Identification and Functional Analysis of a Novel CTNNB1 Mutation in Pediatric Medulloblastoma

Medulloblastoma is the primary malignant tumor of the Central Nervous System (CNS) most common in pediatrics. We present here, the histological, molecular, and functional analysis of a cohort of 88 pediatric medulloblastoma tumor samples. The WNT-activated subgroup comprised 10% of our cohort, and all WNT-activated patients had exon 3 CTNNB1 mutations and were immunostained for nuclear β-catenin. One novel heterozygous CTNNB1 mutation was found, which resulted in the deletion of β-catenin Ser37 residue (ΔS37). The ΔS37 β-catenin variant ectopically expressed in U2OS human osteosarcoma cells displayed higher protein expression levels than wild-type β-catenin, and functional analysis disclosed gain-of-function properties in terms of elevated TCF/LEF transcriptional activity in cells. Our results suggest that the stabilization and nuclear accumulation of ΔS37 β-catenin contributed to early medulloblastoma tumorigenesis.

Pediatric medulloblastoma express immune checkpoint B7-H3

Purpose Medulloblastomas (MB) are highly malignant brain tumors that predominantly occur in young infants. Immunotherapy to boost the immune system is emerging as a novel promising approach, but is often hampered by inhibitory immune checkpoints. In the present study, we have studied immune checkpoint B7-H3 expression in a tissue cohort of human pediatric MB. Methods Expression of B7-H3 was detected by immunohistochemistry and classified via B7-H3 staining intensity and percentage of B7-H3 positive tumor cells. Subsequently, B7-H3 protein expression was distinguished in MB molecular subtypes and correlated to immune cell infiltrates, patient characteristics, and survival. Results B7-H3 protein expression was found in 23 out of 24 (96%) human pediatric MB cases and in 17 out of 24 (71%) MB cases > 25% of tumor cells had any level of B7-H3 expression. B7-H3 protein expression was more frequent on Group-4 MB as compared with other molecular subtypes (p = 0.02). Tumors with high B7-H3 expression showed less influx of γδT cells (p = 0.002) and CD3+ T cells (p = 0.041). Conclusion Immune checkpoint B7-H3 is differentially expressed by the large majority of pediatric MB. This further warrants the development of novel B7-H3-directed (immuno)therapeutic methods for children with incurable, metastatic, or chemo-resistant MB.

Subgroup-Specific Diagnostic, Prognostic, and Predictive Markers Influencing Pediatric Medulloblastoma Treatment

Medulloblastoma (MB) is the most common malignant central nervous system tumor in pediatric patients. Mainstay of therapy remains surgical resection followed by craniospinal radiation and chemotherapy, although limitations to this therapy are applied in the youngest patients. Clinically, tumors are divided into average and high-risk status on the basis of age, metastasis at diagnosis, and extent of surgical resection. However, technological advances in high-throughput screening have facilitated the analysis of large transcriptomic datasets that have been used to generate the current classification system, dividing patients into four primary subgroups, i.e., WNT (wingless), SHH (sonic hedgehog), and the non-SHH/WNT subgroups 3 and 4. Each subgroup can further be subdivided on the basis of a combination of cytogenetic and epigenetic events, some in distinct signaling pathways, that activate specific phenotypes impacting patient prognosis. Here, we delve deeper into the genetic basis for each subgroup by reviewing the extent of cytogenetic events in key genes that trigger neoplastic transformation or that exhibit oncogenic properties. Each of these discussions is further centered on how these genetic aberrations can be exploited to generate novel targeted therapeutics for each subgroup along with a discussion on challenges that are currently faced in generating said therapies. Our future hope is that through better understanding of subgroup-specific cytogenetic events, the field may improve diagnosis, prognosis, and treatment to improve overall quality of life for these patients.

Integrated microRNA Analysis Identifies miR-512-3p as a Potential Biomarker of Poor Outcome in Pediatric Medulloblastoma

Background: Medulloblastoma, a genetically heterogeneous tumor, is the most frequent malignant brain tumor in children. Although several studies have been carried out, the molecular mechanism underlying medulloblastoma tumorigenesis is not completely known. microRNA (miRNA) expression profiles have been associated with development, progression, and prognosis of human cancers, including medulloblastoma. However, the role of miRNAs in pediatric medulloblastoma has been poorly explored.Methods: Global miRNA expression in 24 microdissected medulloblastoma specimens (19 pediatric and 5 adult specimens) was evaluated by microarray assay. miR-512-3p, the most differentially expressed miRNA in these two groups, was analyzed by qRT-PCR in a cohort of 51 consecutive pediatric medulloblastoma samples and 7 pediatric non-neoplastic cerebellum control samples, and its clinical significance was assessed. Further in silico miRNA prediction of target genes was performed with bioinformatics tools.Results: Compared to the controls, miR-512-3p was significantly downregulated in the pediatric medulloblastoma samples. Higher miR-512-3p was associated with incomplete degree of resection, high risk group classification, and poor overall survival. In silico analysis in an independent cohort of medulloblastoma identified that some of the miR-512-3p target genes (SMAD9, SSX2IP, MAPK10, PTCH1, CCDC6, and BMPR2) were statistically correlated with overall survival, metastasis, and death.Conclusions: For the first time, our results have shown that miR-512-3p is significantly associated with poor clinical outcome in pediatric medulloblastoma, suggesting that miR-512-3p is a potential biomarker of prognosis.

Identification of an Immunogenic Medulloblastoma-Specific Fusion Involving EPC2 and GULP1

Medulloblastoma is the most common malignant brain tumor in children. Immunotherapy is yet to demonstrate dramatic results in medulloblastoma, one reason being the low rate of mutations creating new antigens in this entity. In tumors with low mutational burden, gene fusions may represent a source of tumor-specific neoantigens. Here, we reviewed the landscape of fusions in medulloblastoma and analyzed their predicted immunogenicity. Furthermore, we described a new in-frame fusion protein identified by RNA-Seq. The fusion involved two genes on chromosome 2 coding for the enhancer of polycomb homolog 2 (EPC2) and GULP PTB domain containing engulfment adaptor 1 (GULP1) respectively. By qRT-PCR analysis, the fusion was detected in 3 out of 11 medulloblastoma samples, whereby 2 samples were from the same patients obtained at 2 different time points (initial diagnosis and relapse), but not in other pediatric brain tumor entities. Cloning of the full-length sequence indicated that the fusion protein contains the N-terminal enhancer of polycomb-like domain A (EPcA) of EPC2 and the coiled-coil domain of GULP1. In silico analyses predicted binding of the neoantigen-derived peptide to HLA-A*0201. A total of 50% of the fusions described in the literature were also predicted to produce an immunogenic peptide. The EPC2-GULP1 fusion peptide was able to induce a de novo T cell response characterized by interferon gamma release of CD8+ cytotoxic T cells in vitro. While the functional relevance of this fusion in medulloblastoma biology remains to be clarified, our data support an immunotherapeutic approach for pediatric medulloblastoma patients carrying the EPC2-GULP1 fusion and other immunogenic fusions.

TMOD-23. MODELING PEDIATRIC BRAIN CANCER WITH HUMAN ORGANOIDS

Among children/infant brain tumors, Medulloblastoma (MB) is the most common and stands as a cause for a high percentage of morbidity and mortality among patients During the past few years, studies on human MB have uncovered the existence of four major MB groups: WNT, SHH, Group3 and Group4. Patients with Group3 MB currently have the worst outcome among the four groups, and nearly 50% are metastatic at the time of diagnosis. In the last 3 years in our laboratory, we have developed a novel pediatric Medulloblastoma organoid model. We generated human iPSC-derived cancer organoids upon c-MYC/OTX2 and C-MYC/Gfi1 overexpression, mimicking human MB genetic alterations. Furthermore, the use of DNA methylation signature in combination with MB-specific markers analysis indicates that our organoid-based cancer model recapitulates several features of human MB. Now, we are taking advantage of this technology to produce novel brain cancer organoids that we are using to address cancer biology questions.

924 The spatial hierarchy of primary and recurrent medulloblastoma tumor ecosystems

BackgroundMedulloblastoma recurrence occurs in approximately 30% of patients and is universally fatal, presenting one of the most significant unmet clinical challenges in pediatric oncology. It is now clear that medulloblastomas are complex ecosystems, evolving under selective pressure from their microenvironment and cell of origin. Different tumor-immune cell interactions, including, but not limited to, tumor-infiltrating lymphocytes and various tumor suppressive myeloid cell populations, significantly hamper effective treatment strategies for primary, metastatic, and recurrent tumors. Recurrent medulloblastomas are rarely biopsied making biological material for interrogation scarce. Research has assumed that recurrent and primary medulloblastoma tumors have similar biological composition and therefore will respond to the same therapeutic regimens, however, therapies designed using primary biopsies, but tested in Phase I/II trials on children with recurrent disease, have been largely unsuccessful. We hypothesize that there are select immunosuppressive population differences within primary vs. recurrent tumor microenvironments (TME) that need to be elucidated in order to improve treatment modalities and outcomes in pediatric patients.MethodsUsing Akoya’s MOTiFTM PD-1/PD-L1 Panel: Auto Melanoma Kit, the staining protocol was adapted for optimal staining performance on human brain tissue. Following this, 24-formalin-fixed, paraffin embedded pediatric medulloblastoma samples (primary and recurrent biopsies from 12 patients) were stained for the following markers on the Leica BOND RX. Multispectral images were acquired using the Vectra Polaris, and five regions of interest selected on each image. An analysis algorithm was developed using inForm tissue analysis software, and samples were batch processed and data exported. Cell counts, densities, and spatial parameters were generated using the R-script package phenoptrReports to produce outputs of the image analysis data.ResultsFollowing spectral unmixing and autofluorescence isolation, no signal crosstalk was observed. The average signal intensity counts for all markers was found to be within the recommended ranges of 10–30, with a coefficient of variation of ≤15%, indicating successful and consistent staining of the medulloblastoma samples. Comparison between primary vs. recurrent tissues revealed distinctive spatial differences between immune-tumor cell interactions.ConclusionsWe have demonstrated successful adaptation of the MOTiF PD-1/PD-L1 Melanoma panel kit in conjunction with the Phenoptics workflow to support examination of the TME in patient-matched primary and recurrent pediatric medulloblastoma tumor biopsies. Our study provides the first insight into distinctive spatial interactions between primary vs. recurrent tissues, which may improve strategies to comprehend cancer progression, immune surveillance, and ultimately the development of rational, targeted therapeutics based on the differences between the tumor compartments and their immune-microenvironment.Ethics ApprovalEthical approval obtained by Brain UK, ref: 20/008. All participants gave consent to use of their material.

Gene-Specific DNA Methylation Profiles in Pediatric Medulloblastomas

Introduction Medulloblastoma is the most common pediatric central nervous tumor of high malignancy that has been classified into both histological subtypes and molecular subgroups by the 2016 World Health Organization classification. However, there is a still need to understand the genomic characteristics and predict the clinical course. The aim of the study is to investigate the significance of the methylation profiles in molecular subclassification and precision medicine of the disease. Methods The study enrolled 47 pediatric medulloblastoma patients. DNA methylation levels of KLF4, SPINT2, RASSF1A, EZH2, ZIC2, and PTCH1 genes were analyzed using methylation-specific pyrosequencing. The significance of the statistical relationship between methylation profiles and clinicopathological parameters including molecular subgroups and histological subtypes, the status of metastasis, and event-free survival were analyzed. Results DNA methylation analysis demonstrated that KLF4, PTCH1, and ZIC2 hypermethylation were associated with the SHH-activated subgroup, whereas both SPINT2 and RASSF1A hypermethylation were associated with metastatic disease. EZH2 gene was not methylated in any of the samples. Conclusion We think that customized DNA methylation profiling may be a useful tool in the molecular subclassification of pediatric medulloblastoma and a potential technical approach in precision medicine.