Medulloblastoma Subtypes Research Articles

EXTH-02. MODIFYING CAR T CELL EPIGENETIC PROGRAMS TO IMPROVE THEIR PERSISTENCE AGAINST GROUP 3 MEDULLOBLASTOMAS

Abstract BACKGROUND Safe, effective therapies are desperately needed to improve outcomes for patients with medulloblastoma (MB), a malignant pediatric brain cancer. Chimeric antigen receptor (CAR)-T cells are a promising therapeutic option as they target T-cell cytotoxicity exclusively to tumor associated antigens. B7 homolog 3 (B7-H3) is an ideal target for CAR-T cell therapy as all MB subtypes express B7-H3 but healthy tissues do not. However, a major roadblock to tumor clearance and control is the inability of CAR-T cells to persist, suggesting that improving persistence will improve therapeutic efficacy. The goal of this study was to compare head-to-head the impact of knocking out TET2 and DNMT3A negative epigenetic regulators of CAR-T cell persistence against group 3 MB (G3MB), the most malignant MB subtype. METHODS We generated B7-H3 CAR-T cells with CD28ζ signaling domain and performed CRISPR/Cas9 knockout (KO) of TET2, DNMT3A, and AAVS1 as a control. We compared resulting CAR-T cell persistence via repeat stimulation assays in vitro against G3MB cell lines with varying B7-H3 antigen density (D341low, HDMB03mid, and D425high). We further compared KO CAR-T cells in vivo against D341low, HDMB03mid, and D425high orthotopic xenografts. RESULTS Our data shows that DNMT3A KO, but not TET2 KO, consistently improves the persistence and expansion of CAR-T cells against G3MB in vitro. In vivo, KO of epigenetic regulators, improves CAR-T cell therapy against highly aggressive D341low, HDMB03mid, and D425high but is insufficient to elicit curative responses. CONCLUSIONS Our study demonstrates that genetic KO of negative epigenetic regulators of CAR-T cell persistence can improve anti-tumor function against G3MB. We conclude that for the B7-H3.CD28ζ CAR, DNMT3A KO is superior for improving persistence over TET2 KO, but that DNMT3A KO is insufficient in vivo.

EXTH-49. DEVELOPMENTAL ANTIGENS CAN TARGET EMBRYONAL PEDIATRIC BRAIN CANCERS IN ADOPTIVE CELLULAR THERAPY

Abstract Mutations in tumor neoantigens have shown promise in immunotherapies for many cancers, yet pediatric brain tumors, which typically have a lower mutational burden, offer fewer opportunities to capitalize on these targeted therapies. Studies indicate that medulloblastoma (MB) and brain stem gliomas (BSG) originate from abnormal reactivation of post-natal developmental processes. This led us to investigate whether proteins expressed during the early post-natal development of the mouse cerebellum and brainstem could be powerful antigens to fight MB and BSG, respectively. We performed both in vitro and in vivo studies in mice with the RNA of developmental antigens from the brain stem and cerebellum in an adoptive cellular therapy (ACT) platform. We evaluated the reactivity and therapeutic efficacy of these treatments as sustainable antigenic targets against BSG and MBs. We demonstrate that T cells activated towards these non-mutated, tissue-specific developmental antigens can recognize distinct subtypes of MB and BSG, and activate specifically without cross-reacting to the normal brain. This conferred a survival benefit in established orthotopic models of these pediatric brain tumor types. Our research exhibits the value developmental antigens can offer when serving as tumor rejection antigens in MB and BSG.

Tenovin-6 exhibits inhibitory effects on the growth of Sonic Hedgehog (SHH) medulloblastoma, as evidenced by both in vitro and in vivo studies

Medulloblastoma (MB) is the most common malignant brain tumor in children. Within MB, tumors driven by the Sonic Hedgehog (SHH) pathway represent the most heterogeneous subtype, known as SHH subtype medulloblastoma (SHH-MB).Tenovin-6, a recognized p53 activator, has been demonstrated to inhibit autophagy and modulate sirtuin activity, underscoring its potential as a novel therapeutic agent across various malignancies. However, its efficacy in treating SHH-MB remains unexplored. This study aims to investigate the inhibitory effects of tenovin-6 on SHH-MB and elucidate its underlying signaling pathways.We assessed the impact of tenovin-6 on cell proliferation through the CCK-8 and colony formation assays. The scratch and transwell invasion assays were utilized to evaluate the drug’s effects on metastasis. Apoptosis and reactive oxygen species (ROS) levels were measured using flow cytometry. Potential signaling pathways were identified via transcriptomics and quantitative PCR (qPCR).Our in vivo studies involved a mouse xenograft model to explore tenovin-6′s anticancer efficacy against SHH-MB. The findings indicate that tenovin-6 not only inhibits cell proliferation and metastasis in SHH-MB cell lines but also promotes apoptosis, which is closely linked to its proliferation-inhibiting properties. Additionally, animal experiments confirmed that tenovin-6 suppresses MB growth in vivo. We discovered that tenovin-6 reduces intracellular ROS levels and inhibits autophagy in SHH-MB by disrupting the fusion of autophagosomes with lysosomes, likely through inducing autophagosome formation.

A single-cell transcriptomic map of the developing Atoh1 lineage identifies neural fate decisions and neuronal diversity in the hindbrain

Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.

Survival-Related Genes on Chromosomes 6 and 17 in Medulloblastoma.

Survival of Medulloblastoma (MB) depends on various factors, including the gene expression profiles of MB tumor tissues. In this study, we identified 967 MB survival-related genes (SRGs) using a gene expression dataset and the Cox proportional hazards regression model. Notably, the SRGs were over-represented on chromosomes 6 and 17, known for the abnormalities monosomy 6 and isochromosome 17 in MB. The most significant SRG was HMGA1 (high mobility group AT-hook 1) on chromosome 6, which is a known oncogene and a histone H1 competitor. High expression of HMGA1 was associated with worse survival, primarily in the Group 3γ subtype. The high expression of HMGA1 was unrelated to any known somatic copy number alteration. Most SRGs on chromosome 17p were associated with low expression in Group 4β, the MB subtype, with 93% deletion of 17p and 98% copy gain of 17q. GO enrichment analysis showed that both chromosomes 6 and 17 included SRGs related to telomere maintenance and provided a rationale for testing telomerase inhibitors in Group 3 MBs. We conclude that HMGA1, along with other SRGs on chromosomes 6 and 17, warrant further investigation as potential therapeutic targets in selected subgroups or subtypes of MB.

Immunoexpression of Survivin and P53 in the Histological Subtypes of Medulloblastoma: A Cross-Sectional Observational Study.

Medulloblastoma (MB) is a common malignant intracranial neoplasms in children. The treatment and prognosis of this tumor depends on histology and molecular subtypes. Survivin, implicated in various malignancies, may hold prognostic significance. We investigated survivin and p53 immunoreactivity in different histological subtypes in 20 MB cases from January 2018 to June 2021. Immunohistochemistry revealed survivin expression in 75% (15/20) of cases, with cytoplasmic (10 cases), nuclear (four cases), or combined expression (one case). p53 nuclear expression was present in 35% (7/20) of cases. Classical variant MB exhibited predominant p53 and cytoplasmic survivin expression. Given the association of survivin and p53 expression with poor prognosis, especially in the prevalent classical variant, targeted therapies may hold promise for MB treatment advancement.

IMMU-02. MODIFYING CAR T CELL EPIGENETIC PROGRAMS TO IMPROVE THEIR PERSISTENCE AGAINST GROUP 3 MEDULLOBLASTOMAS

Abstract BACKGROUND Safe, effective therapies are desperately needed to improve outcomes for patients with medulloblastoma (MB), a malignant pediatric brain cancer. Chimeric antigen receptor (CAR)-T cells are a promising therapeutic option as they target T-cell cytotoxicity exclusively to tumor associated antigens. B7-homolog 3 (B7-H3) is an ideal target for CAR-T cell therapy as all MB subtypes express B7-H3 and healthy tissues do not. However, a roadblock to tumor clearance is the inability of B7-H3 CAR-T cells to persist, suggesting that improving persistence will benefit anti-tumor efficacy. The goal of this study was to compare head-to-head knocking out TET2 or DNMT3A negative epigenetic regulators of CAR-T cell persistence against group 3 MB (G3MB), the most malignant MB subtype. METHODS We generated B7-H3 CAR-T cells with CD28ζ signaling domain and performed CRISPR/Cas9 knockout (KO) of TET2, DNMT3A, and AAVS1 as a control. We compared resulting CAR-T cell persistence via repeat stimulation assays in vitro against G3MB cell lines with varying B7-H3 antigen density (D341low, HDMB03mid, and D425high). We further compared KO CAR-T cells in vivo against D341low, HDMB03mid, and D425high orthotopic xenografts. RESULTS Our data shows that DNMT3A KO, but not TET2 KO, consistently improves the persistence and expansion of CAR-T cells against G3MB in vitro. In vivo, KO of epigenetic regulators moderately improves CAR-T cell therapy against highly aggressive D341low, HDMB03mid, and D425high but is insufficient to elicit curative responses. CONCLUSIONS Our study demonstrates that genetic KO of negative epigenetic regulators of CAR-T cell persistence can improve anti-tumor function against G3MB. We conclude that for the B7-H3.CD28ζ CAR, DNMT3A KO is superior for improving persistence over TET2 KO, but that DNMT3A KO is insufficient in vivo. We believe this is due to the aggressive nature of G3MB and we are optimizing in vivo studies for better representation of CAR-T cell persistence.

MDB-55. LYMPHOID ENHANCER-BINDING FACTOR 1 (LEF1): AN IMMUNOHISTOCHEMICAL PREDICTIVE MARKER FOR WNT-ACTIVATED MEDULLOBLASTOMA

Abstract AIM To evaluate lymphoid enhancer-binding factor 1 (LEF1) immunohistochemical expression in various molecular subtypes of medulloblastoma. MATERIALS AND METHODS Molecular subtyping of medulloblastomas was based on real-time PCR target 12 gene panel. LEF1 immunohistochemistry was performed and its expression was correlated with histological and molecular subtypes. RESULTS The study cohort included 65 cases; comprising 17 WNT-activated, 20 SHH-activated, and 28 non-WNT/non-SHH [group 3: 11, group 4: 10, non-WNT/non-SHH NOS: 7] medulloblastomas. The age range was 1-40 years (≤18 years=54 [WNTa:15, SHHa:13, non-WNT/non-SHH:26], 19-40 years=11) with a median age of 9, and an interquartile range (IQR) of 5-14.5. Forty-one were males and 24 were females (M:F=1.7:1). LEF1 immunostaining was diffuse (≥50% tumour cells) in 13 cases, focal (5-49% tumour cells) in 11 cases, and negative in 41 cases. All cases with diffuse LEF1 immunostaining (n=13) were WNT-activated with classic histology. Twelve of the 13 cases had CTNNB1 mutation on Sanger sequencing and monosomy 6 by fluorescence in-situ hybridization (FISH) while 1 case was uninterpretable for CTNNB1 Sanger sequencing and chromosome 6 FISH. LEF1 staining intensity was strong in 9 cases while heterogenous in 4 cases. No differential staining pattern was observed for paediatric and adult cases. Diffuse LEF1 immunostaining was significantly associated with the WNT-activated subtype of medulloblastoma (p=<0.01). Of 11 focal LEF1 positive cases, 4 were WNT-activated (all having classic histology and CTNNB1 mutation) and 7 were SHH-activated, TP53-wildtype (classic: 2 and desmoplastic nodular [D/N]: 5). None of the WNT-activated, 12 SHH-activated, TP53-wildtype (6 classic, 6 D/N), 1 SHH-activated, TP53-mutant (large-cell/Anaplastic [LC/A]) and all (n=28) non-WNT/non-SHH (classic: 25, LC/A: 3) were LEF1 negative. CONCLUSION Classic histology with diffuse (≥50% tumor cells; including heterogeneous intensity) LEF1 immunostaining is a good predictive marker for the WNT-activated molecular group of medulloblastoma, however focal staining is not.

MDB-17. A COMPARISON OF MAGNETIC RESONANCE IMAGING AND CEREBROSPINAL FLUID CYTOLOGY FOR DETECTING LEPTOMENINGEAL METASTASIS IN PATIENTS WITH MEDULLOBLASTOMA

Abstract BACKGROUND For medulloblastoma, CSF cytology and MRI together are considered to be superior to either modality alone in identifying leptomeningeal dissemination (LMD). However, MRI has vastly improved since the initial assessment of this approach. METHODS A retrospective medical record review of 130 patients (36.2% female) with medulloblastoma was performed. CSF samples and MR images (6.3% utilized FIESTA) obtained within 1 month of each other from 2000-2023 were analyzed. There were 302 CSF-MRI pairs (range: 1-12 pairs per patient) in total. A 3-tier system (negative, atypical/suspicious, positive) was utilized to account for equivocal findings. RESULTS The mean (SD) age at surgery was 13.2 (13.0) years. Mean (SD) postoperative follow-up time was 7.0 (5.2) years. 71.5% underwent gross or near-total resection, 80.8% received radiotherapy, and 93.1% received chemotherapy. The molecular subtypes were: SHH (24.6%), WNT (4.6%), non-WNT/non-SHH (38.5%), unknown (32.3%). MRI had an 89.8% sensitivity and CSF cytology had a 22.7% sensitivity for detecting LMD that was positive on CSF and/or MRI. MRI was positive in 23.1% of CSF negative cases. CSF was positive in 2.2% of MRI negative cases. For all CSF positive-MRI negative pairs, the CSF samples and MR images were obtained within 10 days of surgery. MRI was positive in 38.9% of CSF atypical cases. CSF was positive in 11.9% of MRI suspicious cases. MRI demonstrated greater or equal ability to CSF in 94.7% of pairs. MRI was more likely to exceed CSF at 3T versus 1.5T (51.3% vs. 27.4%; p<0.01). The overall concordance between CSF cytology and MRI was 61.3%, and this differed by subtype (75.4% SHH vs. 80.0% WNT vs. 52.3% NWNS; p<0.01). CONCLUSIONS Understanding the ability of CSF cytology and MRI to detect LMD is important. While MRI is superior overall, LMD detection varies by MRI Tesla, medulloblastoma subtype, and time since surgery.

MDB-105. CONSTRUCTION OF AN INTERACTIVE MEDULLOBLASTOMA VISUALIZATION PORTAL FROM MOLECULAR AND CLINICAL DATA DERIVED FROM THREE MULTI-INSTITUTIONAL PROTOCOLS (ACNS0331, SJMB03, ACNS0332) TO COMPARE, CONTRAST, AND INVESTIGATE COHORTS AS A MEANS TO REFINE THERAPY

Abstract BACKGROUND Medulloblastoma (MB) patients display a heterogeneous mix of molecular and clinical characteristics that influence tumor behavior and survival. We hypothesized that if these multiple characteristics could be incorporated into an organized, searchable, and interactive dataset, this would advance our understanding and improve treatment of this disease. Consequently, we organized multifaceted datasets into an interactive portal equipped with cutting-edge visualization tools. METHODS Clinical and molecular data from the ACNS0331, SJMB03, and ACNS0332 protocols were collated and harmonized. A data dictionary was built to capture demographics, treatment, histology, methylation classification, mutations, chromosomal copy number variations and outcomes. To display the data, a software framework was applied. Five visualization methods were implemented (summary plots, sample view, scatter plots, sample matrix, and survival plots) in an interconnected manner such that these “cross-talk” with each other. RESULTS Data from 898 patients with MB were incorporated: 131 WNT, 151 SHH, 220 G3, 396 G4. The portal features enabled the creation of custom cohorts that could be instantly queried by any of the dictionary terms. For example, cohorts could be customized to include only cases with a mutation of the user’s choice and analyzed by molecular group, gender, age at diagnosis, or treatment given. Survival of patients with SHH-MB harboring TP53 mutations could be compared to patients with WNT-MB harboring TP53 mutations. Moreover, the influence of MB subtype (e.g. G34_I-VIII), focal amplifications (MYC, MYCN) and whole chromosome aberration phenotype on survival could be queried across treatment regimens accounting for metastatic status and/or additional variables of the user’s choice. CONCLUSION The integration of molecular and clinical data from multiple trials into an interactive visualization data portal presents an unprecedented resource for research and clinical practice. This portal facilitates answering highly specific questions about mutational frequency and patient outcome by molecular and clinical risk factors.

MDB-60. CDK7 INHIBITION DISRUPTS ACQUIRED RESISTANCE MECHANISMS IN MYC-MB

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for 20% of all pediatric brain tumors. Medulloblastoma manifests as a clinically heterogeneous tumor with varying prognoses depending on the molecular subgroup. Group 3 tumors have the worst overall prognosis, and are associated with MYC gene amplification, higher rates of metastasis, and increased likelihood of recurrence. Our prior work demonstrated that WEE1 is upregulated in all MB subtypes and is a critical mediator of MB cell viability. We have established that Myc-driven MB is particularly sensitive to the WEE1 inhibitor AZD1775. Although AZD1775 showed promising results in combination with other genotoxic agents, tumor cells frequently acquire resistance to small molecule inhibitors. Thus, to identify regulators of resistance in Myc-MB we performed a Kinome scale RNAi screen in isogenic parental and resistant cells and identified CDK7 as a key mediator of AZD1775 resistance that restored sensitivity to WEE1 inhibition when depleted. CDK7 controls transcriptional initiation by phosphorylating the c-terminal domain of RNA Pol II. CDK7 has been implicated in several Myc-associated malignancies and is frequently enriched at Super Enhancers. Enhancer-promoter interactions (EPIs) are dynamic and can facilitate resistance mechanisms by inducing additional enhancer elements to promote transcription. Here, we investigated 3D chromatin interactions by HiChIP and further assessed CTCF and H3K27ac patterns after treatment with the CDK7 inhibitor, THZ1, by Cut and Run. Our analysis suggests that AZD1775-resistant cells are reorganized to establish alternate cis-acting elements that reinforce transcription of DNA repair and proliferation genes, circumventing AZD1775 treatment. We further examine cisplatin-resistant MB cells to determine if the mechanism is consistent in Myc-MB cell lines or therapy specific. Overall, we suggest that reorganized chromatin structure aids resistance development and that inhibition of CDK7 intervenes in this process, restoring sensitivity to standard therapies.

MODL-14. TARGETING GL1/GL2-DNMT1-UHRF1 EPIGENETIC COMPLEX DISRUPTION VIA BET INHIBITION IMPAIRS SHH MB DEVELOPMENT

Abstract BACKGROUND Sonic Hedgehog Medulloblastoma (SHH MB) shares 30% among all molecular subtypes of medulloblastomas (MBs). Constitutive activation of SHH signaling in the cerebellum’s granular cell precursors (GCPs) is critical for this MB development. When SHH receptor PATCHED-1 (PTCH1) is mutated, stimulation of this receptor by SHH ligand binding leads to derepression of heterotrimeric GTP-binding protein–coupled receptor Smoothened (SMO) eventually altering the activity, location, and stability of the three downstream glioma associated oncogene (GLI) family members: GLI1, GLI2, and GLI3. GLI1 and GLI2 mostly function as transcriptional activators, while GLI3 mainly plays a repressor role. The bromodomain and extra-terminal domain (BET) family of proteins comprises four conserved members (Brd2, Brd3, Brd4, and Brdt), which are critical regulators of GLI1 and GLI2-mediated transcription. METHODS In the present study, we pharmacologically inhibited BETs with a previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, discovered by our lab. We treated human SHH MB cells (ONS76 cells) in vitro and ex vivo in mouse SHH MB cells (from Ptch1 conditional knockout mice) with minimum effective concentrations of these inhibitors. We screened for GLI1/GLI2-DNMT1-UHRF1 epigenetic complex components expressions by immunoblotting and assessed the effectiveness of inhibiting their interactions by performing PLA assays. RESULTS In this study, we show that pharmacological inhibition of BETs leads to attenuated GLI1 and GLI2 levels. This GLI1 and GLI2 inhibition eventually disrupts the previously reported GL1/GL2, DNMT1-UHRF1 epigenetic complex, which is crucial for SHH MB development. A previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, synthesized and characterized by our lab can inhibit BETs. This could be a potential therapeutic approach targeting GLI-associated epigenetic complexes to treat SHH medulloblastoma.

MDB-98. HISTOPATHOLOGY IS A ROBUST TOOL TO DIFFERENTIATE BETWEEN SHH AND WNT MEDULLOBLASTOMA VERSUS NON-SHH/NON-WNT MEDULLOBLASTOMA: COMPARATIVE ANALYSIS OF HISTOPATHOLOGY, CHROMOSOMAL MICROARRAY, NANOSTRING BASED 22-GENE ASSAY, AND DNA METHYLATION FOR MEDULLOBLASTOMA SUBGROUP ASSIGNMENT ON “HEAD START” 4 CLINICAL TRIAL

Abstract BACKGROUND “Head Start-4” (HS-4) is a prospective, randomized clinic trial that tailors treatment based on medulloblastoma molecular subgroups (WNT, SHH, Group 3, and Group 4) and response to induction chemotherapy, and compares the efficacy of one versus three (tandem) cycles of myeloablative therapy. Here we compare different methodologies used to distinguish between WNT/SHH medulloblastoma (low-risk arm) and non-SHH/non-WNT medulloblastoma (high-risk arm) during the course of the trial. METHODS When HS-4 trial began enrolling patients in 2015, in the absence of a CAP-CLIA certified test for methylation and gene expression profile, we utilized histopathology/immunochemistry (HP/IHC) and chromosomal microarray (CMA) via OncoScanTM (Thermo Fisher) to classify medulloblastoma samples into either WNT, SHH, or non-WNT/non-SHH (Subgroups 3 and 4) at the time of diagnosis. Retrospectively, we performed both NanoString based 22-gene assay and DNA methylation profiling on all patient samples. RESULTS We have HP/IHC, CMA, NanoString and methylation profiling for 54 infants and young children with medulloblastoma enrolled on HS-4. While indeterminate result occurred with CMA in three cases and NanoString in two cases, HP/IHC successfully assigned samples to SHH/WNT and non-SHH/non-WNT arms of the study in all 54 cases. We have HP/IHC, CMA and DNA methylation profiling for additional 33 patients. While pathology/IHC was indeterminate in two cases (one WNT and one group 3 MB), remaining cases accurately categorized medulloblastoma methylation subtype as WNT/SHH and non-WNT/non-SHH. CONCLUSION Due to the long turnaround time, waiting for medulloblastoma molecular subtype confirmation by DNA methylation array may delay treatment initiation. HS-4 data displays robust prediction of WNT/SHH versus non-WNT/non-SHH molecular subtypes by HP/IHC in comparison to DNA methylation profiling and provides a platform to initiate treatment based on HP/IHC while awaiting molecular information. In addition, these data support relying on histopathology for medulloblastoma subtypes in resource poor countries where methylation profiling is not readily available.

Central pathological review and molecular classification of medulloblastoma in countries with limited resources: A commitment to equitable access.

10062 Background: Medulloblastoma, the most common malignant pediatric brain tumor, exhibits molecular heterogeneity influencing prognosis and treatment decisions. This study focuses on feasibility the pathological review and molecular classification with cost-effective methods. Methods: From December 2018 to November 2023, we analysed samples of patients referred to our laboratory. Data were collected on patient demographics (age, gender, origin), pathological review and specific medulloblastoma subtypes identified. The study was performed without costs for parents or institutions. Results: During the study period, 146 patients were included. The predominant age groups being 0 to 4 years (20.1%) and 5 to 9 years (36.1%) The mean age was 10.2 years. 55.6% of patients were female and 43.8% were male. Ninety-three patients were from Brazil and 7% from Latin America. Of the 117 samples that could be analyzed, 43% was classified as group 4, 37% were sonic hedgehog (SHH) group, 10% were group 3 and 4% were WNT group. Five percent of the samples had inconclusive results, with two patients had diagnoses other than medulloblastoma. Seventy percent of samples had a turnaround time for molecular classification results around 30 days. Conclusions: This study highlights the importance of central review pathology and molecular classification in medulloblastoma management. Our results emphasize the feasibility of centralized pathology review and molecular classification in countries with limited resources and provide equity for all patients. Based on this experience, we have expanded for other brain and pediatric tumors. Our effort contributes to ensuring that every patient benefits from the advancements in personalized medicine and this represents a real step to offer the same chances of cure for everyone.

Abstract 4889: Integrative multi-omic analysis reveals novel prognostic biological entities in pediatric medulloblastoma

Abstract Medulloblastoma is a cerebellar tumor for which relapses are associated with poor survival rates of less than 5%. Molecular heterogeneity and dose-limiting toxicities that occur with standard of care approaches, which include surgical resection, craniospinal irradiation, and chemotherapy, complicate the treatment of both primary and recurrent medulloblastoma due to adverse long-term sequela. While the integration of molecular analyses into the histopathology of pediatric medulloblastomas has changed the way these diseases are diagnosed, classified, and treated, there remains an unmet need to further understand the underpinnings of the disease to improve clinical outcomes and minimize neuronal, cognitive, and hormonal toxicities that occur with standard-of-care therapies. We therefore aimed to investigate novel subgroups of pediatric medulloblastoma as a basis for further defining biological properties and targets for the disease through multi-omic characterization. We derived single-nucleotide variant, copy number variant, expression, alternative splicing, and methylation array data from pediatric medulloblastomas profiled as part of the Children’s Brain Tumor Network (CBTN) and the Open Pediatric Cancer (OpenPedCan) projects. Using non-negative matrix factorization across the five datasets derived from genomic, transcriptomic, and epigenomic profiling, we identified 14 clusters found to be prognostically significant by Kaplan-Meier analysis of overall survival (p < 0.0001). Our model further subdivided sonic hedgehog (SHH) and Group 3/4 tumors, but not Wnt-driven tumors, suggesting that the latter forms a homogeneous molecular subgroup. Among our subgroups, we recapitulated the four classically defined medulloblastoma subtypes known to be associated with mutations in KDM6A, KMT2C, CTNNB1, PTCH1, TP53, KBTBD4, MYC, and MYCN. Cascading biological relationships across the copy number, methylation, and expression domains were observed among novel subgroups, potentially driving migratory, immuno-regulatory, GPCR-related, growth factor-related, and histone methylation biological programs. A comparison of subgroup expression profiles to transcriptional signatures from the Library of Integrated Network-Based Cellular Signatures (LINCS) program identified canonical oncogenic pathways unique to each subgroup as potentially targetable, including, but not limited to, MAPK, VEGFA, inflammatory, fatty acid metabolic, and PI3K/AKT signaling. Our work uncovers novel biological entities in pediatric medulloblastoma potentially driven by higher order mechanisms across the genome, transcriptome, and epigenome, with the possibility of informing novel precision medicine approaches for the disease. Citation Format: Komal Rathi, Varun Kesherwani, Ammar S. Naqvi, Yuankun Zhu, Alex Sickler, Xiaoyan Huang, Bo Zhang, Brian Rood, Adam C. Resnick, Adam A. Kraya. Integrative multi-omic analysis reveals novel prognostic biological entities in pediatric medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4889.

Development and validation of a 23-gene expression signature for molecular subtyping of medulloblastoma in a long-term Chinese cohort.

Medulloblastoma is the most common childhood malignant brain tumor andis a leading cause of cancer-related death in children. Recent transcriptional studies have shown that medulloblastomas comprise at least four molecular subgroups, each with distinct demographics, genetics, and clinical outcomes. Medulloblastoma subtyping has become critical for subgroup-specific therapies. The use of gene expression assays to determine the molecular subgroup of clinical specimens is a long-awaited application of molecular biology for this pediatric cancer. In the current study, we established a medulloblastoma transcriptome database of 460 samples retrieved from three published datasets (GSE21140, GSE37382, and GSE37418). With this database, we identified a 23-gene signature that is significantly associated with the medulloblastoma subgroups and achieved a classification accuracy of 95.2%. The 23-gene signature was further validated in a long-term cohort of 142 Chinese medulloblastoma patients. The 23-gene signature classified 21 patients as WNT (15%), 41 as SHH (29%), 16 as Group 3 (11%), and 64 as Group 4 (45%). For patients of WNT, SHH, Group 3, and Group 4, 5-year overall-survival rate reached 80%, 62%, 27%, and 47%, respectively (p < 0.0001), meanwhile 5-year progression-free survival reached 80%, 52%, 27%, and 45%, respectively (p < 0.0001). Besides, SHH/TP53-mutant tumors were associated with worse prognosis compared with SHH/TP53 wild-type tumors and other subgroups. We demonstrated that subgroup assignments by the 23-gene signature and Northcott's NanoString assay were highly comparable with a concordance rate of 96.4%. In conclusion, we present a novel gene signature that is capable of accurately and reliably assigning FFPE medulloblastoma samples to their molecular subgroup, which may serve as an auxiliary tool for medulloblastoma subtyping in the clinic. Future incorporation of this gene signature into prospective clinical trials is warranted to further evaluate its clinical.

FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

Developmental basis of SHH medulloblastoma heterogeneity

Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies.

Cdc14B/Cyclin B1 signaling modulates the pathogenesis of sonic hedgehog subtype medulloblastoma.

Medulloblastoma (MB) is a severe malignancy of the central nervous system that predominantly occurs in the cerebellum of children. Overactivation of the sonic hedgehog (Shh) signaling pathway is the primary cause of the development and progression of Shh subtype MB, although the detailed mechanisms underlying this process remain largely elusive. In this study, we discovered that Shh can promote proliferation in MB cells through non-canonical Hedgehog signaling. This involves Shh binding to Patched 1, disrupting its interaction with Cyclin B1, allowing for nuclear translocation of Cyclin B1, and inducing the activation of genes involved in cell division. Furthermore, we observed that deregulation of Cdc14B leads to the stabilization of the Cyclin B1/CDK1 complex in MB cells through activating Cdc25C, a phosphatase known to help maintain Cyclin B1 stability. Our findings highlight the role of Cdc14B/Cdc25C/CDK1/Cyclin B1 in mediating Hedgehog signaling-driven pathogenesis in MB and have implications for identifying potential therapeutic targets.

EXTH-46. SCHLAFEN11 IS A POWERFUL BIOMARKER OF CHEMOSENSITIVITY IN MEDULLOBLASTOMAS

Abstract Non-biased and biased screening for prognostic factors in medulloblastomas showed that Schlafen family member 11 (SLFN11) is a powerful prognostic marker. SLFN11 is a putative DNA/RNA helicase, whose expression improves response to DNA damaging agents in some cancers, but its role in medulloblastoma has not been reported. SLFN11 protein and mRNA level were found to be expressed exclusively in WNT-activated and a proportion of SHH-activated medulloblastoma. WNT pathway activation did not acutely induce SLFN11, rather, its expression correlated with a specific hypomethylation site on the SLFN11 promoter. Genetic introduction of SLFN11 into medulloblastoma cell lines with no baseline expression dramatically increased their response to the DNA damaging agents cisplatin and SN-38 in vitro. Intracranial xenograft studies also showed markedly increased sensitivity to cisplatin after induced SLFN11 overexpression in medulloblastoma cells. In addition, CRISPR-mediated deletion of SLFN11 in a medulloblastoma cell line with high baseline expression reduced the response to chemotherapy. SLFN11 could also be upregulated pharmacologically by the histone deacetylase-inhibitor RG2833, supporting the importance of epigenetic control. Importantly, markedly increased sensitivity to cisplatin and SN-38 was seen in initially SLFN11-negative medulloblastoma cells also treated with RG2833, suggesting an approach by which more aggressive medulloblastoma subtypes might be targeted. Our findings suggest a novel mechanism for the increased chemosensitivity of some medulloblastoma subtypes linked to a specific biomarker, as well as a novel combinatorial chemotherapeutic strategy for the treatment of more aggressive medulloblastoma subtypes (Groups 3 and 4) lacking SLFN11.