranial xenograft surgical resection models. RESULTS The hydrogel was both biocompatible and effected cytotoxicity following drug release. In vivo efficacy experiments against MB3 indicated a comparable median survival in treatment arms receiving radiotherapy or CHIR99021 and PG545 loaded LDDS. However, combined radiotherapy and LDDS conferred a significant survival enhancement, including long-term survivors. Against AT/RT, the median survival of arms receiving radiotherapy was comparable to that of the CHIR99021 and ribavirin loaded LDDS, with long-term survivors observed only in the latter arm. CONCLUSION The application of LDDSs against cerebellar brain tumours offers a promising novel therapeutic alternative. For MB3, the combination of radiotherapy and a LDDS significantly enhances survival compared to radiotherapy alone. For AT/RT, the comparable survival of the LDDS and radiotherapy alone is encouraging and indicates the possibility of circumventing radiation-induced adverse effects for young children impacted by this disease.

ENTPD1 (CD39) and NT5E (CD73) expression in human medulloblastoma: an in silico analysis.

Medulloblastoma is the most common malignant tumor in the pediatric population. Its classification has incorporated key molecular variations alongside histological characterization. CD39 (also known as ENTPD1) and CD73 (also known as NT5E), enzymes of the purinergic signaling pathway, act in synergy to generate extracellular adenosine, creating an immunosuppressive tumor microenvironment. Our study examined the expression of mRNA of these genes in previously described transcriptome data sets of medulloblastoma patient samples from the Cavalli Cohort (n = 763). Survival distribution was estimated according to the Kaplan-Meier method using a median cut-off and log-rank statistics (p ≤ 0.05). In non-WNT and non-SHH medulloblastoma Group 4 (n = 264), the high expression of ENTPD1 and NT5E was significantly related to a lower overall survival (p = 2.7e-04; p = 2.6e-03). In the SHH-activated group (n = 172), the high expression of ENTPD1 was significantly related to lower overall survival (p = 7.8e-03), while the high expression of NT5E was significantly related to greater overall survival (p = 0.017). In the WNT group (n = 63), the expressions of ENTPD1 and NT5E were not significantly correlated with overall survival (p = 0.212; p = 0.101). In non-WNT and non-SHH medulloblastoma Group 3 (n = 113), the high expression of ENTPD1 was significantly related to greater survival (p = 0.034), while expression of NT5E was not significantly related to survival of patients (p = 0.124). This in silico analysis indicates that ENTPD1 (CD39) and NT5E (CD73) can be seen as potential prognostic markers and therapeutic targets for primary medulloblastomas in non-WNT and non-SHH Group 4.

Identification of a prognosis-related phagocytosis regulator gene signature in medulloblastoma

ObjectivesThe aims of this study were to screen for phagocytosis regulator-related genes in tissue samples from children with medulloblastoma (MB) and to construct a prognostic model based on those genes. MethodsDifferentially expressed genes between the MB and control groups were identified using the GSE50161 dataset from the Gene Expression Omnibus database. Prognosis-related phagocytosis regulator genes were selected from the GSE85217 dataset. Intersecting genes of the two datasets (differentially expressed prognosis-related phagocytosis regulator genes) were submitted to unsupervised cluster analysis to identify disease subtypes, after which the association between the subtypes and the immune microenvironment was analyzed. A prognostic risk score model was constructed, and functional, immune-related, and drug sensitivity analyses were performed. ResultsIn total, 23 differentially expressed prognosis-related phagocytosis regulator genes were identified, from which two disease subtypes (clusters 1 and 2) were classified. The prognoses of the patients in cluster 2 were significantly worse than those of the patients in cluster 1. The immune microenvironment differed significantly between the two subtypes. Finally, 10 genes (FAM81A, EZR, NDUFB9, RCOR1, FOXO4, NHLRC2, KIF23, PTPN6, SMAGP, and MED13) were selected to establish the prognostic risk score model. The prognosis in the low-risk group was better than that in the high-risk group. The model genes NDUFB9 and PTPN6 were positively correlated with M2 macrophages. ConclusionTen key phagocytosis regulator genes were screened to construct a prognostic model for MB. These genes may serve as key biomarkers for predicting the prognosis of patients with this type of brain cancer.

MDB-106. MEDULLOBLASTOMA TUMOR MICROENVIRONMENTS SHOW INTRA- AND INTER-TUMORAL HETEROGENEITY ON SPATIAL TRANSCRIPTOMICS

Abstract BACKGROUND Medulloblastoma defines a heterogeneous set of neuroepithelial neoplasms of the posterior fossa. Four groups of medulloblastoma are recognized: wingless (WNT), sonic hedgehog (SHH), group 3, and group 4. The tumor microenvironment (TME) influences tumor progression and response to therapy and has emerged as a growing target for the study of novel therapeutic approaches. METHODS We show spatial gene expression architecture through 10X Visium Spatial Sequencing on 16 formalin-fixed paraffin-embedded (FFPE) samples representing all molecular groups of medulloblastoma. Spatial transcriptomics data is correlated with clinical parameters, including M-stage at diagnosis, tumor histological classification, and outcomes data including survival and relapse status. RESULTS Spatial sequencing demonstrates both intra- and inter-tumoral TME heterogeneity across molecular subgroups. Unsupervised clustering and uniform manifold approximate projection illustrate clusters of distinct spatial gene expression representing cell states from different components of the TME, including tumor-associated astrocytes (TAA), macrophages (TAM), and vascular endothelium. Medulloblastoma cells constitute the majority of cells and express marker genes representing different stages of neuronal differentiation and cell cycle. Pathway analysis reveals the enrichment of pathways associated with cell motility (RHO GTPase, MET, MAPK signaling), heat shock response, growth factor signaling, and adaptive immune response (TLR, MHC-II signaling). In addition, molecular pathways known in medulloblastoma, including NF-kB and TP53 regulatory pathways, were also enriched. Neighborhood-enrichment analysis reveals the co-localization of TAMs and TAAs in close spatial relationship with mitotic progenitor-like medulloblastoma cells. Additionally, these two cell types constitute a greater proportion of the TME in patients at relapse compared to initial diagnosis. CONCLUSION Spatial transcriptomics enables new insight into the heterogeneity of the medulloblastoma TME. Notably, our analysis elucidates the spatial association of TAMs and TAAs with proliferating tumor cells and an increased abundance of these cell types following relapse. These initial results support the role of TAMs and TAAs in medulloblastoma progression.

MDB-12. NANOPORE SEQUENCING AS A CUTTING-EDGE TECHNOLOGY FOR MEDULLOBLASTOMA CLASSIFICATION

Abstract INTRODUCTION Medulloblastoma (MB) is one of the most prevalent embryonal malignant brain tumors, divided into four distinct molecular subgroups (WNT, SHH, group 3, and group 4) with its individual prognosis. A more extensive classification of MB has recently been provided, identifying numerous subtypes, some with poor prognosis, leading to a significant number of patients succumbing to the disease while many survivors experience long-term side effects, highlighting the need for efficient subgrouping methods. Distinct genome-wide DNA methylation profiles characterize each MB subgroup, and the advent of Nanopore DNA sequencing has positioned itself as a reliable technique for comprehensive copy number and methylation profiling. MATERIAL AND METHODS We evaluated the ability of Nanopore sequencing to provide clinically relevant methylation and copy number profiles of MB, on a discovery cohort of 45 frozen MB samples, benchmarked against the gold standard EPIC array, and subsequently validated on a cohort of 116 MB samples. RESULTS The majority of MB in both the discovery cohort (43/45; 95.6%) and the validation cohort (110/116; 94.8%) were accurately subgrouped by Nanopore sequencing. Flongle flow cells for 18 MB allowed for a more rapid and cost-effective analysis, with 94.4% (17/18) being correctly classified. Additionally, Nanopore sequencing allowed us to correctly subtype 24/30 (80.0%) within MB groups. DISCUSSION We provided proof of concept that Nanopore sequencing can subgroup MB using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples and cell-free DNA obtained from cerebrospinal fluid. This first large-scale study establishes the proof of concept that this modern and innovative technology is well-suited for MB classification. We confirm the use of Nanopore sequencing on circulating DNA and present an initial exploration of its application on DNA extracted from FFPE samples. Its ability to deliver quick and cost-effective results firmly establishes Nanopore sequencing as a game-changer in the field of MB classification.

METB-15. ROLE OF DNA METHYLATION PROFILING IN THE MANAGEMENT OF PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS: A SINGLE INSTITUTIONAL SERIES

Abstract BACKGROUND DNA methylation profiling has now been integrated into the WHO classification of CNS tumors and serves as confirmatory means to diagnose CNS tumors in conjunction with genetic sequencing/microarray analysis. The practical impact on patient care in the retrospective and prospective settings is less known. METHODS Methylation analysis was performed on a cohort of previously and newly diagnosed children with CNS tumors at Children’s Hospital Orange County from 2022-2023. Data regarding histologic diagnosis, integrated molecular diagnosis, methylation classification, timing of result completion and functional impact of methylation results on patient diagnosis/management was evaluated. RESULTS Forty patients (21 boys, ages 5 weeks-17 years) with previously diagnosed (N=11) or newly diagnosed (N=21) CNS tumors were evaluated by methylation analysis at one or more institutions. The most common tumor types were diffuse type pediatric high-grade glioma (N=6) and ependymoma PFA (n=4). Methylation analysis revealed no match in two cases (ALK-EML4 fusion embryonal tumor and recurrent low grade glioneuronal tumor) and had insufficient DNA extraction in one patient with low grade glioneuronal spinal cord tumor. Among the 5 tumors with duplicate methylation analyses at two institutions, three Ependymoma PFA subgroups were concordant while two showed differing classifications (diffuse type pediatric high-grade glioma RTK1 subtype vs. non- classifiable and medulloblastoma group 4 vs. non-WNT, non-SHH). In patients with previously diagnosed CNS tumors, methylation either led to a change in classification or modification of diagnosis in 7 out of 11 patients. The average time from surgery to the receipt of methylation results for newly diagnosed patients was 24 days (range 4-90 days). CONCLUSION Methylation analysis was beneficial in both the previously diagnosed and newly diagnosed setting in patients with previously difficult to classify tumors following conventional molecular analysis. Changes in tumor classification or modifications in diagnosis were observed in the majority of patients.

PATH-08. PRE-OPERATIVE TUMOUR CLASSIFICATION USING NANOPORE SEQUENCING OF CEREBROSPINAL FLUID

Abstract BACKGROUND The introduction of tumour classification by analysis of DNA methylation has changed the landscape of paediatric neuro-oncology. With the launch of Oxford Nanopore sequencing, generation of sequencing data is possible in real-time, including information on base-modifications. Diverse classification tools for tumour tissue have been developed recently. While this is an important step towards optimization of surgical treatment, pre-operative knowledge on tumour entity would further facilitate surgical planning. Therefore, we investigated the possibility of liquid biopsy for rapid pre-operative tumour classification. METHODS CSF samples from patients with pre-operative extra-ventricular drain (EVD) (n = 5) as well as samples obtained via Ommaya reservoirs in cases of suspected recurrence (n = 3) were analysed. Additionally, samples from non-tumour patients (epilepsy, inflammation) who had lumbar puncture were available as controls. Samples were sequenced on MinIon devices (Oxford Nanopore) and subsequently classified with previously published tumour tissue classification pipelines (NanoDx, crossNN, Sturgeon). RESULTS Even though CSF derived from EVD has been stored on room temperature for up to 72 hours before processing, cfDNA isolation was possible with the advantage of higher CSF input amounts. DNA fragments showed a distribution characteristic for cell-free DNA, with a maximum peak at 170bp. The amount of total input DNA ranged from 0.35ng to 12ng, with higher input amounts generating more sequencing data but not resulting in better classification. Classification by sturgeon and crossNN/nanoDx using CSF derived cfDNA was possible in exemplary cases, such as a medulloblastoma patient with a new inoperable lesion, where CSF analysis resulted in medulloblastoma Group 4 within the first hour of sequencing. In total, 38% of samples were correctly classified. CONCLUSION Liquid biopsy as a method to pre-operatively classify tumours and thereby enable risk-adjusted surgery is feasible. Ongoing efforts focus on optimisation of ctDNA based tumour classification by extension of the cohort.

SURG-02. INTRAOPERATIVE APPLICATION OF A POLYMER-BASED HYDROGEL AGAINST MEDULLOBLASTOMA GROUP 3 AND ATYPICAL TERATOID/RHABDOID TUMOURS

Abstract BACKGROUND Local drug delivery systems (LDDSs) applied during neurosurgery offer a means of overcoming poor blood brain barrier (BBB) permeability and have been extensively investigated for the treatment of adult gliomas. To the best of our knowledge, they have not been investigated for the treatment of paediatric cerebellar tumours, including medulloblastoma group 3 (MB3) and atypical teratoid/rhabdoid tumours (ATRT), both of which are associated with poor prognoses. METHODS We prepared and characterised an injectable and biodegradable poly(ethyleneglycol)-poly(caprolactone)-poly(ethyleneglycol) (PECE) hydrogel, which has been investigated broadly throughout the literature with an array of therapeutic agents against numerous cancer types. Here, we loaded the PECE hydrogel with chemotherapeutics previously identified as being effective against primary MB3 and ATRT in vitro, but which do not cross the BBB in vivo, namely CHIR99021, ribavirin and PG545. Biocompatibility and cytotoxicity of drug loaded hydrogels was assessed in vitro. LDDSs safety and efficacy was evaluated using patient derived MB3 and ATRT intracranial xenograft resection models. RESULTS The hydrogel was both biocompatible and effected cytotoxicity following drug release. In vivo efficacy experiments against MB3 indicated a comparable median survival in treatment arms receiving radiotherapy or CHIR99021 and PG545 loaded LDDS. However, combined radiotherapy and LDDS conferred a significant survival enhancement including long-term survivors. Against ATRT, the median survival of arms receiving radiotherapy was comparable to that of the CHIR99021 and ribavirin loaded LDDS, with long-term survivors observed only in the latter arm. CONCLUSIONS The application of LDDSs against cerebellar brain tumours offers a promising novel therapeutic alternative. For MB3, the combination of radiotherapy and a LDDS significantly enhances survival compared to radiotherapy alone. For ATRT, the comparable survival of the LDDS and radiotherapy alone is encouraging and indicates the possibility of circumventing radiation-induced adverse effects for young children impacted by this disease.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

Development of a high-throughput screening platform to identify new therapeutic agents for Medulloblastoma Group 3

Pediatric brain tumors (PBTs) represent about 25 % of all pediatric cancers and are the most common solid tumors in children and adolescents. Medulloblastoma (MB) is the most frequently occurring malignant PBT, accounting for almost 10 % of all pediatric cancer deaths. MB Group 3 (MB G3) accounts for 25–30 % of all MB cases and has the worst outcome, particularly when associated with MYC amplification. However, no targeted treatments for this group have been developed so far.Here we describe a unique high throughput screening (HTS) platform specifically designed to identify new therapies for MB G3. The platform incorporates optimized and validated 2D and 3D efficacy and toxicity models, that account for tumor heterogenicity, limited efficacy and unacceptable toxicity from the very early stage of drug discovery. The platform has been validated by conducting a pilot HTS campaign with a 1280 lead-like compound library. Results showed 8 active compounds, targeting MB reported targets and several are currently approved or in clinical trials for pediatric patients with PBTs, including MB. Moreover, hits were combined to avoid tumor resistance, identifying 3 synergistic pairs, one of which is currently under clinical study for recurrent MB and other PBTs.

Metabolite profiles of medulloblastoma for rapid and non-invasive detection of molecular disease groups

The malignant childhood brain tumour, medulloblastoma, is classified clinically into molecular groups which guide therapy. DNA-methylation profiling is the current classification 'gold-standard', typically delivered 3-4 weeks post-surgery. Pre-surgery non-invasive diagnostics thus offer significant potential to improve early diagnosis and clinical management. Here, we determine tumour metabolite profiles of the four medulloblastoma groups, assess their diagnostic utility using tumour tissue and potential for non-invasive diagnosis using invivo magnetic resonance spectroscopy (MRS). Metabolite profiles were acquired by high-resolution magic-angle spinning NMR spectroscopy (MAS) from 86 medulloblastomas (from 59 male and 27 female patients), previously classified by DNA-methylation array (WNT (n=9), SHH (n=22), Group3 (n=21), Group4 (n=34)); RNA-seq data was available for sixty. Unsupervised class-discovery was performed and a support vector machine (SVM) constructed to assess diagnostic performance. The SVM classifier was adapted to use only metabolites (n=10) routinely quantified from invivo MRS data, and re-tested. Glutamate was assessed as a predictor of overall survival. Group-specific metabolite profiles were identified; tumours clustered with good concordance to their reference molecular group (93%). GABA was only detected in WNT, taurine was low in SHH and lipids were high in Group3. The tissue-based metabolite SVM classifier had a cross-validated accuracy of 89% (100% for WNT) and, adapted to use metabolites routinely quantified invivo, gave a combined classification accuracy of 90% for SHH, Group3 and Group4. Glutamate predicted survival after incorporating known risk-factors (HR=3.39, 95% CI 1.4-8.1, p=0.025). Tissue metabolite profiles characterise medulloblastoma molecular groups. Their combination with machine learning can aid rapid diagnosis from tissue and potentially invivo. Specific metabolites provide important information; GABA identifying WNT and glutamate conferring poor prognosis. Children with Cancer UK, Cancer Research UK, Children's Cancer North and a Newcastle University PhD studentship.

Mitochondrial subtype MB-G3 contains potential novel biomarkers and therapeutic targets associated with prognosis of medulloblastoma

BackgroundMedulloblastoma is the most common malignant brain tumor in children. There are four groups, each with different causal mutations, affected pathways and prognosis. Here, we investigated the role of mitochondria in medulloblastoma and whether there are differences between the different groups.MethodsWe compared the gene expression levels in the four different medulloblastoma groups (MB-WNT, MB-SHH, MB-G3 and MB-G4), with the focus on genes associated with mitochondria. We used several tools including Salmon, Tximeta, DESeq2, BiomaRt, STRING, Ggplot2, EnhancedVolcano, Venny 2.1 and Metscape.ResultsA total of 668 genes were differentially expressed and the most abundant genes were associated with cell division pathway followed by modulation of chemical synaptic transmission. We also identified several genes (ABAT, SOX9, ALDH5A, FOXM1, ABL1, NHLH1, NEUROD1 and NEUROD2) known to play vital role in medulloblastoma. Comparative expression analysis revealed OXPHOS complex-associated proteins of mitochondria. The most significantly expressed genes in the MB-SHH and MB-G4 groups were AHCYL1 and SFXN5 while PAICS was significantly upregulated in MB-WNT group. Notably, MB-G3 contained the most downregulated genes from the OXPHOS complexes, except COX6B2 which was strongly upregulated.ConclusionsWe show the importance of mitochondria and compare their role in the four different medulloblastoma groups.

Outcomes Following Radiation Therapy (RT) for Very Young Age CNS Embryonal Tumors on COG ACNS0334 According to Molecular-Confirmed Diagnosis

The outcomes of upfront or relapse radiation therapy (RT) for the Children's Oncology Group ACNS0334 protocol based on molecular diagnosis were assessed. Therapy included maximal safe surgery, high-dose chemotherapy with stem cell rescue, randomization for inclusion of high dose methotrexate (MTX) and optional RT. There were 24 patients that received RT on COG ACNS0334 of 77 evaluable patients with a diagnosis of either high-risk medulloblastoma (MB) or supratentorial primitive neuroectodermal tumor (SPNET). RT was a recommendation (M0: Focal, M+: CSI 18 Gy) given young patient age <36 months at enrollment. Seven RT patients were excluded for ineligible pathology (1 ATRT, 1 HGG) or insufficient tissue. The aim of this report is to review outcomes of 17 patients on ACNS0334 receiving RT (8 Upfront, 9 at relapse) with a molecular diagnosis that included MB, Pineoblastoma (PB), or Embryonal tumor with multilayered rosettes (ETMR). In the MB group, there were 9 patients irradiated with MB (Group 3 = 8, SHH = 1). 5-year OS for MB Group 3 receiving RT (median primary dose 54 Gy) was 62.5% with no difference observed comparing 6 patients treated with upfront RT versus 2 treated at relapse (p = 0.27). All upfront RT for MB Group 3 had initial partial response (PR) to 0334 chemotherapy. RT delivery for upfront RT MB Group 3 included craniospinal radiation (CSI) in 5 patients and 1 patient who received focal RT to the primary (50 Gy) and metastatic site (44 Gy). Eighty percent of CSI for upfront RT in Group 3 was 18 Gy or 23.4 Gy. Relapse RT for MB Group 3 (2 patients) utilized full dose CSI (36 Gy, 39.6 Gy) and both patients are survivors with 5+ years follow-up. CSI dose for Group 3 MB was higher for relapse RT (mean 37.8 Gy) as compared to upfront RT (mean 19.8 Gy, p = 0.013). Use of MTX was 50% in both upfront RT and relapse RT Group 3 MB cohorts. One patient with MB SHH (classic histology) underwent upfront focal RT (54 Gy) after initial PR to systemic therapy (without MTX) and is surviving 5+ years. PB: Of 4 PB patients (median primary dose 48.8 Gy) 1 had RT upfront (CSI 18 Gy) and 3 had RT at relapse (1 patient received CSI, 21 Gy). All patients with PB expired within 2 years. MTX was given in 75% (including 1 upfront RT PB). Two of 3 patients treated at relapse had prior complete response (CR). ETMR: All 4 patients (median primary dose 54 Gy) with ETMR were treated at relapse, with CSI given in 1 patient (23.4 Gy). All patients with ETMR expired within 2 years, and 2 (50%) had received MTX. Two patients (50%) had initial CR. The RT cohort for Group 3 MB on ACNS0334 exhibited long-term survival both for both upfront and relapse RT, however relapsed Group 3 MB received higher dose CSI. RT upfront for MB, including one surviving MB SHH patient receiving focal RT, was solely given for incomplete initial chemotherapy response. There were no survivors for either PB or ETMR when the majority (88%) were treated at relapse.

EpiGe: A machine-learning strategy for rapid classification of medulloblastoma using PCR-based methyl-genotyping

Molecular classification of medulloblastoma is critical for the treatment of this brain tumor. Array-based DNA methylation profiling has emerged as a powerful approach for brain tumor classification. However, this technology is currently not widely available. We present a machine-learning decision support system (DSS) that enables the classification of the principal molecular groups-WNT, SHH, and non-WNT/non-SHH-directly from quantitative PCR (qPCR) data. We propose a framework where the developed DSS appears as a user-friendly web-application-EpiGe-App-that enables automated interpretation of qPCR methylation data and subsequent molecular group prediction. The basis of our classification strategy is a previously validated six-cytosine signature with subgroup-specific methylation profiles. This reduced set of markers enabled us to develop a methyl-genotyping assay capable of determining the methylation status of cytosines using qPCR instruments. This study provides a comprehensive approach for rapid classification of clinically relevant medulloblastoma groups, using readily accessible equipment and an easy-to-use web-application.t.

Mitochondrial DNA mutations in Medulloblastoma

To date, several studies on genomic events underlying medulloblastoma (MB) biology have expanded our understanding of this tumour entity and led to its division into four groups—WNT, SHH, group 3 (G3) and group 4 (G4). However, there is little information about the relevance of pathogenic mitochondrial DNA (mtDNA) mutations and their consequences across these. In this report, we describe the case of a female patient with MB and a mitochondriopathy, followed by a study of mtDNA variants in MB groups. After being diagnosed with G4 MB, the index patient was treated in line with the HIT 2000 protocol with no indications of relapse after five years. Long-term side effects of treatment were complemented by additional neurological symptoms and elevated lactate levels ten years later, resulting in suspected mitochondrial disease. This was confirmed by identifying a mutation in the MT-TS1 gene which appeared homoplasmic in patient tissue and heteroplasmic in the patient’s mother. Motivated by this case, we explored mtDNA mutations across 444 patients from ICGC and HIT cohorts. While there was no statistically significant enrichment of mutations in one MB group, both cohorts encompassed a small group of patients harbouring potentially deleterious mtDNA variants. The case presented here highlights the possible similarities between sequelae caused by MB treatment and neurological symptoms of mitochondrial dysfunction, which may apply to patients across all MB groups. In the context of the current advances in characterising and interpreting mtDNA aberrations, recognising affected patients could enhance our future knowledge regarding the mutations’ impact on carcinogenesis and cancer treatment.

Low Expression of the NRP1 Gene Is Associated with Shorter Overall Survival in Patients with Sonic Hedgehog and Group 3 Medulloblastoma.

Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor. Neuropilin-1 (NRP1), encoded by the NRP1 gene, is a transmembrane glycoprotein overexpressed in several types of cancer. Previous studies indicate that NRP1 inhibition displays antitumor effects in MB models and higher NRP1 levels are associated with poorer prognosis in MB patients. Here, we used a large MB tumor dataset to examine NRP1 gene expression in different molecular subgroups and subtypes of MB. We found overall widespread NRP1 expression across MB samples. Tumors in the sonic hedgehog (SHH) subgroup showed significantly higher NRP1 transcript levels in comparison with Group 3 and Group 4 tumors, with SHH samples belonging to the α, β, Δ, and γ subtypes. When all MB subgroups were combined, lower NRP1 expression was associated with significantly shorter patient overall survival (OS). Further analysis showed that low NRP1 was related to poorer OS, specifically in MB subgroups SHH and Group 3 MB. Our findings indicate that patients with SHH and Group 3 tumors that show lower expression of NRP1 in MB have a worse prognosis, which highlights the need for subgroup-specific investigation of the NRP1 role in MB.

Group-specific cellular metabolism in Medulloblastoma

BackgroundCancer metabolism influences multiple aspects of tumorigenesis and causes diversity across malignancies. Although comprehensive research has extended our knowledge of molecular subgroups in medulloblastoma (MB), discrete analysis of metabolic heterogeneity is currently lacking. This study seeks to improve our understanding of metabolic phenotypes in MB and their impact on patients’ outcomes.MethodsData from four independent MB cohorts encompassing 1,288 patients were analysed. We explored metabolic characteristics of 902 patients (ICGC and MAGIC cohorts) on bulk RNA level. Moreover, data from 491 patients (ICGC cohort) were searched for DNA alterations in genes regulating cell metabolism. To determine the role of intratumoral metabolic differences, we examined single-cell RNA-sequencing (scRNA-seq) data from 34 additional patients. Findings on metabolic heterogeneity were correlated to clinical data.ResultsEstablished MB groups exhibit substantial differences in metabolic gene expression. By employing unsupervised analyses, we identified three clusters of group 3 and 4 samples with distinct metabolic features in ICGC and MAGIC cohorts. Analysis of scRNA-seq data confirmed our results of intertumoral heterogeneity underlying the according differences in metabolic gene expression. On DNA level, we discovered clear associations between altered regulatory genes involved in MB development and lipid metabolism. Additionally, we determined the prognostic value of metabolic gene expression in MB and showed that expression of genes involved in metabolism of inositol phosphates and nucleotides correlates with patient survival.ConclusionOur research underlines the biological and clinical relevance of metabolic alterations in MB. Thus, distinct metabolic signatures presented here might be the first step towards future metabolism-targeted therapeutic options.Graphical

Transcriptome analysis stratifies second-generation non-WNT/non-SHH medulloblastoma subgroups into clinically tractable subtypes.

Medulloblastoma (MB), one of the most common malignant pediatric brain tumor, is a heterogenous disease comprised of four distinct molecular groups (WNT, SHH, Group 3, Group 4). Each of these groups can be further subdivided into second-generation MB (SGS MB) molecular subgroups, each with distinct genetic and clinical characteristics. For instance, non-WNT/non-SHH MB (Group 3/4) can be subdivided molecularly into eight distinct and clinically relevant tumor subgroups. A further molecular stratification/summarization of these SGS MB would allow for the assignment of patients to risk-associated treatment protocols. Here, we performed DNA- and RNA-based analysis of 574 non-WNT/non-SHH MB and analyzed the clinical significance of various molecular patterns within the entire cohort and the eight SGS MB, with the aim to develop an optimal risk stratification of these tumors. Multigene analysis disclosed several survival-associated genes highly specific for each molecular subgroup within this non-WNT/non-SHH MBcohort with minimal inter-subgroup overlap. These subgroup-specific and prognostically relevant genes were associated with pathways that could underlie SGS MB clinical-molecular diversity and tumor-driving mechanisms. By combining survival-associated genes within each SGS MB, distinct metagene sets being appropriate for their optimal risk stratification were identified. Defined subgroup-specific metagene sets were independent variables in the multivariate models generated for each SGS MB and their prognostic value was confirmed in a completely non-overlapping validation cohort of non-WNT/non-SHH MB (n = 377). In summary, the current results indicate that the integration of transcriptome data in risk stratification models may improve outcome prediction for each non-WNT/non-SHH SGS MB. Identified subgroup-specific gene expression signatures could be relevant for clinical implementation and survival-associated metagene sets could be adopted for further SGS MB risk stratification. Future studies should aim at validating the prognostic role of these transcriptome-based SGS MB subtypes in prospective clinical trials.

IGFBP2 promotes proliferation and cell migration through STAT3 signaling in Sonic hedgehog medulloblastoma

Medulloblastoma (MB) is the most common pediatric brain malignancy and is divided into four molecularly distinct subgroups: WNT, Sonic Hedgehog (SHHp53mut and SHHp53wt), Group 3, and Group 4. Previous reports suggest that SHH MB features a unique tumor microenvironment compared with other MB groups. To better understand how SHH MB tumor cells interact with and potentially modify their microenvironment, we performed cytokine array analysis of culture media from freshly isolated MB patient tumor cells, spontaneous SHH MB mouse tumor cells and mouse and human MB cell lines. We found that the SHH MB cells produced elevated levels of IGFBP2 compared to non-SHH MBs. We confirmed these results using ELISA, western blotting, and immunofluorescence staining. IGFBP2 is a pleiotropic member of the IGFBP super-family with secreted and intracellular functions that can modulate tumor cell proliferation, metastasis, and drug resistance, but has been understudied in medulloblastoma. We found that IGFBP2 is required for SHH MB cell proliferation, colony formation, and cell migration, through promoting STAT3 activation and upregulation of epithelial to mesenchymal transition markers; indeed, ectopic STAT3 expression fully compensated for IGFBP2 knockdown in wound healing assays. Taken together, our findings reveal novel roles for IGFBP2 in SHH medulloblastoma growth and metastasis, which is associated with very poor prognosis, and they indicate an IGFBP2-STAT3 axis that could represent a novel therapeutic target in medulloblastoma.

Medulloblastoma: The latest major advances

Medulloblastoma (MB) is a malignant brain tumor that mainly affects children. It is rarely found in adults. Among the four groups of MB defined today according to molecular characteristics, group 3is the least favorable with an overall survival rate of 50%. Current treatments, based on surgery, radiotherapy, and chemotherapy, are not sufficiently adapted to the different characteristics of the four MB groups. However, the use of new cellular and animal models has opened new doors to interesting therapeutic avenues. In this review, we detail recent advances in MB research, with a focus on the genes and pathways that drive tumorigenesis, with particular emphasis on the animal models that have been developed to study tumor biology, as well as advances in new targeted therapies.